Note: Descriptions are shown in the official language in which they were submitted.
CA 02404763 2002-10-04
WO 01/34645 PCT/US00/31044
MODULATING IL-13 ACTIVITY USING MUTATED IL-13
MOLECULES THAT ARE ANTAGONISTS OR AGONISTS OF
IL-13
RELATED APPLICATIONS
This is a continuation-in-part of U.S. Provisional Application Serial No.
60/165,236, filed November 11, 1999, the contents of which are incorporated by
reference.
STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH
Not applicable.
FIELD OF THE INVENTION
This invention relates to mutated forms of IL-13 which have higher
binding affinity for the IL-13 receptor than does wild-type IL-13. The
invention further
relates to uses and medicaments of various of these mutants which are
antagonists or
agonists, respectively, of IL-13 mediated activity. The invention further
relates to
targeted delivery of agents to cells overexpressing receptors to IL-13 by
using certain of
these IL-13 mutants as targeting moieties.
BACKGROUND OF THE INVENTION
IL-13 is a pleiotropic cytokine which plays a major role in immune
response and inflammation. (Minty, A. et al., Nature 362:248-250 (1993);
Michel, G. et
al., J. Invest. Derm. 103:433-433 (1994); McKenzie, A.N.J. et al., Proc Natl
Acad Sci
(USA) 90:3735-3739 (1993). It can inhibit production of proinflammatory
cytokines IL-
1, IL-6, TNF-alpha and downregulate the expression of CD14 on monocytes.
(Cosentino,
G. et al., J. Immunol. 155:3145-3151 (1995)). CD14 is a LPS receptor which is
important
for monocyte functions. (Lauer, R.P. et al, J. Immunol. 145:1390 (1990);
Wright, S.D. et
al, Science 249:1431 (1990)). IL-13 also plays a major role in B cells. It can
upregulate
CD23, CD72, MHC class II, surface IgM on B cells; drive IgE class switch and
induce
CA 02404763 2002-10-04
WO 01/34645 PCT/US00/31044
production of immunoglobulins by B cells. (Punnonen, J. et al., Proc Natl Acad
Sci
(USA) 90:3730-3734 (1993)).
Recent studies have demonstrated IL-13 plays a prominent role in atopic
dermatitis (Akdis, M. et al., J. Immunol. 159:4611-4619 (1997); Katagiri, K.
et al., Clin.
Exp. Immunol. 108:289-294 (1997)), allergic rhinitis (Pawankar, R U. et al.,
Am. J.
Respir. Crit. Care Med. 152:2059-2067 (1995)) and pulmonary asthma (Wills-
Karp, M.
et al., Science 282:2258-2261 (1998); Grunig, G. et al., Science 282:2261-2263
(1998)).
Targeted pulmonary expression of IL-13 can cause severe pulmonary pathology
including
mononuclear and eosinophilic inflammatory response, mucus cell metaplasia,
airway
fibrosis and obstruction, among other things (Zhou, Z. et al., J. Clin.
Invest. 103:779-788
(1999)), as well as hepatic fibrosis induced by Schistosomiasis (Chiaramonte,
M. G. et
al., J Clin Investig 104:777 (1999)), and susceptibility to Leishmania major
infection
(Matthews, D. J. et al., Jlmmunol 164:1458 (2000)). IL-13 also plays a major
role in
malignancies. It has been shown to be produced by various cancer cell types
including
renal cell carcinoma (Obiri, N. I. et al., Clin. Cancer Res. 2:1743-1749
(1996)). IL-13
has also been shown to be an autocrine growth factor for Hodgkin/Reed-
Sternberg tumor
cells (Kapp, U. et al., J. Exp. Med. 189:1939-1945 (1999); Fricker, J. Mol.
Med. Today
5:463-463 (1999)).
The receptors for IL-13 have been identified on a variety of normal and
malignant cell types (Vita, N. et al., J. Biol. Chem. 270:3512-3517 (1995);
Hilton, D. J. et
al., Proc. Natl. Acad. Sci. U.S.A. 93:497-SO1 (1996); Zurawski, S. M. et al.,
J. Biol.
Chem. 270:13869-13878 (1995); Debinski, W. et al., Clin. Cancer Res. 1:1253-
1258
(1995); Debinski, W. et al., J. Biol. Chem. 270:16775-16780 (1995); Obiri, N.
I. et al., J.
Biol. Chem. 270:8797-8804 (1995); Murata, T. et al., J. Immunol. 156:2972-2978
(1996);
Puri, R K. et al., Blood 87:4333-4339 (1996); Debinski, W. et al., J. Biol.
Chem.
271:22428-22433 (1996); Aman, M. J. et al., J. Biol. Chem. 271:29265-29270
(1996);
Murata, T. et al., Biochem. Biophys. Res. Commun. 238:90-94 (1997); Obiri, N.
I. et al., J.
Biol. Chem. 272:20251-20258 (1997); Husain, S. R et al., Clin. Cancer Res.
3:151-156
(1997); Murata, T. et al., Int. J Cancer 70:230-240 (1997); Obiri, N. I. et
al., J. Immunol.
158:756-764 (1997); Maini, A. et al., J. Urol. 158:948-953 (1997); Debinski,
W. et al.,
Int. J. Cancer 76:547-551 (1998); Murata, T. et al., Int. J. Mol. Med. 1:551-
557 (1998);
Debinski, W. et al., Nat. Biotechnol. 16:449-453 (1998); Murata, T. et al.,
Blood 91:3884-
3891 (1998); Joshi, B. H. et al., Cancer Res. 60:1168-1172 (2000)). IL-13
receptors (IL-
2
CA 02404763 2002-10-04
WO 01/34645 PCT/US00/31044
13R) are over expressed on human solid cancer cell lines including renal cell
carcinoma,
AIDS-associated Kaposi's sarcoma, ovarian carcinoma, prostate cancer, and
malignant
glioma
The function of IL-13 is accomplished through interaction with its plasma
membrane receptors. The IL-13 receptor ("IL-13R") complex appears to exist in
three
different types. In the first type, IL-13 forms a complex with the IL-4
receptor (3 chain
(also known as IL4Ra), IL13R a' (also known as IL13Ra1) and IL-13 R a (also
known
as IL13Ra2). This type of receptor complex is expressed on limited non-
hematopoietic
tumor cell lines such as renal cell carcinoma, AIDS-associated Kaposi's
sarcoma and
glioblastoma multiforme. (Puri, R.K. et al., Blood 87:4333-4339 (1996);
Husain, S.R. et
al., Clin Cancer Res 3:151-156 (1997); Debinski, W. et al., Clin Cancer Res
1:1253-1258
(1995); Husain, S.R. et al., Cancer Res 58:3649-3653 (1998); Husain, S.R. et
al., Nature
Med 5:817-822 (1999)).
In the Type II IL-13 receptor complex, the IL-l3Ra chain is not present
and IL13 binds to IL-4R~i and IL-l3Ra' chains. The Type II receptor complex is
reported to be expressed on some non-hematopoietic malignant cells such as
A431, PA-1
and HT-29. In the third type of IL 13R, IL4R(3 and IL 13Ra' chains may
associate with
the IL-2R gamma chain (which is known as "gammac"), which is also present in
the
IL4R, IL7R, IL9R and IL15R systems. (Miyajima, A. et al., Ann Rev Immunol
10:295-
331 (1992); Kishimoto, T. et al., Cell 76:253-262 (1994); Giri, J.G. et al.,
Embo Journal
13:2822-2830 (1994). Type III IL13R is present on hematopoietic cells such as
human
erythroleukemia cell line TF-1 and healthy human primary monocytes. Although
the
gammac chain does not interact with IL-13R directly, it modulates IL13R
function
through downregulation of IL-l3Ra, and to some extent a', chains. (Kuznetsov,
V.A. et
al., Biophysical Journal 77:154-172 (1999)).
The IL-4R system, which is related to the IL13R system, also exists in
three different types. In type I IL4R, the IL4R~i chain forms a complex with
gammac, in
type II receptors, the IL4R(3 chain forms a complex with the ILl3Ralpha' chain
and, in
type III receptors, all three chains are present. (Murata, T. et al., (1997)
supra). From
these studies, it has been concluded that the IL4R~i and ILl3Ra' chains are
shared
between the IL4R and IL13R systems. (Murata, T. et al., Intl JMoI Med 1:551-
557
3
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WO 01/34645 PCT/US00/31044
( 1998)).
Several studies suggest that blocking the effect of IL-13 by soaking up IL-
13 with the a chain of the IL-13 receptor (IL-13R), a protein which binds IL-
13, can
provide therapeutic benefit in various inflammatory diseases in marine models
including
bronchial asthma (Wills-Karp, M. et al., Science 282:2258-2261 (1998); Grunig,
G. et al.,
Science 282:2261-2263 (1998)), hepatic fibrosis induced by schistosomiasis
(Chiaramonte, M. G. et al., J. Clin. Invest. 104:777-785 (1999)), as well as
decrease
susceptibility to Leishmania major infection (Matthews, D. J. et al., J.
Immunol.
164:1458-1462 (2000)). In the past, IL-4 antagonists have been used to block
the effect
of IL-4 in various marine models of inflammatory diseases (Grunewald, S. M. et
al., .l.
Biol. Chem. 272:1480-1483 (1997); Grunewald, S. M. et al., J. Invest.
Dermatol.
110:501-SO1 (1998); Grunewald, S. M. et al., J. Immunol. 160:4004-4009 (1998);
Carballido, J. M. et al., J. Cellular Biochem.,114:114 (1995); Carballido, J.
M. et al., J.
Immunol. 155:4162-4170 (1995)). At least two inhibitors of IL-4, including an
IL-4
1 S antagonist and a soluble IL-4R~3 chain, are in the clinic for the
treatment of bronchial
asthma (Smith, L. J. Ann. Intern. Med. 130:531-532 (1999); Asthmatics, B. G.-
Q.
(http://www.Bayer.com/webzine/asthma/kstudie en.html) In). Because IL-4
inhibitors
not only block the effect of IL-4 but also the effect of IL-13 through shared
receptors on
some cell types, it is hypothesized that IL-4 mutant can also block the effect
of IL-13 in
certain systems (Zurawski, S. M. et al., EMBO J. 12:2663-2670 (1993)).
However, since
the IL-l3Ra chain is not shared with the IL-4R, and since this chain binds IL-
13 with
stronger affinity than does the IL-4R, it has been predicted that IL-4 mutants
will not be
very useful in blocking the effect of IL-13 on every cell type (Murata, T. et
al., Blood
91:3884-3891 (1998)). To overcome this problem, a soluble extracellular domain
of IL-
l3Ra has been fused to Fc portion of human immunoglobulin and the resulting
protein
(IL-l3Ra/Fc chimera) has been found to block the effect of IL-13 in vitro and
in many
models of inflammatory diseases in vivo (Donaldson, D. D. et al., J. Immunol.
161:2317-
2324 (1998)). Because IL-13 can signal in the absence of IL-l3Ra chain,
however, it is
not likely that soluble receptor fusion protein might not be able to block the
effect of IL-
13 in every system.
A variety of human solid tumor cells express elevated levels of IL13
receptors. (Obiri, N.I. et al., JClin Invest 91:88-93 (1993); Obiri, N.I. et
al., Clin Exp
Immunol 95:148-155 (1994); Puri, R. Cytokines: Interleukins and Their
Receptors, 143-
4
CA 02404763 2002-10-04
WO 01/34645 PCT/US00/31044
186 (1995); Debinski, W. et al., JBiol Chem 270:16775-16780 (1995)). To target
these
receptors, a chimeric protein composed of IL13 and a mutated form of
Pseudomonas
exotoxin (PE38) was produced. (Debinski, W. et al., Clin Cancer Res 1:1253-
1258
(1995); Debinski, W. et al., JBiol Chem (1995) supra). This cytotoxin is
highly
cytotoxic to IL13R-positive malignancies. (Debinski, W. et al., Clin Cancer
Res (1995)
supra; Debinski, W. et al., JBiol Chem (1995) supra). The results of some of
this work
are also embodied in U.S. Patents No. 5,614,191 and 5,919,456, both of which
are
incorporated herein by reference. Unfortunately, the binding affinity of IL13-
PE38 was
times lower than native, or "wild-type" IL13. (Puri, R.K. et al., Blood
87:4333-4339
10 ( 1996))
IL-13 and IL-4 share receptors on normal cells. Debinski et al., Nature
Biotechnology 16:449-453 (1998) (hereafter, "Debinski et al., 1998" or
"Debinski
1998"). It appears that human IL-13 ("hIL-13" or "hILl3") may possess at least
two
receptor recognition sites, one that recognizes one of the chains of the IL-4
receptor, and
1 S another which recognizes the IL-13 receptor (or "IL-13R"). Id. Although
the crystal
structure of IL-13 is not known, recent work has attempted to draw structural
analogies
from the IL-4 receptor (or "IL-4R") to the IL-13 receptor. Thus, noting that a
mutation
changing the glutamic acid at position 9 to lysine in IL-4 impaired binding to
the IL-4R,
and that the glutamic acid at this position was conserved between IL-4 and IL-
13,
Debinski et al., 1998 created a mutated IL-13 in which the glutamic acid at
position 13
(which corresponds to position 9 of IL-4) was changed to lysine. This
molecule, styled
hIL13.E13K, was found to have only weak proliferative activity (or mitogenic)
activity.
When coupled to a modified Pseudomonas exotoxin, PE4E, the cytotoxin
(hIL13.E13K-
PE4E) exhibited decreased cytotoxicity to normal cells but increased
cytotoxicity to
tumor cells expressing IL-13R when compared to native, or wild-type, IL-13-
PE4E. In
competition assays against wild-type IL-13, hIL13.E13K was found to bind to U-
251MG
cells with much greater affinity than did wild type hILl3. Debinski et al.,
1998. The IL-
13E13K mutation, and certain other mutations which increase affinity for the
IL-13R, are
also discussed in International Application Publication No. WO 99/51643.
SUMMARY OF THE INVENTION
The invention relates to the uses of mutated forms of IL-13 with higher
affinity than that of native IL-13 for the IL-13 receptor. The invention
relates to the
5
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WO 01/34645 PCT/US00/31044
discovery that some of the mutants are antagonists of IL-13 mediated activity,
and that
some are agonists of such activity. These discoveries permit the activity of
IL-13 to be
upregulated or downregulated according to the particular use intended.
Further, the
invention relates to the use of certain of the mutants as targeting moieties
for
S immunoconjugates, including immunotoxins directed at cells of cancers which
overexpress the IL-13 receptor.
More specifically, the invention provides a method for preventing, or for
reducing the severity of, a disease which is mediated by the activity of IL-
13, said method
comprising administering a mutated IL-13 which is an antagonist of IL-13
activity in an
amount effective to prevent, or to reduce the severity of, said disease. In
general, the
antagonist is a mutated IL-13 in which a glutamic acid residue at position 13
of the IL-13
amino acid sequence is replaced by a neutrally charged or a positively charged
amino acid
residue. Tn preferred embodiments, the amino acid substituted for the glutamic
acid
residue is positively charged. The positively charged residue may be a lysine,
an
arginine, or a histidine, although arginine is less preferred. Mimetics of the
natural amino
acids may also be used so long as the mutated IL-13 continues to function as
an
antagonist of IL-13 activity by, for example, the assays set forth herein.
Diseases and
conditions that can be prevented or ameliorated by the use of the antagonists
include any
condition in which IL-13 causes, enhances, mediates, or prolongs the
condition. In
particular, the disease condition can be bronchial asthma, atopic dermatitis,
allergic
rhinitis, schistosomiasis, Leishmania, Hodgkin's disease, renal cell
carcinoma, Kaposi's
sarcoma, or another cancer in which IL-13 serves as a growth factor.
The invention further relates to the use of the antagonists of the invention
for the manufacture of a medicament for the prevention or treatment of a
disease which is
mediated by the presence of IL-13. In a preferred form, the antagonist is a
mutated IL-13
in which a glutamic acid residue at position 13 of IL-13 is replaced by a
neutrally charged
or a positively charged amino acid residue. The medicament can be used for
diseases
such as bronchial asthma, atopic dermatitis, allergic rhinitis,
schistosomiasis, Leishmania,
Hodgkin's disease, renal cell carcinoma, Kaposi's sarcoma and any other cancer
in which
IL-13 functions as a growth factor.
The invention further relates to the discovery that some mutants of IL-13
are agonists of IL-13 activity. The invention therefore provides a method for
augmenting
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WO 01/34645 PCT/US00/31044
an IL-13-mediated response in a cell, said method comprising contacting said
cell with a
mutated IL-13, which mutated IL-13 has one or more mutations selected from the
group
consisting of replacing an arginine residue at position 112 of IL-13 by a
neutrally charged
or a negatively charged amino acid residue, replacing a glutamic acid residue
at position
110 of IL-13 with a neutrally charged or a positively charged residue,
replacing an
arginine at position 109 of IL-13 with a neutrally charged or a negatively
charged residue,
replacing a glutamic acid residue at position 92 of IL-13 with a neutrally
charged or a
positively charged residue, replacing a positively charged residue at position
69 with a
neutrally charged or a negatively charged residue, and replacing positively
charged
residue at position 66 with a neutrally charged or a negatively charged
residue, with the
proviso that a position of said mutated IL-13 corresponding to position 13 of
wild-type
IL-13 is not occupied by a lysine residue. In preferred forms, the method
includes use of
an IL-13 mutated in one or more of the following ways: the arginine at
position 112 of
IL-13 is replaced by a residue selected from the group consisting of glutamic
acid and
aspartic acid, the glutamic acid at position 110 of IL-13 is replaced by a
residue selected
from the group consisting of lysine and arginine, the arginine at position 109
of IL-13 is
replaced by a residue selected from the group consisting of glutamic acid and
aspartic
acid, the glutamic acid position 92 of IL-13 is replaced by a residue selected
from the
group consisting of lysine and arginine, a positively charged residue at
position 69 of IL-
13 is replaced by a residue selected from the group consisting of glutamic
acid and
aspartic acid, or a positively charged residue at position 66 of IL-13 is
replaced by a
residue selected from the group consisting of glutamic acid and aspartic acid.
The
contacting of the cell occurs in vitro or in vivo.
The invention further provides for the use of agonists of IL-13 in the
manufacture of a medicament for pretreating bone marrow stem cell donors. The
mutated
IL-13 may have one or more mutations selected from the group consisting of
replacing an
arginine residue at position 112 of IL-13 by a neutrally charged or a
negatively charged
amino acid residue, replacing a glutamic acid residue at position 110 of IL-13
with a
neutrally charged or a positively charged residue, replacing an arginine at
position 109 of
IL-13 with a neutrally charged or a negatively charged residue, replacing a
glutamic acid
residue at position 92 of IL-13 with a neutrally charged or a positively
charged residue,
replacing a positively charged residue at position 69 with a neutrally charged
or a
negatively charged residue, and replacing positively charged residue at
position 66 with a
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WO 01/34645 PCT/US00/31044
neutrally charged or a negatively charged residue; with the proviso that a
position of said
mutated IL-13 corresponding to position 13 of wild-type IL-13 is not occupied
by a lysine
residue. In preferred forms, the mutated IL-13 has one or more of the
following
mutations: the arginine at position 112 of IL-13 is replaced by a residue
selected from the
group consisting of glutamic acid and aspartic acid, the glutamic acid at
position 110 of
IL-13 is replaced by a residue selected from the group consisting of lysine
and arginine,
the arginine at position 109 of IL-13 is replaced by a residue selected from
the group
consisting of glutamic acid and aspartic acid, the glutamic acid position 92
of IL-13 is
replaced by a residue selected from the group consisting of lysine and
arginine, a
positively charged residue at position 69 of IL-13 is replaced by a residue
selected from
the group consisting of glutamic acid and aspartic acid, and the arginine at
position 66 of
IL-13 is replaced by a residue selected from the group consisting of glutamic
acid and
aspartic acid, provided that the glutamic acid residue at position 13 is not
replaced by a
positively charged residue. In less preferred forms, the glutamic acid may be
replaced by
a neutrally charged residue.
In another group of embodiments the invention provides an IL-13-receptor
binding molecule selected from an IL-13, a circularly permuted IL-13, and a
molecule
with at least about 85% identity to IL-13, which IL-13-receptor binding
molecule has a
binding affinity for an IL-13 receptor at least about three times higher than
that of wild-
type IL-13, and which comprises one or more mutations selected from the group
consisting of changing an amino acid corresponding to a glutamic acid at
position 110 of
IL-13 to a neutrally charged or to a positively charged amino acid, and
changing an amino
acid corresponding to a glutamic acid at position 92 of IL-13 to a neutrally
charged or to a
positively charged amino acid. The present invention also provides molecules
with
increased binding affinity to the IL-13R compared to wild type IL-13. The
molecules
have at least about 85% identity to IL-13, preferably 90% identity, and more
preferably
95% identity. The molecules further bind to the IL-13R with at least about
three times
the affinity with which wild type IL-13 binds to the receptor and in preferred
forms bind
with 5 times the affinity. In the most preferred forms, the molecules bind
with about 10
times the affinity of wtIL-13. The molecules further comprise one or both
mutations
selected from the group consisting of changing an amino acid corresponding to
a glutamic
acid at position 110 of IL-13 to a positively charged amino acid and changing
an amino
acid corresponding to a glutamic acid at position 92 of IL-13 to a positively
charged
CA 02404763 2002-10-04
WO 01/34645 PCT/US00/31044
amino acid. In preferred embodiments, the IL-13R binding molecules are an IL-
13 or
cpIL-13 with one or both of the mutations noted.
The IL-13R binding molecules of the invention have markedly higher
biological activity than does wild type (wt) IL-13. In preferred forms, the
molecules have
at least about twice that of wtIL-13, and in even more preferred forms will
have activity
at least about 5 times that of wtIL-13. In the most preferred forms, the
molecules have
about 10 times the activity of wtIL-13. In a preferred assay, the biological
activity is
measured by conducting a proliferation assay in TF-1 cells.
The invention fiirther provides chimeric molecules in which the IL-13R
binding molecules described above serve as a targeting moiety. In particular,
the
invention provides chimeric molecules comprising which IL-13R binding
molecules and
one or more effector molecules. The IL-13R binding molecule can be an IL-13 or
cpIL-
13 comprising one or both mutations selected from the group consisting of
changing an
amino acid corresponding to a glutamic acid at position 110 of IL-13 to a
positively
charged amino acid and changing an amino acid corresponding to a glutamic acid
at
position 92 of IL-13 to a positively charged amino acid. The cpIL-13 can be a
cpIL-13 in
which the native IL-13 is opened between residues 43 and 44 (Gly and Met
respectively)
to produce a cpIL-13 having Met44 as the amino terminus and G1y43 as its
carboxyl
terminus that specifically binds an IL-13 receptor.
The effector molecule can be a cytotoxin, a label, a radionuclide, a drug, a
liposome, a ligand, or an antibody. The cytotoxin can be a Pseudomonas
exotoxin, a
Diphtheria toxin, ricin, saponin, gelonin, abrin, or ribosome inactivating
protein. In
preferred embodiments, the cytotoxin is a Pseudomonas exotoxin (PE) or a
Diphtheria
toxin (DT) which has been modified to reduce or eliminate its binding
capacity. In
particularly preferred forms, the PE is PE38, PE38QQR, PE38KDEL, and PE4E. The
chimeric molecules can be a single chain fusion protein.
In a preferred embodiment, this invention provides for improved methods
for specifically delivering an effector molecule to a tumor cell bearing an IL-
13 receptor.
The method involves providing a chimeric molecule comprising an effector
molecule
attached to an IL-13R binding molecule of the invention, and contacting the
tumor with
the chimeric molecule resulting in binding of the chimeric molecule to the
tumor cell.
In another embodiment, this invention provides a method for impairing the
growth of tumor cells, more preferably solid tumor cells, bearing an IL-13
receptor. In
preferred forms, the tumor cell is selected from the group consisting of a
renal cell
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WO 01/34645 PCT/US00/31044
carcinoma cell, a glioma cell, a medulloblastoma cell, a head and neck cancer
cell, a
pancreatic cancer cell, and a Kaposi's sarcoma cell. The method involves
contacting the
tumor cell with a chimeric molecule comprising an effector molecule selected
from the
group consisting of a cytotoxin, a radionuclide, a ligand and an antibody. The
effector
molecule is attached or linked to an IL-13 receptor binding molecule of the
invention, as a
targeting molecule, to form the chimeric molecule. Preferred cytotoxic
effector
molecules include Pseudomonas exotoxin, Diphtheria toxin, ricin and abrin.
Pseudomonas exotoxins, such as PE38, PE38QQR, PE38KDEL, and PE4E, are
particularly preferred. The targeting molecule may be conjugated or fused to
the effector
molecule with attachment by fusion preferred for cytotoxic effector molecules.
The
tumor growth that is impaired may be tumor growth in a human. Thus the method
may
further comprise administering the chimeric molecule to a human by any of
various
parenteral means, such as administration into a vein, into a body cavity, or
into a lumen or
an organ. The method may further comprise direct injection or administration
into the
central nervous system, such as the brain or the spinal fluid.
In yet another embodiment, this invention provides for a method of
detecting the presence or absence of a tumor. The method involves contacting
the tumor
with a chimeric molecule comprising a detectable label attached to an IL-13R
binding
molecule and detecting the presence or absence of the label. In a preferred
embodiment,
the label is selected from the group consisting of a radioactive label, an
enzymatic label,
an electron dense label, and a fluorescent label.
This invention also provides for vectors comprising a nucleic acid
sequence encoding a chimeric polypeptide fusion protein comprising an IL-13R
binding
molecule of the invention', attached to a second polypeptide. The chimeric
polypeptide
fusion protein specifically binds to a tumor cell bearing an IL-13 receptor. A
preferred
vector encodes an IL-13R binding molecule-PE or cpIL-13R binding molecule-PE
fusion
protein. In more preferred forms, the PE moiety is PE38QQR, PE4E, PE38KDEL, or
PE4E.
This invention also provides for host cells comprising a nucleic acid
sequence encoding a chimeric polypeptide fusion protein comprising an IL-13R
binding
molecule attached to a second polypeptide. A preferred host cell comprises a
nucleic acid
encoding an IL-13R binding molecule attached to a PE, such as PE38, PE38QQR,
PE38KDEL, or PE4E, and even more preferably encodes them as a fusion protein.
The
CA 02404763 2002-10-04
WO 01/34645 PCT/US00/31044
encoded fusion protein specifically binds to a tumor cell bearing an IL-13
receptor.
Particularly preferred host cells are bacterial host cells, especially E. coli
cells.
In still yet another embodiment, this invention provides chimeric
molecules that specifically bind a tumor cell bearing an IL-13 receptor. In
one preferred
embodiment, the chimeric molecule comprises a cytotoxic molecule attached to
an IL-13
receptor binding molecule of the invention. The IL-13 receptor binding
molecule may be
conjugated or fused to the cytotoxic molecule. In a preferred embodiment, the
IL-13
receptor binding molecule is fused to the cytotoxin, thereby forming a single-
chain fusion
protein. Preferred cytotoxic molecules include Pseudomonas exotoxin,
Diphtheria toxin,
ricin, and abrin, with Pseudomonas exotoxins (especially PE38, PE38KDEL,
PE38QQR,
or PE4E) being most preferred.
The invention additionally provides for pharmacological compositions
comprising a pharmaceutically acceptable carrier and a chimeric molecule where
the
chimeric molecule comprises and effector molecule attached to an IL-13
receptor binding
molecule. The IL-13 receptor binding molecule and effector molecules may be
conjugated or fused to each other. Particularly preferred effector molecules
include
cytotoxins, labels, radionuclides, drugs, liposomes, ligands, and antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Homology of IL-13 among species.
Homology of mature IL-13 among species calculated using the "PILEUP"
function of the of GCG program (see Example 2, infra). The numbering shown
here may
differ from that usually used for one or more species due to inserted gaps
during the
homology pileup. Tyrosine is generally classified as an aromatic acid rather
than as a
charged one. However, since tyrosine as well as glutamic acid or aspartic acid
can make
hydrogen ion bonds in acidic conditions, it was considered here as a
negatively charged
group. Four cysteine residues are completely conserved between the four
species. Many
charged groups are also conserved.
Figure 2. Effect of wt IL-13 and IL-13R112D on the proliferation of
hematopoietic cells. TF-1 [Fig. 2A] and B9 [Fig. 2B] cells were incubated at
37°C with
various concentrations of wt IL-13 or IL-13R112D as described in the Examples.
Data
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are represented as mean cpm of quadruplicate determinations ~ SD. The
experiment was
repeated 9 times.
Figure 3. Effect of wt IL-13 and IL-13R112D on CD14 expression on
primary monocytes. Elutriated monocytes were cultured in medium containing 0-
50
ng/ml wt IL-13 or IL-13R112D for 48 hr. Cells were then stained with anti CD14-
FTC
conjugated antibody or isotype control and analyzed for CD14 expression by
FACScan
equipment.
Fig. 3A. Fluorescence intensity is shown on the x axis as mean channel
number on a log scale.
Fig. 3B. Mean fluorescence intensities of each concentration of ligand
were shown as a table.
Fig. 3C. Mean fluorescence intensities of each ligand were shown as a
graph. The suppressive effect of CD14 with 1 ng/ml of IL-13R112D is seen to be
comparable to that of 10 ng/ml of wt IL-13.
Figure 4. Inhibition of binding of ['zsI]IL-13 and ['zsI]IL-4 on PM-RCC
cells.
Cells (1 x 106) were incubated with S00 pM ['zsI]IL-13 [Fig. 4A] or 500
pM [125'zsI]IL-4 [Fig. 4B] with or without increasing concentrations (up to 10
nM) of wt
IL-13 or IL-13R112D. Bound radioactivity was determined as described in the
Examples. Data are presented as the mean percentage of maximal specific
binding
without unlabeled ILs. Total'zsI-IL-13 [Fig. 4A] and'zsI-IL-4 [Fig. 4B] bound
to PM-
RCC cells was 2962 ~ 123 and 1706 ~ 141, cpm ~ SD, respectively. Data are
represented
as mean t SD of duplicate determination. The SD are shown when they are larger
than
the symbols.
Figure 5. Cytotoxicity of IL-13PE38 on PM-RCC cells.
Figure 5A. Cells were cultured with various concentrations of IL-
13PE38 (0, 0.1 -1000 ng/ml) with or without 2000 ng/ml of wt IL-13 or IL-
13R112D.
Figure SB. Cells were cultured with various concentrations of wt IL-13 or
IL-13R112D (0, 0.2-2000 ng/ml) with IL-13PE38 as described in the Examples.
The data
were obtained from the mean of quadruplicate determinations, and the assay was
repeated
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several times. The concentration of IL-13-PE38 at which SO % inhibition of
protein
synthesis occurred (IC50) was calculated. Data are means; bars represent the
SD.
Figure 6. SDS-PAGE analysis of purified human IL-13 and its mutants.
S Approximately five hundred nanograms of each purified cytokine was
loaded per sample. Proteins were detected using a Coomassie Blue stain. M;
MultiMarkTM Multi-Colored Standard (Novex, San Diego, CA).
Figure 7. Competition for the binding of lasl-IL-13 by wtIL-13 and
double mutein IL-13E13KR112D.
2x105 U251 cells were incubated with S00 pM lzsl-IL-13 with various
concentration of unlabeled wtIL-13 or IL-13E13KR112D. The cell-bound
radioactivity
was determined with a gamma counter. The error bars represent the standard
deviation of
duplicate determinations.
Figure 8. Proliferation or TF-1 cells induced with human IL-13 or its
mutants.
Panel A. Ten to twenty thousand TF-1 cells per well were cultured in the
presence or absence of various concentration of wtIL-13 or its mutants for 52 -
54 h.
Panel B. The cells were cultured with or without 3 ng/ml wtIL-13 and
presence or absence of 300 ng/ml or 1~g/ml E13KR112D. The cell proliferation
was
determined by the uptake of 3H-thymidine in the dividing cells. The reported
data are the
average of triplicate or quadruplicate samples with error bars representing
the standard
deviation within a data set. Experiments were repeated several times. DM:
double
mutein, IL-13E13KR112D. * represent statistically significant at p < 0.01
level from no
IL-13 control; ** p < 0.01 vs. wtIL-13; *** not significant vs. no IL-13
control and p <
0.01 vs wtIL-13.
Figure 9. Down modulation of CD14 expression on monocytes
Primary elutriated monocytes (1x10' /tube) were cultured with or without
1 ng/ml wtIL-13 in presence or absence of 1 ~,g/ml E13KR112D for 48 hr. Then
cells
were washed and stained as described in Example 16. Gated mean fluorescence
intensity
(NIF'I) number are indicated in each panel.
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Figure 10. Activation of STATE by IL-13 or its mutants in various cell
lines.
Panel A. Photos of gels showing results of EBV-B, THP-1 or KSY-1 cells
incubated with 0, 1, 10 or SO ng/ml wtIL-13 or its mutants for 15 minutes.
Panel B. THP-1 cells were incubated with 0 or 10 ng/ml wtIL-13 or with
ng/ml wtIL-13 and 100 or S00 ng/ml IL-13E13KR112D for 15 minutes.
WtIL-13 and IL-13E13KR112D were added simultaneously. Position of
STATE on each gel is shown by a line (panel A) or by an arrowhead (panel B).
Cells
were processed and electrophoretic mobility-shift assay (EMSA) were performed
as
10 described in Examples 16 and 17.
Figure 11. IL-13 and double mutein block the cytotoxic activity of IL-
13PE38QQR on U251 cells and PM-RCC cells. A thousand cells per well were
cultured
in leucine-free media. All cultures were incubated over night prior to the
addition of 1
1 S ~.Ci tritiated leucine. Cells were then incubated for 4 h, harvested and
radioactivity
counted with beta counter. The data are the average of quadruplicate
determinations with
the error bars representing the standard deviation within a data set.
Experiments were
repeated twice.
Panel A. PM-RCC cells in various concentration of an IL-13-
immunotoxin (IL-13PE38QQR) and 1 ~g/ml wtIL-13 or its muteins. The legend in
panel
A refers to the wells in which a competitor was present (the three data lines
at the top of
the panel). A fourth line, with open triangles denoting the data points, shows
the results
for cells exposed to the IL-13 immunotoxin in the absence of wtIl-13 or its
muteins, as
denoted by the legend "No competitors." Y-axis is 3H-Leucine incorporation in
counts
per minute (CPM).
Panel B. Graphs of U251 cells (top graph of panel) and PM-RCC cells
(bottom graph of panel) in the presence of 1 ng/ml IL-13PE38QQR with or
without
various concentrations of wtIL-13 or of its mutants.
Figure 12. SDS-PAGE analysis of purified IL-13 and its mutants.
Approximately five hundred nanograms of each purified cytokine was
loaded per sample. Proteins were detected using Coomassie Blue stain. M;
MultiMarkTM
Multi-Colored Standard (Novex, San Diego, CA). Legend: wt IL-13: wild-type IL-
13.
E13K:IL-13E13K. R112D:IL-13R112D.
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Figure 13. Competition for the binding of lzsI-IL-13 by wtIL-13 and IL-
13E13K
5x10s PM-RCC cells (shown in panel A) or U251 cells (shown in panel B)
per tube were incubated with 200 pM or 500 pM lzsl-IL-13, respectively, and
with
various concentrations of unlabeled wtIL-13 or IL-13E13K. Data presented are
total cell
bound lzsl_IL-13 with error bars representing the standard deviation of
duplicate
determinations.
Figure 14. Proliferation of TF-1 cells induced by IL-13 or IL-13E13K.
Panel A. Ten thousand TF-1 cells per well were cultured in the presence
or absence of various concentration of wtIL-13 or IL-13E13K for 52 - 54 h.
Panel B. The cells were cultured with or without 1 ~g/ml 1L-13E13K and
various concentrations of wtIL-13 for 52 h.
Panel C. The cells were cultured with or without 3 ng/ml wtIL-13 and in
the presence or absence of 300 ng/ml or lpg/ml IL-13E13K.
For all panels, cell proliferation was determined by the uptake of 3H-
thymidine in the dividing cells. Data presented are percentage of count
without cytokine
stimulation and the average of triplicate or quadruplicate samples with error
bars
representing the standard deviation within a data set. Experiments were
repeated several
times. E13K:IL-13E13K.
Figure 15. Effect of IL-13E13K on IL-13 induced down modulation of
CD 14 expression on monocytes
Primary elutriated monocytes (1x10' /tube) were cultured with or without
1 ng/ml wtIL-13 in the presence or absence of 1 pg/ml IL-13E13K (shown as
"E13K") for
48 hr. Cells were then washed and stained as described in material and
methods. Gated
mean fluorescence intensity (MFI) number is indicated in each panel.
Panel A: Cells cultured without cytokine stimulation.
Panel B: Cells cultured in presence of wtIL-13, but absence of IL-
13E13K.
Panel C: Cells cultured in presence of wtIL-13 and in presence of IL-
13E13K.
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Figure 16. Activation of STAT6 by IL-13 or IL-13E13K in various cell
lines.
Panel A: EBV-B, THP-1 or KSY-1 cells were incubated with 0, 1, 10 or
SO ng/ml wtIL-13 or IL-13E13K for 15 minutes.
Panel B: THP-1 cells were incubated with 0 or 10 ng/ml wtIL-13 or with
ng/ml wtIL-13 plus 100 or 500 ng/ml IL-13E13K for 15 minutes.
For both panels, wtIL-13 and IL-13E13K were added simultaneously.
Cells were processed and EMSA was performed as described in Example 18.
Location of
STAT6 on each gel is noted.
Figure 17. Predicted model for IL-13 interaction with its receptors.
Homology model of the CRH domain of IL-l3Ra', IL-4Ra chains and IL-13. This
model is designed to,show the type II IL-13R complex. The 3-dimensional model
is
shown as a ribbon diagram. A and D indicate a-helix A and D of the IL-13
molecule,
respectively. The figure was prepared using the InsightII program.
Figure 18. Suppression of HD cell proliferation by an exemplary IL-13
antagonist.
Panel A. L1236 cells (2X104/well) were cultured in RPMI with or
without 2 pg/ml IL-13E13K (Antagonist) in a COZ incubator for 48 hr or 72 hr.
Viable
cells were counted after trypan blue exclusive staining (Life Technologies,
Inc., Grand
Island, NY). Data is shown as mean t standard deviation of cell number per
well. * p
<0.01
Panel B. 1x105/ml L1236 cells (left two bars) or L428 cells (right two
bars) were cultured with or without 7.5 pg/ml IL-13 antagonist for 48 hr in
37°C
humidified 5% COZ incubator. After 1 ~,Ci 3H-thymidine pulse per well, cells
were
incubated for additional 9 hr and then harvested. Data is shown as mean +
standard
deviation of CPM. Symbol: **; p = 0.03, N.S.; not significant.
Panel C. 1x105/ml L1236 or L428 cells were incubated in RPMI
containing 0.1 ng/ml wild-type IL-13 with or without various concentration of
IL-13
antagonist for 48 hrs in a 37°C, humidified, 5% COZ incubator. After
lp,Ci 3H-thymidine
pulse per well, cells were incubated for additional 9 hrs and then harvested
and counted.
Data is shown as mean + standard deviation of percent CPM.
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Panel D. 1x105/ml L1236 or L428 cells were incubated in RPMI
containing 0.1 ng/ml wild-type IL-13 with or without various concentration of
IL-13-BP.
Remainder of protocol was as in Panel C.
Figure 19. Expression of IL-13 and functional receptor complex in
Hodglin's disease cell lines.
Panel A. Rt-PCR assay was performed as described in Example 20. The
rt-PCR reaction was performed as follows: initial reverse transcription step
at 48°C for 45
min followed by inactivation of reverse transcriptase and denaturation step at
94°C for 2
min; 40 cycles at 94°C 30 sec; 54°C 60 sec, 68°C 120 sec,
respectively. The products
were electrophoresed in 1 % agarose gel and stained by ethidium bromide.
Panel B. Dot blot analysis for secretion of IL-13 protein by HD cell lines.
Panel C. Growth of L1236, L591, and L428 HD cell lines in absence or in
presence ofIL-13.
Panel D. Growth of L1236 or L428 HD cells in absence or in presence of
immunotoxin IL-13-PE38QQR.
DETAILED DESCRIPTION
I. Introduction
The invention provides methods of modulating the effect of IL-13 on cells
and on illnesses. First, the invention concerns the fact that certain mutants
of IL-13
which have higher binding affinity for the IL-13 receptor ("IL-13R") than does
IL-13
function as antagonists of IL-13 activity, while other mutants act as strong
agonists of IL-
13. Use of the agonists permits the activity of IL-13 to be upregulated when
that is
desirable, such as when activating dendritic cells in vitro, while use of the
antagonists
permits the activity of IL-13 to be downregulated when that effect is
desirable, such as
decreasing the effect of IL-13 in inflammatory diseases such as asthma.
Additionally, the
invention provides new mutants with a high affinity for the IL-13R. These
mutants can
be fused to an effector molecule to form a chimeric molecule known as an
immunoconjugate. Where the fused effector molecule is a toxin, the construct
is known
as an immunotoxin; immunotoxins targeted by the high affinity IL-13 mutants of
the
invention can be used to specifically kill cells overexpressing the IL-13R.
Since the cells
of many cancers, such as gliomas, overexpress the IL-13R, the new mutants
offer new
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immunotoxins with which to attack these cells. The mutants can be used, for
example, to
purge cell cultures of IL-13-overexpressing cancer cells.
The discussion below first discusses IL-13 mutants which have higher
binding affinity for the IL-13R than that of IL-13. The mutants comprise
changes of the
glutamic acid ("E", in single letter code) at position 110 of IL-13, or the
glutamic acid at
position 92 of IL-13, or both, to an amino acid which is positively charged at
physiological pH, such as lysine or arginine.
The discussion then discusses the biological activity of mutants of IL-13.
In particular, mutants of IL-13 in which the arginine at position 112 is
altered to a neutral
amino acid or, more preferably, to an acidic amino acid, such as glutamic acid
or aspartic
acid, are strong agonists of IL-13 activity. That is, such mutants activate IL-
13 mediated
activity more strongly than does native (also called "wild-type" or "wt") IL-
13. Mutating
the arginine at position 112 to aspartic acid has previously been suggested to
create a
mutant which binds more specifically to the IL-13R, rendering the mutant
useful as the
targeting portion of an immunotoxin directed to IL-13-expressing cells. The
increased
biological activity of the mutant and its utility by itself as an agent,
independent of the
activity of an effector molecule fused to it, have not previously been
identified.
The discussion then turns to the discovery that mutating the glutamic acid
("E") at position 13 to a neutral amino acid, or more preferably an amino acid
which
carries a positive charge at physiological pH, results in a mutant that is an
antagonist of
IL-13. That is, in the presence of such a mutant, the activity of endogenous
IL-13 is
reduced or wholly blocked. This permits the alleviation of conditions in which
IL-13 is
implicated as a causative or enhancing agent. Such conditions include, for
example,
asthma, allergic rhinitis, certain cancers, such as Hodgkin's Disease,
Kaposi's sarcoma ,
and renal cell carcinoma, and susceptibility to Leishmaniasis. Remarkably, the
presence
of a mutation to a neutral amino acid or, more preferably, to a basic acidic
acid at position
13 of IL-13 causes the mutant to be an antagonist of IL-13 activity even if
the molecule
contains other mutations, such as changing the arginine at position 112 to
aspartic acid,
which would otherwise cause the mutant to be a strong agonist of IL-13
activity. For
example, the double mutant IL-13E13KR112D is an antagonist of IL-13 activity
even
though the mutant IL-13R112D is a strong agonist of IL-13-mediated activity.
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A. Molecules with Increased Affinity for the IL-13 Receptor
As noted in the Background, the crystal structure of IL-13 is not known.
IL-13 does, however, have some homology to IL-4 and binds to one or more
chains of the
IL-4 receptor ("IL-4R"). In the absence of direct information, inferences
about the
structure and binding positions of IL-13 are made by analogy to the structure
of IL-4.
Previous work has shown that mutating a residue of glutamic acid in human IL-4
(hIL-4)
at position 9 to lysine severely impaired the binding of hIL-4 to the IL-4a
chain. Kruse et
al., EMBO J 12:5121-5129 (1993). This residue is conserved between IL-4 and IL-
13 (in
which the residue is predicted to fall at position 13), and it was thought the
residue might
be involved in binding of hIL-13 to chains common to both the IL-13R and the
hIL-4R.
As reported in Debinski et al., 1998, mutating this residue to lysine (that
is, changing a
negatively charged residue to a positively charged residue), increased its
binding affinity
of the mutated IL-13 to hIL-13R, but decreased the ability of the interleukin
to stimulate
the growth of cells exposed to it.
Based on the crystallographic structure of IL-4 and the homology of the
amino acid sequences, it has been predicted that IL-13 has four alpha helices
(styled alpha
helices A, B, C, and D) and two beta sheets. See, Bamborough, P. et al.,
Protein
Engineering 7:1077-1082 (1994). The residue mutated in the work reported in
Debinski
et al., 1998, was in the putative A alpha helix of the molecule. Debinski did
not report
examination as to the effect of mutating residues in other helices of IL-13.
Mutating the charge of a different residue in a different putative helix of
IL-13, the arginine at position 112 in helix D, results in a molecule with a
sharply
increased affinity for the IL-13R compared to wtIL-13. In competitive binding
assays,
the mutated IL-13 (IL-13R112D) showed an affinity for the IL-13R some 5 to 10
times
greater than that of wild type IL-13 (wtIL-13). See, e.g., Figure 4 and
Examples 7 and 13.
In preferred embodiments, the arginine at position 112 can be mutated to a
neutral amino
acid. In more preferred embodiments, it can be mutated to an aspartic acid or
a glutamic
acid, with aspartic acid being the most preferred. Other negatively charged
amino acids
or mimetics can, however, be used. Any construct or derivative which does not
bind to
the IL-13 receptor with at least about 3 times the affinity of wtIL-13 is not
within the
scope of the claimed invention.
Because the results noted above demonstrate that amino acid residues in
the putative D helix of IL-13 are involved in interactions with the IL-13R,
changing the
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charge of two other, nearby charged residues predicted to be in helix D,
arginine at
position 109 and the glutamic acid at position 110, will likewise result in
increased
affinity for the IL-13R. The preferred mutations for the arginine at position
109 are the
same as those discussed above with regard to the arginine at position 112.
With respect
S to the glutamic acid at position 110, since glutamic acid is negatively
charged at
physiological pH, the preferred mutations are those which replace the glutamic
acid
residue with a neutral or with a positively charged amino acid. In preferred
embodiments,
the glutamic acid is mutated to a positively charged residue such as a lysine
or an
arginine, although other positively charged natural amino acids, such as
tyrosine,
synthetic amino acids, or mimetics can be used, so long as the resulting
molecule retains
at least about 3 fold higher binding affinity for the IL-13 receptor compared
to wild type
IL-13.
Based on studies of residues conserved among species, the consideration
that charged residues are more likely to be exposed on the surface of the
protein, and a
review of the chains which are shared between the IL-4 and the IL-13 receptors
and those
which are not, changing the negatively charged glutamic acid at position 92 of
IL-13 to a
neutral residue or to a positively charged residue will also increase its
binding to the IL-
13R. In preferred embodiments, the amino acid substituted for the glutamic
acid at
position 92 is a lysine or an arginine, although other positively charged
natural amino
acids, such as tyrosine, synthetic amino acids, or mimetics can be used, so
long as the
resulting molecule retains at least about 3 fold higher binding affinity for
the IL-13
receptor compared to wild type IL-13.
It is understood that variations of IL-13 can be made which retain the
capacity to bind to the IL-13 receptor but which do not follow the amino acid
sequence of
wild type IL-13. In the simplest form, for example, a molecule might be made
which
contains one, two, or more conservative substitutions, particularly at
positions not
expected to be implicated in binding to the IL-13 receptor. Such variations
are intended
to be encompassed within the invention. Specifically, the invention
encompasses IL-13
receptor binding molecules with at least about 85% identity to IL-13 and which
have
about 3 times the binding affinity for the IL-13 receptor of wild type IL-13
or more and
which have a charge-changing mutation at one, two, three, or all four of the
positions
specified above. More preferably, the IL-13 receptor binding molecules have at
least
about 90 % identity to IL-13 and, even more preferably, have about 95%
identity to the
wild type sequence. In the most preferred embodiments, the IL-13 receptor
binding
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molecules are wild type IL-13 with a charge-changing mutation at residue 112,
residue
110, residue 109, residue 92, or any two of these positions, or at three, or
at all four.
The numbers of the residues set forth above are to the accepted sequence
for mature human IL-13 as shown in Figure 1. As discussed in more detail
below,
however, IL-13 can be permuted to create so-called circularly permuted II,-13,
or "cpIL-
13." Further, the sequence of native IL-13 can be altered by, for example,
removing
terminal residues, conjugation, or the like. Each of these variations can
change the
numbering of the residues. It is therefore understood that reference herein to
residues
"112," "110," "109" and "92," refers in its first sense to the amino acid
residues in those
position of the residues in mature wild-type human IL-13, as set forth, for
example, in
Figure 1, but also refers, as appropriate in context, to residues in
variations of IL-13 or
cpIL-13 which correspond to residues 112, 110, 109, and 92 of the mature, wild-
type IL-
13 sequence, so long as the mutation of the corresponding residues results in
at least
about a 3 fold increase in binding affinity of the molecule to the IL-13
receptor compared
to the binding affinity of IL-13. To clarify that these higher binding
affinity molecules
are not necessarily merely wild type IL-13 with mutations in these particular
residues, the
molecules provided by the invention have been termed "IL-13 receptor binding
molecules."
B Higher Affinity IL-13R Binding Molecules Can Also Have Increased
Biological Activity
In marked contrast to the results reported in Debinski et al., 1998, in which
the mutated IL-13 lost virtually all biological activity, IL-13R112D showed
increased, not
decreased, biological activity. As discussed in Examples 10, 11, and 12,
below, the
mutated IL-13 showed approximately 10-fold higher activity than did wtIL-13 in
standard
assays for the activity of this interleukin. In this regard, IL-13R112D showed
10-fold
higher activity in the inhibition of CD 14 expression in primary monocytes,
and 10-fold
higher activation of STATE on EBV-immortalized B cells and the THP-1 monocytic
cell
line. See, Figure 3 and Example 12, infra. STATE is a signal transduction
molecule and
activator of transcription which is known to be activated by IL-13. Whereas
the mutation
reported by Debinski et al., 1998 showed a marked decrease in the ability of
the
interleukin to induce proliferative activity, IL-13R112D induced a 10-fold
increase in
proliferation of TF-1 and B9 cell lines. See, Figure 2A and B. And, IL-13R112D
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interacted with the IL-13 receptor with greater activity than wtIL-13 in all
cell types
tested.
Given these results with the mutation of arginine to the negatively charged
aspartic acid residue, it is expected that mutating the arginine to a residue
with a neutral
charge or to another residue with a negative charge at physiological pH, such
as glutamic
acid, will likewise result in an IL-13 mutant with an activity as an agonist
stronger than
that of native IL-13. In more preferred embodiments, the residue substituted
for the
arginine at position 112 is negatively charged. Any particular residue or
mimetic can be
tested for its effect as an agonist by the assays set forth in the Examples
below. Aspartic
acid is the most preferred residue to substitute for the arginine at position
112.
As noted in the Background section, IL-13 is a potent activator of the
immune system. In particular, IL-13 is an activator of dendritic cells, which
are
professional antigen-presenting cells ("APCs"), and can be used, in
conjunction with
GM-CSF, to cause differentiation of monocytes into dendritic cells. Due to the
increased
biological activity of the IL-13 receptor binding molecules of the invention,
they can be
substituted for IL-13 or IL-4 in in vitro protocols to activate dendritic
cells or other APCs.
While APCs activated by these protocols have several uses, in one important
embodiment, they are often used in procedures to "pulse" or to "load"
dendritic cells with
antigen prior to reinfusing the APCs into a patient to augment or to induce an
immune
response against that antigen.
The agonists of the invention can also be used in vitro to reduce the
occurrence of Graft Versus Host Disease ("GVHD"). GVHD is a significant
concern
after allogeneic stem cell transplantation. Various studies have demonstrated
that type-1
T lymphocytes (secreting interleukin("IL")-2 and interferon-gamma) in
harvested donor
cells mediate acute GVHD, whereas type-2 T lymphocytes (secreting IL-4 and IL-
10) can
prevent acute GVHD. Type-2 T cells also produce IL-13. It is clinically well
known that
pretreatment of donors with granulocyte colony-stimulating factor (G-CSF)
suppresses
the severity of GVHD even though there are many lymphocytes contaminants in
the
harvested stem cells.of the G-CSF-treated donor. This is because pretreatment
of donors
with G-CSF polarizes the donor's T lymphocytes toward type-2 cytokine
production. In
marine models, this has been shown to reduce severity of experimental GVHD
(see, e.g.,
Blood, 86(12):4422-4429 (1995)). As wild type IL-13 is shown to polarize type-
2 T cells,
it is predicted that IL-13R112D may more powerfully polarize type-2 T cells of
donors
resulting in prevention of acute GVHD.
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C. IL-13E13KR112D is a powerful IL-13 antagonist
The double mutant IL-13E13KR112D produced by site-directed
mutagenesis of two amino acids in the predicted a helix A and D of the IL-13
molecule,
is an antagonist of IL-13 activity. The antagonistic activity of IL-13 mutein
IL-
13E13KR112D was determined based on the inhibition of wtIL-13 induced
proliferation
of TF-1 cells, wtIL-13 induced down modulation of CD14 in primary monocytes,
inhibition of wtIL-13 induced activation of STAT6 in B cells, monocytes and
cancer
cells, displacement of l2sl-IL-13 binding on cancer cells and neutralization
of cytotoxicity
mediated by IL13-PE38QQR in cancer cells. Thus, the antagonistic activities of
IL-
13E13KR112D were evident in cells that expressed Type I and type II/III IL-13
receptors.
In an initial study, reported in Section I B, above, a single IL-13 mutein,
IL-13R112D, was created by substituting aspartic acid (D) for arginine (R) at
position
112. Because the position of amino acid 112 in IL-13 was predicted to
correspond to
position 124 in the IL-4 molecule, and since mutation of position 124 of IL-4
resulted in
an IL-4 antagonist (IL-4Y124D) (Kxuse, N. et al., Embo J. 11:3237-3244
(1992)), it was
predicted that IL-13R112D would be an IL-13 antagonist. Surprisingly, the
opposite
effect was observed. IL-13R112D turned out to be a powerful IL-13 agonist with
5-10
fold improved binding affinity to IL-13 receptors, as reported in Section I B.
A mutation at position 13 of IL-13, which is predicted to be located in the
a helix A of the IL-13 molecule has also been produced In this IL-13 mutein,
the
glutamic acid at position 13 was changed to lysine (Debinski, W. et al., Nat.
Biotechnol.
16:449-453 (1998)). This mutant protein, IL-13E13K, reported 50-fold higher
binding
affinity to IL-13 receptors compared to wtIL-13 on U251 cell lines. Id.. To
further
improve the binding affinity of IL-13, a double mutant, IL-13E13KR11D, was
produced.
It was predicted that this molecule would have a much higher binding affinity
to IL-13
receptor compared to wtIL-13, IL-13R112D and IL-13E13K. This molecule was
expressed in Escherichia coli and homogeneously purified material was found to
bind IL-
13 receptors. Surprisingly, this double mutein IL-13 when tested in various
biological
assays turned out to be an antagonist of IL-13, rather than an agonist. Based
on this
result, an IL-13 mutated to a neutral or to a negatively charged residue at
position 112 are
agonists unless the glutamic acid residue at position 13 has been mutated to a
positively
charged residue.
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D. IL-13E13K is also a powerful antagonist of IL-13 activity
IL-13E13K, a powerful antagonist of IL-13, which bound well to the IL-
13R, was produced by site-directed mutagenesis of an amino acid in the
predicted a-helix
A of the IL-13 molecule. Without wishing to be bound by theory, the results
demonstrate
that G1u13 in IL-13 is of crucial importance for the potency of the cytokine
for signal
generation, but not for the binding of IL-13 to its receptors. The
antagonistic activity of
IL-13 mutein IL-13E13K was determined based on: a) 4 to 8-fold better
displacement of
l2sl-IL-13 binding on cancer cells; b) the inhibition of wtIL-13 induced
proliferation of
TF-1 cells; c) neutralization of wtIL-13 induced down modulation of CD 14
expression in
primary monocytes; and d) inhibition of wtIL-13 induced activation of STATE in
B cells,
monocytes and cancer cells. Thus, the antagonistic activities of IL-13E13K
were evident
in cells that expressed Type I, Type II and Type III IL-13 receptors.
In view of this result, and those reported above for the double mutant IL-
13E13KR112D, it is evident that mutating the acidic residue at position 13
causes the
mutant to be an antagonist of IL-13 activity. Thus, mutants of IL-13 in which
the
glutamic acid at position 13 is changed to a residue with a neutral charge
will also act as
antagonists of IL-13 activity. In preferred embodiments, the glutamic acid at
position 13
is changed to a residue which is positively charged at physiological pH. For
example, the
glutamic acid residue at position 13 can be mutated to lysine, arginine or
histidine.
Arginine is somewhat less preferred as a positively charged residue; in more
preferred
embodiments, the glutamic acid is mutated to lysine or histidine. A mutation
to lysine is
the most preferred embodiment.
IL-13 receptors belong to a large family of cytokine receptors, including
receptors (or receptor subunits) for interleukins-2, 3, 4, 5, 6, granulocyte
macrophage
colony-stimulating factor, granulocyte colony-stimulating factor,
erythropoietin, growth
hormone and prolactin (Bazan, J. F. Proc Natl Acad Sci, USA 87:6934 (1990)).
All these
proteins show particular homology in the amino acid sequence of their
postulated or
proven ligand binding CRH domains. These receptors also belong to the
immunoglobulin
superfamily which consists of many proteins characterized by immunoglobulin-
like beta
sandwich structure. Id. Despite similarities in receptors, their cognate
ligands show only
limited homology at the amino acid sequence level. However, crystallographic
analyses
or computer prediction of the topology of ligands indicate that they have
similar tertiary
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WO 01/34645 PCT/US00/31044
structure in that they are composed of four major a-helixes arranged in up-up
& down-
down directions. Based on these studies, the critical interface residues for
IL-4 were
found to be located in a-helix A and D of the ligand. Similarly, the amino
acid residue
which is critical for receptor binding and signal transduction for marine GM-
CSF is
reported to be G1u21 which is located in a-helix A (Altmann, S. W. et al.,
JBiol Chem
270:2233 (1995); Altmann, S. W. et al., Growth Factors 12:251 (1995)). The
receptor
binding domains of growth hormone and interleukin-6 are reported to be located
in a-
helix A and C (DeVos, A. M. et al., Science 255:306 (1992); Savino, R. et al.,
EMBO
Journal 13:1357 (1994); Savino, R. et al., EMBO Journal 13:5863 (1994)).
Without
wishing to be bound by theory, our studies suggest that residue Arg112,
predicted to be
located in the C-terminal end of a-helix D of hIL-13, is one of the critical
residues of
receptor binding. The results reported here suggest that residue G1u13,
predicted to be
located in a-helix A, is also critical for receptor interaction. Based on
these studies, we
predict that the receptor interface of IL-13 may be located in a-helix A and
D. These
predictions are supported by the proposed model of IL-13 and IL-13R complex.
Thus,
this model may be an useful tool to investigate receptor binding sites in the
IL-13
molecule.
Since IL-13 has been shown to be involved in many inflammatory diseases
including bronchial asthma, it is expected that IL-13E13K and IL-13E13KR112D
and
other antagonists of the invention will be able to neutralize or reduce the
effect of IL-13,
reducing the severity or occurrence of asthma symptoms. Similarly, since IL-13
is
involved in allergic rhinitis and atopic dermatitis, it is expected that IL-
13E13K, IL-
13E13KR112D and other antagonists will be useful in mitigating these
conditions.
E. Antagonists to IL-13 Slow the Growth of Hodgkin's Disease, Renal Cell
Carcinoma, and Kaposi's Sarcoma cells
Cytokines may play a critical role in the survival and proliferation of
Hodgkin/Reed-Sternberg ("H/RS" or "HD/RS") cells. Histopathological studies
have
demonstrated that a large number of (reactive) lymphocytes and some
eosinophils
surround H/RS cells. The symptoms such as fever, night sweat or weight loss
seen in
Hodgkin's Disease ("HD") patients have been hypothesized to be caused by
cytokines
produced by H/RS cells themselves or surrounding cells (Drexler, HG, Leukemia
and
Lymphoma, 8:283-313 (1992) ("Drexler 1992"); Drexler, HG, Leukemia and
Lymphoma,
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9: 1-25 (1993) ("Drexler 1993")). While as much as thirteen cytokines were
postulated to
be autocrine/paracrine growth factors for H/RS cells, it has not been
clarified which
cytokine(s) is essential for pathogenesis of I~ (Drexler, 1992; Drexler 1993).
Recently by using microarray technology it was discovered that IL-13
might be an autocrine growth factor for H/RS cells (Kapp et al., JExp Med,
189:1939-
1945 (1999)). In two cell lines tested, one cell line grew in autocrine
fashion and
neutralizing IL-13 monoclonal antibody blocked the proliferation of these
cells. Id. The
mechanism of selective inhibition of cell growth was not demonstrated.
As reported above, the present invention provides IL-13 mutants with
altered bioactivities, such as antagonist or super agonist properties. In
particular, a novel
IL-13 antagonist, IL-13E13K, in which glutamic acid at position 13 was
substituted by
lysine in the IL-13 molecule, was produced. This mutant binds to IL-13R with
up to 8
fold higher affinity than that of wild-type IL-13. Moreover, it antagonizes
the
proliferative activity of IL-13 on TF-1 erythroleukemia cells, reverses the IL-
13 induced
down modulation of CD 14 expression on monocytes and suppresses the IL-13
induced
STAT-6 activation in Ebstein-Barr virus immortalized B-cells, THP-1 monocytic
cell line
and in KSY-1 Kaposi's sarcoma cells. Because IL-13 has been shown to be
autocrine
growth factor on H/RS cells, the IL-13 antagonist was examined to determine
whether it
could inhibit the autocrine/paracrine proliferation of these cells. In
addition, the subunit
composition of IL-13R was examined to determine why some H/RS are responsive
to IL-
13 and IL-13 antagonists.
All H/RS cell lines express mRNA for IL-13R components but only one of
two-expressed functional IL-13R as determined by IL-13 induced proliferation
and
internalization. It is of interest to note that in the previous study,
suppression of IL-13
effect using antibody was seen in only one of two H/RS cell lines even though
both cell
lines expressed IL-13 (Kapp et al., 1999, supra). Thus, the Kapp 1999 study
confirms
that some H/RS cells may not express functional IL-13R.
The studies reported in the Examples also indicate that IL-13-PE38QQR
can be used for the eradication of H/RS cells in vitro and in vivo. The
concentration of
IL-13-PE38QQR that causes maximal inhibition of protein synthesis is
clinically
achievable. Preclinical studies have suggested that high serum level of IL-
13PE38QQR
can be safely achieved in monkeys when given intravenously (Cmax; 5027 t 1583
ng/ml). Thus, sensitivity of H/RS cell may be within a therapeutic range in
vivo.
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The mechanisms) of different sensitivity of H/RS cells to IL-13 and IL-13
antagonist is still not clear. The lack of response to IL-13 on L428 may be
explained by
low level expression of IL-13Ra2 chain. IL-13 Ra2 chain has been shown to bind
to IL-
13 with high affinity but it does not signal by itself. Similarly, cell lines
that express IL-
13Ra2 chain do not seem to respond to stimulatory growth effect of IL-13.
Alternative
mechanisms) may also be operational for example, disbalanced expression of
different
receptor chains, inappropriate receptor subunits aggregation or mutation of
receptor
chains.
Taken together, the results show that IL-13E13K is a useful agent for the
therapy of HD in which H/RS cells express functional IL-13R. The results with
IL-
13E13KR112D indicate that the presence of the E13K mutation, or the mutation
of E13
to a neutral residue or, preferably, to arginine, will likewise result in an
IL-13 antagonist.
The antagonists of the invention are superior to anti-IL-13 antibody (Kapp et
al., 1999,
supra) because the ICSO of antibody is 1000 times higher than that of IL-
13E13K. The
1 S antagonists of the invention are also better than using the a chain of the
IL-13R
(sometimes called the "IL-13 binding protein," or "IL-13BP", see, Zhang et
al., J. Biol.
Chem. 272: 9474-9480 (1997)). IL-13BP comprises a soluble extracellular domain
of the
IL-13 receptor which is normally membrane bound. In order to produce the
domain in a
form in which it can be secreted by recombinant cells, it is fused to the Fc
portion of
human immunoglobulin. The resulting IL-l3Ra/Fc chimera has been found to block
the
effect of IL-13 in vitro and to block the effect of IL-13 in vivo in animal
models of
inflammatory disease. The antagonists of the invention are smaller than IL-
13BP fusion
constructs, and therefore achieve better biodistribution and better
bioavailability.
Accordingly, they are better agents for blocking IL-13 effects both in vitro
and in vivo.
In recent studies, we have found that Kaposi's sarcoma cells secrete IL-13.
Studies with antibodies to IL-13 showed that blocking IL-13 with antibodies
inhibited the
growth of Kaposi's sarcoma cells, therefore showing that IL-13 functions as a
growth
factor for these cells. Since the antagonists of the invention react
specifically with the IL-
13R and bind with at least an order of magnitude higher affinity than
antibodies bind to a
typical antigen, the antagonists are expected to strongly inhibit the growth
of Kaposi's
sarcoma cells. Similarly, renal cell carcinoma cells have been found to
secrete IL-13 and
can be inhibited in the same fashion. Thus, IL-13E13K, IL-13E13KR112D, and
other
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antagonists of the invention can be used to slow or stop the growth of
Kaposi's sarcoma
and renal cell carcinoma cells.
The fact that IL-13 serves as an autocrine factor in three such disparate
cancers suggests that IL-13 is an autocrine growth factor for other cancers,
and
particularly those which overexpress the IL-13 receptor. Based on the results
above, we
expect that IL-13 antagonists can be used to slow the growth other cancers for
which IL-
13 serves as an autocrine factor. We have recently discovered that some
pancreatic
cancers and approximately one-quarter of head and neck cancers overexpress the
IL-13
receptor. Growth of these IL-13R-overexpressing cancers can be slowed by
contacting
them with the antagonists of the invention.
II. Definitions
Units, prefixes, and symbols are denoted in their Systeme International de
Unites (SI) accepted form. Numeric ranges are inclusive of the numbers
defining the
range. Unless otherwise indicated, nucleic acids are written left to right in
5' to 3'
orientation; amino acid sequences are written left to right in amino to
carboXy orientation.
The headings provided herein are not limitations of the various aspects or
embodiments
of the invention, which can be had by reference to the specification as a
whole.
Accordingly, the terms defined immediately below are more fizlly defined by
reference to
the specification in its entirety.
As used herein, "wild type IL-13" and "native IL-13" are synonymous and
refer to the mature form of IL-13. The nucleotide and amino acid sequences of
IL-13
have been publicly available since at least 1993. See, McKenzie, et al., Proc
Natl Acad
Sci USA 90:3735-3739 (1993). The amino acid and nucleotide sequences are
available
from GenBank under Accession No. L06801. See also, SEQ ID NO:1 and discussion
in
section VI A, infra.
The term, "modulating," when used in the context of the biological activity
of a molecule, means to upregulate or to downregulate the biological activity
of the
molecule as desired to achieve an intended end, such as a therapeutic result.
Thus, as
used herein, modulating the activity of IL-13 refers to increasing or
decreasing the effect
normally caused by IL-13 in a particular context by use of an agent which acts
as an
agonist or an antagonist of IL-13.
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As used herein, the term "mediate" in connection with the effect of IL-13
on a condition or disease, means that IL-13 causes, contributes to,
aggravates, enhances,
or prolongs the existence or severity of the condition or disease.
"Negatively charged," in reference to an amino acid, refers to those amino
acids, amino acid derivatives, amino acid mimetics and chemical moieties that
are
negatively charged at physiological pH. Negatively charged amino acids
include, for
example aspartic acid and glutamic acid, which are preferred negatively
charged amino
acids for use in the invention. An "acidic" residue is a residue that is
negatively charged
at physiological pH.
"Positively charged," in reference to an amino acid, refers to those amino
acids, amino acid derivatives, amino acid mimetics and chemical moieties that
are
positively charged at physiological pH. Positively charged amino acids
include, for
example, lysine and arginine, which are preferred positively charged amino
acids for use
in the invention. A "basic residue" is a residue that is positively charged at
physiological
pH.
The term "residue" as used herein refers to an amino acid that is
incorporated into a polypeptide. The amino acid may be a naturally occurnng
amino acid
and, unless otherwise limited, may encompass known analogs of natural amino
acids that
can fixnction in a similar manner as naturally occurnng amino acids.
The term "position," with respect to an amino acid residue in a
polypeptide, refers to a number corresponding to the numerical place that
residue holds in
the polypeptide. By convention, residues are counted from the amino terminus
to the
carboxyl terminus of the polypeptide. Thus, position 13 of IL-13 would be the
thirteenth
residue from the amino terminus of the IL-13 sequence.
The term "occupy" or "occupied" with respect to a position in a
polypeptide sequence refers to the amino acid residue in the particular
position described.
Thus, position 13 of IL-13 is usually "occupied" by a glutamic acid residue.
In a mutant
in which the glutamic acid is replaced by a lysine residue (typically by site-
directed
mutagenesis), the position would be said to be occupied by a lysine.
Mutations of an amino acid at a particular position are stated according to
convention. Under the convention for stating changes in a protein, the
original amino
acid is listed first (in standard single letter code), followed by the
position at which it
occurs in the protein or polypeptide, and then by the amino acid replacing the
original
amino acid. Thus, the replacement of an arginine at position 112 by an
aspartic acid is
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designated by the notation: Rl 12D. The term "IL-13R112D therefore designates
an IL-
13 protein in which the arginine which is normally found at position 112 has
been
mutated to aspartic acid.
A "fusion protein" refers to a polypeptide formed by the joining of two or
more polypeptides through a peptide bond formed between the amino terminus of
one
polypeptide and the carboxyl terminus of another polypeptide. The fusion
protein may be
formed by the chemical coupling of the constituent polypeptides or it may be
expressed as
a single polypeptide from nucleic acid sequence encoding the single contiguous
fusion
protein. A single chain fusion protein is a fusion protein having a single
contiguous
polypeptide backbone.
A "chimeric molecule" is a single molecule created by joining two or more
molecules that exist separately in their native state. The single, chimeric
molecule has the
desired functionality of all of its constituent molecules. Frequently, one of
the constituent
molecules of a chimeric molecule is a "targeting molecule" or "targeting
moiety." The
targeting molecule is a molecule such as a ligand or an antibody that
specifically binds to
its corresponding target, for example a receptor on a cell surface. Thus, for
example,
where the targeting molecule is a cytokine such as IL-13, the chimeric
molecule will
specifically bind (target) cells and tissues bearing an IL-13 receptor.
Another constituent of the chimeric molecule may be an "effector
molecule." The effector molecule refers to a molecule (or, in the context of a
chimeric
molecule, the portion or "moiety") that is to be specifically transported to
the target to
which the chimeric molecule is specifically directed. The effector molecule
typically has
a characteristic activity that is desired to be delivered to the target cell.
Effector
molecules include cytotoxins, labels, radionuclides, ligands, antibodies,
drugs, liposomes
(including liposomes loaded with a drug whose delivery to the target cell is
desired), and
the like.
The term "specifically deliver" as used herein refers to the preferential
association of a molecule with a cell or tissue bearing a particular target
molecule or
marker and not to cells or tissues lacking that target molecule. It is, of
course, recognized
that a certain degree of non-specific interaction may occur between a molecule
and a non-
target cell or tissue. Nevertheless, specific delivery, may be distinguished
as mediated
through specific recognition of the target molecule. Typically specific
delivery results in
a much stronger association between the delivered molecule and cells bearing
the target
molecule than between the delivered molecule and cells lacking the target
molecule.
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Specific delivery typically results in greater than 2 fold, preferably greater
than 3 fold,
more preferably 10 fold or greater and most preferably greater than 100 fold
increase in
amount of delivered molecule (per unit time) to a cell or tissue bearing the
target
molecule as compared to a cell or tissue lacking the target molecule or
marker.
A "spacer" as used herein refers to a peptide that joins the proteins
comprising a fusion protein. Generally a spacer has no specific biological
activity other
than to join the proteins or to preserve some minimum distance or other
spatial
relationship between them. However, the constituent amino acids of a spacer
may be
selected to influence some property of the molecule such as the folding, net
charge, or
hydrophobicity of the molecule.
A "ligand", as used herein, refers generally to all molecules capable of
reacting with or otherwise recognizing or binding to a receptor on a target
cell.
Specifically, examples of ligands include, but are not limited to, antibodies,
lymphokines,
cytokines, receptor proteins such as CD4 and CDB, solubilized receptor
proteins such as
soluble CD4, hormones, growth factors, and the like which specifically bind
desired
target cells.
The term "cpIL-13" is used to designate a circularly permuted (cp) IL-13.
Circular permutation is functionally equivalent to taking a straight-chain
molecule, fusing
the ends (directly or through a linker) to form a circular molecule, and then
cutting the
circular molecule at a different location to form a new straight chain
molecule with
different termini.
A "conservative substitution", when describing a protein refers to a change
in the amino acid composition of the protein that does not-substantially alter
the protein's
activity. Thus, "conservatively modified variations" of a particular amino
acid sequence
refers to amino acid substitutions of those amino acids that are not critical
for protein
activity or substitution of amino acids with other amino acids having similar
properties
(e.g., acidic, basic, positively or negatively charged, polar or non-polar,
etc.) such that the
substitutions of even critical amino acids do not substantially alter
activity. Conservative
substitution tables providing functionally similar amino acids are well known
in the art.
Such substitutions preferably are made in accordance with the following list,
which
substitutions may be determined by routine experimentation provide modified
structural
and functional properties of a synthesized polypeptide molecule, while
maintaining the
receptor binding, or inhibiting or mimicking biological activity, of IL-13, as
determined
by, for example, competitive binding, proliferation, and cytotoxicity assays.
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Original Exemplary
Residue Substitution
Ala Gly; Ser
Arg Lys
S Asn Gln; His
Asp Glu
Cys Se
Gln Asn
Glu Asp
Gay Ala; Pro
His Asn; Gln
Ile Leu; Val
Leu Ile; Val
Lys Arg; Gln; Glu
1 S Met Leu; Tyr; Ile
Phe Met; Leu; Tyr
Se Thr
Thr Se
Trp Tyr
Tyr Trp; Phe
Val Ile; Leu
Put differently, the following six groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (I~, Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton (1984) PROTEINS, W.H. Freeman and Company. In
addition, individual substitutions, deletions or additions which alter, add or
delete a single
amino acid or a small percentage of amino acids in an encoded sequence are
also "
conservatively modified variations".
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The term "substantial identity" or "substantial similarity" in the context of
a polypeptide indicates that a polypeptides comprises a sequence which can
have 40%
sequence identity to a reference sequence, or preferably 70%, or more
preferably 85%
sequence identity to the reference sequence, or even more preferably 90%
identity over a,
comparison window of about 10-20 amino acid residues.
"Percentage amino acid identity" or "percentage amino acid sequence
identity" refers to a comparison of the amino acids of two polypeptides which,
when
optimally aligned, have approximately the designated percentage of the same
amino
acids. For example, "95% amino acid identity" refers to a comparison of the
amino acids
of two polypeptides which when optimally aligned have 95% amino acid identity.
The
IL-13R binding molecules of the invention have at least about 85% identity to
wtIL-13,
preferably about 90% identity, and more preferably about 95% identity. For
convenience
of reference herein, "identity" and "identical" in the context of comparing an
IL-13
receptor binding molecule to wild type IL-13 in percentage terms refers to
their
percentage amino acid identity or percentage amino acid sequence identity.
Preferably, residue positions which are not identical (other than the
specific mutations noted herein) differ by conservative amino acid
substitutions. Because
the substituted amino acids have similar properties, the substitutions do not
change the
functional properties of the polypeptides. An indication that two polypeptide
sequences
are substantially identical is that one peptide is immunologically reactive
with antibodies
raised against the second peptide. Thus, a polypeptide is substantially
identical to a
second polypeptide, for example, where the two peptides differ only by a
conservative
substitution. An indication that two nucleic acid sequences are substantially
identical is
that the polypeptide which the first nucleic acid encodes is immunologically
cross
reactive with the polypeptide encoded by the second nucleic acid. Another
indication that
two nucleic acid sequences are substantially identical is that the two
molecules hybridize
to each other under stringent conditions.
For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison
algorithm, test and reference sequences are input into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm program
parameters are
designated. The sequence comparison algorithm then calculates the percent
sequence
identity for the test sequences) relative to the reference sequence, based on
the
designated program parameters.
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Optimal alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482
(1981), by
the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443
(1970), by the search for similarity method of Pearson & Lipman, Proc. Natl.
Acad. Sci.
USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics
Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see
generally
Ausubel et al., supra).
One example of a useful algorithm is PILEUP. PILEUP creates a multiple
sequence alignment from a group of related sequences using progressive,
pairwise
alignments to show relationship and percent sequence identity. It also plots a
tree or
dendogram showing the clustering relationships used to create the alignment.
PILEUP
uses a simplification of the progressive alignment method of Feng & Doolittle,
J. Mol.
Evol. 35: 351-360 (1987). The method used is similar to the method described
by
Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align up to 300
sequences, each of a maximum length of 5,000 nucleotides or amino acids. The
multiple
alignment procedure begins with the pairwise alignment of the two most similar
sequences, producing a cluster of two aligned sequences. This cluster is then
aligned to
the next most related sequence or cluster of aligned sequences. Two clusters
of
sequences are aligned by a simple extension of the pairwise alignment of two
individual
sequences. The final alignment is achieved by a series of progressive,
pairwise
alignments. The program is run by designating specific sequences and their
amino acid
or nucleotide coordinates for regions of sequence comparison and by
designating the
program parameters. For purposes of this invention, a reference sequence can
be
compared to other test sequences to determine the percent sequence identity
relationship
using the following parameters: default gap weight (3.00), default gap length
weight
(0.10), and weighted end gaps.
Another example of algorithm that is suitable for determining percent
sequence identity and sequence similarity is the BLAST algorithm, which is
described in
Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing
BLAST
analyses is publicly available through the National Center for Biotechnology
Information
(http://www.ncbi.nlm.nih.gov~. This algorithm involves first identifying high
scoring
sequence pairs (HSPs) by identifying short words of length W in the query
sequence,
which either match or satisfy some positive-valued threshold score T when
aligned with a
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word of the same length in a database sequence. T is referred to as the
neighborhood
word score threshold (Altschul et al, supra). These initial neighborhood word
hits act as
seeds for initiating searches to find longer HSPs containing them. The word
hits are then
extended in both directions along each sequence for as far as the cumulative
alignment
score can be increased. Extension of the word hits in each direction are
halted when: the
cumulative alignment score falls off by the quantity X from its maximum
achieved value;
the cumulative score goes to zero or below, due to the accumulation of one or
more
negative-scoring residue alignments; or the end of either sequence is reached.
The
BLAST algorithm parameters W, T, and X determine the sensitivity and speed of
the
alignment. The BLAST program uses as defaults a word length (W) of 11, the
BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both strands.
In addition to calculating percent sequence identity, the BLAST algorithm
also performs a statistical analysis of the similarity between two sequences
(see, e.g.,
Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of
similarity provided by the BLAST algorithm is the smallest sum probability
(P(1~),
which provides an indication of the probability by which a match between two
nucleotide
or amino acid sequences would occur by chance. For purposes of this invention,
a
nucleic acid is considered similar to a reference sequence if the smallest sum
probability
in a comparison of the test nucleic acid to the reference nucleic acid is less
than about 0.1,
more preferably less than about 0.01, and most preferably less than about
0.001.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides
and polymers thereof in either single- or double-stranded form. Unless
specifically
limited, the term encompasses nucleic acids containing known analogues of
natural
nucleotides which have similar binding properties as the reference nucleic
acid and are
metabolized in a manner similar to naturally occurnng nucleotides. Unless
otherwise
indicated, a particular nucleic acid sequence also implicitly encompasses
conservatively
modified variants thereof (e.g. degenerate codon substitutions) and
complementary
sequences and as well as the sequence explicitly indicated. Specifically,
degenerate
codon substitutions may be achieved by generating sequences in which the third
position
of one or more selected (or all) codons is substituted with mixed-base and/or
deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);
Ohtsuka et al., J.
Biol. Chem. 260:2605-2608 (1985); and Cassol et al., 1992; Rossolini et al.,
Mol. Cell.
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Probes 8: 91-98 (1994)). The term nucleic acid is used interchangeably with
gene,
cDNA, and mRNA encoded by a gene.
The phrase "a nucleic acid sequence encoding" refers to a nucleic acid
which contains sequence information for a structural RNA such as rRNA, a tRNA,
or the
S primary amino acid sequence of a specific protein or peptide, or a binding
site for a trans-
acting regulatory agent. This phrase specifically encompasses degenerate
codons (i.e.,
different codons which encode a single amino acid) of the native sequence or
sequences
which may be introduced to conform with codon preference in a specific host
cell.
III. Uses of IL-13 Agonists
A. Activation of Dendritic Cells
Recent studies have demonstrated that peripheral blood derived- or bone
marrow derived- dendritic cells ("DC") are very potent professional antigen
presenting
cells ("APCs"). These cells in clinical trials as vaccines to augment immune
responses to
cancer and infection by HIV. Typically, peripheral blood derived monocytes are
collected from a normal donor or a cancer patient and cultured with IL-4 and
granulocyte-
macrophage colony stimulating factor (GM-CSF). After 6-10 days, these cells
are
generally found to be differentiated into dendritic cells, which show unique
surface
phenotypes. For example, they begin to express CD1 lc, CD80, and CD83, and MHC
class II expression is increased. The DCs are then treated in a manner to
"load" them
with an antigen relevant to the condition against which it is desired to
augment the
immune response. Loading is usually accomplished by pulsing the cells with
antigen,
tumor cell lysates, peptide antigens, or apoptotic bodies, by fusing them to
whole tumor
cells, or by transforming the cells with genes which express the desired
antigens, with or
without co-stimulatory molecules. The treated cells are then washed and
injected to
patients at multiple time point schedules to boost the immune response. The
treated DCs
migrate to lymph nodes, where they express the antigen in the context of MHC
class I
molecules, thereby "educating" cytotoxic T cells to recognize the antigen.
These T cells
then circulate and kill cells that express the antigen.
The success of these forms of immune therapy depend on the generation of
mature DCs. Recent studies have shown that IL-13 can replace IL-4 in the
activation of
DCs. As little as 10 ng/ml IL-13, when combined with 10 to 100 ng/ml GM-CSF,
can
generate dendritic cells from peripheral blood monocytes. Since IL-13R112D is
5 to 10
times as potent an agonist of IL-13 activity, it can be used in place of IL-
13, but
considerably smaller amounts need to be used to generate dendritic cells
compared to the
amount of IL-13 which is required. One can use as little as 0.1 ng/ml of IL-
13R112D
along with GM-CSF in the protocol taught in the Examples to activate DCs.
Other
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agonists of IL-13 activity, such as IL-13E110K, IL-13E110R, IL-13R109D, IL-
13R109E,
IL-13E92K, IL-13E92R, and mutants of IL-13 with a D or an E at position 66 or
position
69, can be used in the same manner in the maturation and activation of potent
DCs. Thus,
the agonists of the invention provide a reagent for DC maturation and
activation that
eliminates dependence on the production of large amounts of IL-13.
B. Pretreatment of Bone Marrow Stem Cell Donors
Graft-versus-host disease (GVHD) following allogeneic stem cell
transplantation is still a major obstacle to an otherwise highly useful
treatment. Various
studies have demonstrated that type-1 T lymphocytes (secreting interleukin
("IL")-2 and
interferon-gamma) in harvested donor cells mediate acute GVHD, whereas type-2
T
lymphocytes (secreting IL-4 and IL-10) can prevent acute GVHD. Type-2 T cells
also
produceIL-13.
Pan et al., Blood 86:4422-4429 (1995), reported that, in an animal model,
pretreatment of donors with granulocyte colony-stimulating factor (G-CSF)
suppressed
the severity and incidence of acute GVHD, despite the fact that large numbers
of
lymphocytes remained in the donor infusion. It was noted that type 1 T-cells
(which
secrete IL-2 and interferon gamma) mediate acute GVHD, while type 2 T-cells,
which
secrete IL-4 and IL-10, can prevent GVHD. Pretreatment of donors with G-CSF
polarize
donor T lymphocytes toward type-2 cytokine production, which is associated
with
reduced type 1 cytokine production and reduced severity of experimental GVHD.
Id.
Wild type IL-13 polarizes T-cells towards type-2 cytokine production and
can be used along with G-CSF to decrease the severity of GVHD. Given the
greater
potency of the agonists of the invention, it is expected that the agonists can
be used in
place of wtIL-13 at the same dose with greater effect or in smaller amounts to
achieve the
same effect. Typically. donors will be treated on an alternate day schedule
with IL-
13R112D (doses between 1 to 100 microgram/Kg body weight) for two weeks prior
to the
harvesting of bone marrow cells. The cells collected be tested for type 2
polarization by
determining their cytokine output. Cells with high levels of type 2 cytokines
relative to
type 1 cytokine production will be infused into the recipient. Suitable
recipients include
those in need of transplantation for cancer therapy, or in need of rescue of
marrow after
intensive chemotherapy.
Agonists of IL-13 activity suitable for use in the polarization of T-cells to
decrease GVHD include IL-13E110K, IL-13E110R, IL-13R109D, IL-13R109E, IL-
13E92K, IL-13E92R, and mutants of IL-13 with a D or an E at position 66 or
position 69.
IL-13R112D is the most preferred agonist for this use.
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IV. Use of IL-13 Antagonists in Asthma, Allergic Ithinitis, Atopic Dermatitis,
Cancer, and Other Conditions
A. Asthma
IL-13 is a necessary and sufficient factor for the expression of allergic
asthma. IL-13 induces pathophysiological features of asthma in animals in a
manner that
is independent of IgE, IL-4, and eosinophils and is now considered the central
mediator of
allergic asthma. See, e.g., Wills-Karp, M., et al., Science 282:2258-22621
(1998).
Further studies in patients with asthma have demonstrated that IL-13 is
locally produced
in the bronchoalveolar lavage (BAL) cells when challenged with allergen
(Huang, et al. J.
Immunol. 155:2688, (1995)). Up to 3 ng/ml of IL-13 protein/ml BAL was detected
in
allergen challenged patients.
As noted by Van der Pouw Kraan, T., et al., Clin Exp Immunol 111:129-
135 (1998), IgE antibodies play a crucial role in allergic type 1 reactions. A
study of the
role of IL-13 in IgE synthesis in allergic asthma patients showed IgE
production in
allergic asthma patients is more dependent on IL-13 than in non-patients due
to enhanced
IL-13 production and to enhanced IgE production in response to IL-13.
These results indicate that neutralization of endogenous IL-13 will
diminish the production of IgE and, therefore, the onset or strength of
asthmatic attacks.
Since IL-13 induces pathophysiological features of asthma in animals in a
manner that is
independent of IgE, IL-4, and eosinophils, IL-13 antagonists are helpful in
both IgE
dependent and in IgE-independent asthma. The antagonists of IL-13 of the
invention are
expected to neutralize the effect of IL-13 and thus will be useful for
preventing the onset
of asthma, or for decreasing the severity of asthma once it has developed.
Since local production of IL-13 is important in the pathophysiology of
asthma, the intranasal route is preferred for therapeutic applications. This
route of
administration can be easily performed by patients, as many patients are
already familiar
with the use of inhalers used in connection with other drugs for the treatment
of asthma.
Typically, the amount of wt IL-13 in bronchoalveolar lavage (BAL) cells is
determined
(for example, up to 3 ng/ml BAL) in patients with allergen induced asthma, and
a ten fold
higher amount of antagonist of the invention is administered. Typical
intranasal doses are
30 ng/ml to 30 microgram/ml, administered every alternate day for two weeks.
The
antagonists can also be administered by systemic i.v. administration, 1 to 50
microgram/Kg doses every alternate day for two weeks. In severe cases, or
where
immediate suppression of the asthma is required, a continuous infusion can be
administered, using higher doses (100 micrograms/kg) of antagonist.
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B. Allergies, including Allergic Rhinitis
Studies in an animal model with a mutated IL-4 which is also an
antagonist for IL-13 have indicated that the antagonist inhibited allergic
responses and
allergic symptoms mediated either by IgE or IgGI. E.g., Grunewald, S. et al.,
J. Immunol
160:4004-4009 (1998). Because IL-4 inhibitors not only block the effect of IL-
4 but also
the effect of IL-13 through shared receptors on some cell types, IL-4 mutants
can also
block the effect of IL-13 in certain systems. However, since the IL-l3Ra chain
is not
shared with IL-4R, and since this chain binds IL-13 with strong affinity, IL-4
mutants do
not block the effect of IL-13 on every cell type. The antagonists of the
invention are
therefore more specific than the IL-4/IL-13 antagonists used by Grunewald et
al., and are
expected to inhibit allergic responses and symptoms more completely than did
the
mutated IL-4 antagonist used in the Grunewald et al. study. Accordingly, the
IL-13
antagonists of the invention, such as IL-13E13K and IL-13E13KR112D, are
particularly
useful for reducing or eliminating allergic responses.
C. Atopic Dermatitis
The synthesis of IgE is an important factor in the development and
maintenance of atopic dermatitis in patients. It has been reported that levels
of mRNA for
IL-13 were significantly higher in the peripheral blood mononuclear cells
(PBMC) of
patients with atopic dermatitis than in PBMC of controls. See, Katagiri, K. et
al., Clin
Exp Immunol 108:298-294 (1997). As noted in the subsection on asthma, above,
IL-13 is
implicated in the synthesis of IgE, and IL-13 antagonists can diminish the
synthesis of
IgE. Accordingly, the antagonists of the invention are expected to diminish
the
symptoms and longevity of atopic dermatitis.
D. Inflammatory conditions
IL-13 has been shown to be involved in inflammatory conditions in
addition to those listed above. Specifically, IL-13 is implicated in hepatic
fibrosis
induced by schistosomiasis and in susceptibility to Leishmania major
infection.
Administration of IL-13 antagonists ameliorates the formation of hepatic
fibrosis and
reduces susceptibility to infection by Leishmania major.
E. Cancers
As noted in some detail in Section I E, above, IL-13 has been shown to
fimction as an autocrine factor in Hodgkin's disease. See, e.g., Kapp et al.,
J. Exp. Med.
189:1939-1945 (1999). Neutralizing antibodies to IL-13 were shown to block the
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proliferation of these cells in a dose dependent manner. Id. Additionally,
Kaposi's
sarcoma cells and renal cancer cells have been shown to secrete IL-13, and
antibodies to
IL-13 have been shown to inhibit the growth of Kaposi's sarcoma cells, showing
that IL-
13 has an autocrine effect on these cells as well. Accordingly, the
antagonists of the
invention can be used to slow the growth of HD/RS cells, Kaposi's sarcoma
cells, and
renal cell carcinoma cells.
The antagonists of the invention can further be used to slow or to stop the
growth of other cancers in which IL-13 increases proliferation or acts as an
autocrine
growth factor. Such cancers can be readily identified. For example, Kapp et
al., supra,
describe the confirmation of IL-13 expression by tumor cell lines using
Northern blots
and ELISA assays, as well as assays for showing that an antagonist blocks
proliferation of
such cells in a dose dependent manner. Additional assays are described in
Examples 19
and 20, below. In a typical protocol, the cells are washed to remove any
endogenously-
secreted IL-13 (to provide a baseline), and divided into cultures. IL-13 is
added to all but
one of the cultures (the one to which IL-13 is not added remains as a
control), one culture
is left with only IL-13 added, and an IL-13 antagonist is added to the other
cultures, with
each succeeding culture receiving an increasing amount of antagonist. The
growth of
cancers in which IL-13 is a growth factor will be inhibited by the antagonist
in a dose-
dependent manner, while the growth of cancers in which IL-13 is not a growth
factor is
not affected.
It should be noted that in addition to the cancers mentioned above, a
number of cancers, including gliomas, medulloblastomas, about 25% of head and
neck
cancers, and some pancreatic cancers, overexpress the IL-13 receptor. It is
expected that
these cancers proliferate in the presence of IL-13 and that contacting cells
of these
cancers with the antagonists of the invention will stop or slow the
proliferation of the
cells.
V. Chimeric Molecules Targeted to the IL-13 Receptor.
The present invention provides compositions, including molecules with
higher binding affinity for IL-13R than that of native IL-13, chimeric
molecules which
use these higher affinity molecules as targeting agents coupled to an effector
molecule,
and methods for specifically delivering an effector molecule to a tumor cell.
The
methods involve the use of chimeric molecules comprising a targeting molecule
attached
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to an effector molecule. The chimeric molecules of this invention permit
delivery of
effector molecules which specifically target tumor cells.
The improved specific targeting of this invention is premised, in part, on
previous findings from our laboratory that solid tumors, especially
carcinomas, express
IL-13 receptors at extremely high levels compared to normal tissues. For
example, while
the IL-13 receptors (IL-13R) are overexpressed on tumor cells, expression on
other cells
(e.g. monocytes, B cells, and T cells) appears negligible. Thus, by
specifically targeting
the IL-13 receptor, the present invention provides chimeric molecules that are
specifically
directed to solid tumors while minimizing targeting of other cells or tissues.
In a preferred embodiment, this invention provides for compositions and
methods for impairing the growth of tumors. These methods involve providing a
chimeric molecule comprising a cytotoxic effector molecule attached to a
targeting
molecule which binds to IL-13R with an affinity at least about three times
that of wild
type IL-13. The cytotoxic effector molecule may be a native or modified
cytotoxin such
as Pseudomonas exotoxin (PE), Diphtheria toxin (DT), ricin, abrin, saponin,
gelonin,
ribosome inactivating protein, and the like, or it may be a radionuclide, a
drug, or a
liposome which is itself loaded with a drug or other cytotoxic agent. In a
preferred
embodiment, the cytotoxic molecule (or "moiety") is a cytotoxin. In even more
preferred
embodiments, the cytotoxin is a peptide or protein, which can be expressed as
a single
chain fusion protein with the targeting molecule.
The chimeric cytotoxin is administered to an organism
containing tumor cells which are then contacted by the chimeric molecule. The
targeting
molecule component of the chimeric molecule specifically binds to the
overexpressed IL-
13 receptors on the tumor cells. Once bound to the IL-13 receptor on the cell
surface, the
cytotoxic effector molecule mediates internalization into the cell where the
cytotoxic
molecule inhibits cellular growth or kills the cell.
The use of chimeric molecules comprising a targeting
moiety joined to a cytotoxic effector molecules to target and kill tumor cells
is known in
the prior art. For example, chimeric fusion proteins which include interleukin
4 (IL-4) or
transforming growth factor (TGFa) fused to Pseudomonas exotoxin (PE) or
interleukin 2
(IL-2) fused to Diphtheria toxin (DT) have been tested for their ability to
specifically
target and kill cancer cells (Pastan et al., Ann. Rev. Biochem., 61: 331-354
(1992)).
Prior work from our laboratory showed that although
chimeric IL-4-cytotoxin molecules are known in the prior art, and IL-4 shows
some
sequence similarity to IL-13, cytotoxins targeted by a moiety specific to the
IL-13
receptor had significantly increased efficacy as compared to IL-4 receptor
directed
cytotoxins. As noted in U.S. Patent No. 5,919,456 and 5,614,191, this appears
to be due
to at least three factors.
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First, IL-13 receptors are expressed at much lower levels, if
at all on non-tumor cells (e.g. monocytes, T cells, B cells). Thus cytotoxins
directed to
IL-13 receptors show reduced binding and subsequent killing of healthy cells
and tissues
as compared to other cytotoxins.
Second, the receptor component that specifically binds IL-
13 appears to be expressed at significantly higher levels on solid tumors than
the receptor
component that binds IL-4. Thus, tumor cells bind higher levels of cytotoxic
chimeric
molecules directed against IL-13 receptors than cytotoxic chimeric molecules
directed
against IL-4 receptors.
Third, and finally, IL-4 receptors are up-regulated when
immune system cells (e.g. T-cells) are activated. This results in healthy
cells, for example
T-cells and B-cells, showing greater susceptibility to IL-4 receptor directed
cytotoxins.
Thus, the induction of an immune response (as against a cancer), results in
greater
susceptibility of cells of the immune system to the therapeutic agent. In
contrast, IL-13
receptors have not been shown to be up-regulated in activated T cells. Thus IL-
13
receptor targeted cytotoxins have no greater effect on activated T cells and
thereby
minimize adverse effects of the therapeutic composition on cells of the immune
system.
In another embodiment, this invention also provides for
compositions and methods for detecting the presence or absence of tumor cells.
These
methods involve providing a chimeric molecule comprising an effector molecule,
that is a
detectable label attached to a targeting molecule of the invention. The IL-13
receptor
targeting moiety specifically binds the chimeric molecule to tumor cells with
high
affinity. The cells are then marked by their association with the detectable
label.
Subsequent detection of the cell-associated label indicates the presence of a
tumor cell.
In yet another embodiment, the effector molecule may be
another specific binding moiety such as an antibody, a growth factor, or a
ligand. The
chimeric molecule will then act as a highly specific bifunctional linker. This
linker may
act to bind and enhance the interaction between cells or cellular components
to which the
chimeric molecule binds. Thus, for example, where the "targeting" component of
the
chimeric molecule comprises a polypeptide of the invention that specifically
binds to an
IL-13 receptor and the "effector" component is an antibody or antibody
fragment (e.g. an
Fv fragment of an antibody), the targeting component specifically binds cancer
cells,
while the effector component binds receptors (e.g., IL-2 or IL-4 receptors) on
the surface
of immune cells. The chimeric molecule may thus act to enhance and direct an
immune
response toward target cancer cells.
In still yet another embodiment the effector molecule may
be a pharmacological agent (e.g. a drug) or a vehicle containing a
pharmacological agent.
This is particularly suitable where it is merely desired to invoke a non-
lethal biological
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response. Thus the moiety that specifically binds to an IL-13 receptor may be
conjugated
to a drug such as vinblastine, doxirubicin, genistein (a tyrosine kinase
inhibitor), an
antisense molecule, or other pharmacological agents known to those of skill in
the art,
thereby specifically targeting the pharmacological agent to tumor cells over
expressing
IL-13 receptors.
Alternatively, the targeting molecule may be bound to a
vehicle containing the therapeutic composition. Such vehicles include, but are
not limited
to liposomes, micelles, various synthetic beads, and the like.
One of skill in the art will appreciate that the chimeric
molecules of the present invention may include multiple targeting moieties
bound to a
single effector or conversely, multiple effector molecules bound to a single
targeting
moiety. In still other embodiment, the chimeric molecules may include both
multiple
targeting moieties and multiple effector molecules. Thus, for example, this
invention
provides for "dual targeted" cytotoxic chimeric molecules in which an IL-13
receptor
binding molecule of the invention is attached to a cytotoxic molecule and
another
molecule (e.g. an antibody, or another ligand) is attached to the other
terminus of the
toxin. Such a dual-targeted cytotoxin might comprise a PE substituted for
domain Ia at
the amino terminus of a PE and anti-TAC(Fv) inserted in domain III, between
amino acid
604 and 609. Other antibodies may also be suitable.
VI. The Targeting Molecule.
In a preferred embodiment, the targeting molecule is a molecule that
specifically binds to the IL-13 receptor with at least about 3 times the
affinity of wild type
IL-13. In preferred embodiments, the molecule binds to the IL-13 receptor with
an
affinity at least about 5 times the affinity of wild type IL-13 and, in more
preferred
embodiments, the molecules bind with at least about 10 times the affinity or
more. The
term "specifically binds", as used herein, when referring to a protein or
polypeptide,
refers to a binding reaction which is determinative of the presence of the
protein or
polypeptide in a heterogeneous population of proteins and other biologics.
Thus, under
designated conditions, the specified molecule binds to its particular "target"
protein (e.g.
an IL-13 receptor) and does not bind in a significant amount to other proteins
present in
the sample or to other proteins to which the ligand may come in contact in an
organism.
Assay formats for detecting specific binding of ligands (e.g. an IL-13
receptor binding molecule) with their respective receptors are well known in
the art. The
Examples provide a protocol for assessing the competitive binding of IL-13
receptor
binding molecules of the invention compared to wild type IL-13.
The IL-13 receptor is a cell surface receptor that specifically binds IL-13
and mediates a variety of physiological responses in various cell types as
described below
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in the description of IL-13. The IL-13 receptor may be identified by
contacting a cell or
other sample with labeled IL-13 and detecting the amount of specific binding
of IL-13
according to methods well known to those of skill in the art.
Alternatively, an anti-IL-13 receptor antibody may also be used to identify
IL-13 receptors. The antibody will specifically bind to the IL-13 receptor and
this
binding may be detected either through detection of a conjugated label or
through
detection of a labeled second antibody that binds the anti-IL-13 receptor
antibody.
In a preferred embodiment, the moiety utilized to specifically target the IL-
13 receptor is an IL-13 receptor binding molecule with at least about 3 times
the binding
affinity for the IL-13 receptor of wild type IL-13. In particularly preferred
embodiments,
the moiety is an IL-13 or cpIL-13 mutated as taught herein and that
specifically binds the
IL-13 receptor with an affinity at least about 3 times that of wild type IL-
13.
A) IL-13
As noted above, interleukin-13 (IL-13) is a pleiotropic cytokine that is
recognized to share many of the properties of IL-4, with which it shares
approximately
30% sequence identity. It exhibits IL-4-like activities on
monocytes/macrophages and
human B cells (Minty et al., Nature, 362: 248 (1993), McKenzie et al. Proc.
Natl. Acad.
Sci. USA, 90:3735-3739 (1987) ("McKenzie et al.").
The nucleotide and amino acid sequences of human IL-13 were
determined and set forth in the publication by McKenzie et al., supra, and are
also
available on the Internet at, for example, the Entrez browser of the National
Center for
Biotechnology Information (www.ncbi.nlm.nih.gov) under accession number
L06801.
The first eighteen (18) amino acid residues of the sequence set forth by
McKenzie et al.
(through and including the third alanine) are considered in the art to be a
signal sequence
and the mature IL-13 protein is considered to commence with the nineteenth
residue, a
serine. SEQ ID NO:1 sets forth the translation (including both the signal
sequence and
the mature IL-13 sequence) as deposited by McKenzie et al., in GenBank under
the
accession number noted above. Figure 1 shows the sequence of mature human IL-
13 and
compares it to the sequences of mature IL-13 of other species. References
herein to
particular residues of IL-13, such as residues 92, 110, and 112, and to
percentages of
identity to IL-13, are to the amino acid sequence of mature human IL-13. The
sequence
of mature human IL-13 set forth in Figure 1 (and derivable from McKenzie et
al. by
commencing with the serine residue noted above) is also referred to herein as
"native" or
"wild type" IL-13. SEQ ID N0:2 is the nucleotide sequence for human IL-13,
including
the signal sequence and non-coding regions.
The IL-13R binding molecules of the invention have at least about 3 times
the binding affinity for the IL-13R than does wild type IL-13. Conveniently,
the binding
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affinity of IL-13 can be determined by using recombinant IL-13. Recombinant IL-
13 for
such assays is commercially available from a number of sources (see, e.g. R &
D
Systems, Minneapolis, Minnesota, USA, and Sanofi Bio-Industries, Inc.,
Tervose,
Pennsylvania, USA). Alternatively, a gene or a cDNA encoding IL-13 may be
cloned
S into a plasmid or other expression vector and expressed in any of a number
of expression
systems according to methods well known to those of skill in the art. Methods
of cloning
and expressing IL-13 and the nucleic acid sequence for IL-13 are well known
(see, for
example, Minty et al. (1993) supra, and McKenzie et al., supra). In addition,
the
expression of IL-13 as a component of a chimeric molecule is detailed in
Example 4.
One of skill in the art will appreciate that analogues or fragments of IL-13
bearing will also specifically bind to the IL-13 receptor. For example,
conservative
substitutions of residues (e.g., a serine for an alanine or an aspartic acid
for a glutamic
acid) comprising native IL-13 will provide IL-13 analogues that also
specifically bind to
the IL-13 receptor. Thus, the term "IL-13", when used in reference to a
targeting
molecule, also includes fragments, analogues or peptide mimetics of IL-13 that
also
specifically bind to the IL-13 receptor.
B) Circularly permuted IL-13.
In another embodiment of this invention, the targeting moiety can be a
circularly permuted IL-13 (cpIL-13) mutated by non-conservative changes of
residues
corresponding to residues 92, 110, or 112 of IL-13, or non-conservative
changes to
combinations of these residues. Circular permutation is functionally
equivalent to taking
a straight-chain molecule, fusing the ends (directly or through a linker) to
form a circular
molecule, and then cutting the circular molecule at a different location to
form a new
straight chain molecule with different termini (see, e.g., Goldenberg, et al.
J. Mol. Biol.,
165: 407-413 (1983) and Pan et al. Gene 125: 111-114 (1993)). Circular
permutation
thus has the effect of essentially preserving the sequence and identity of the
amino acids
of a protein while generating new termini at different locations.
Circular permutation of IL-13 provides a means by which the native IL-13
protein may be altered to produce new carboxyl and amino termini without
diminishing
the specificity and binding affinity of the altered first protein relative to
its native form.
With new termini located away from the active (binding) site, it is possible
to incorporate
the circularly permuted IL-13 into a fusion protein with a reduced, or no
diminution, of
IL-13 binding specificity and/or avidity.
It will be appreciated that while circular permutation is described in terms
of linking the two ends of a protein and then cutting the circularized protein
these steps
are not actually required to create the end product. A protein can be
synthesized de novo
with the sequence corresponding to a circular permutation of the native
protein. Thus, the
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term "circularly permuted IL-13 (cpIL-13)" refers to all IL-13 proteins having
a sequence
corresponding to a circular permutation of a native IL-13 protein regardless
of how they
are constructed.
Generally, a permutation that retains or improves the binding specificity
and/or avidity (as compared to the native IL-13) is preferred. If the new
termini interrupt
a critical region of the native protein, binding specificity and avidity may
be lost.
Similarly, if linking the original termini destroys IL-13 binding specificity
and avidity
then no circular permutation is suitable. Thus, there are two requirements for
the creation
of an active circularly permuted protein: 1) the termini in the native protein
must be
favorably located so that creation of a linkage does not destroy binding
specificity and/or
avidity; and 2) there must exist an "opening site" where new termini can be
formed
without disrupting a region critical for protein folding and desired binding
activity (see,
e.g., Thorton et al. J. Mol. Biol., 167: 443-460 (1983)). This invention
establishes that
IL-13 meets these criteria and provides for circularly permuted IL-13 that
having
improved binding characteristics.
When circularly permuting IL-13, it is desirable to use a linker that
preserves the spacing between the termini comparable to the unpermuted or
native
molecule. Generally linkers are either hetero- or homo-bifunctional molecules
that
contain two reactive sites that may each form a covalent bond with the
carboxyl and the
amino terminal amino acids respectively. Suitable linkers are well known to
those of skill
in the art and include, but are not limited to, straight or branched-chain
carbon linkers,
heterocyclic carbon linkers, or peptide linkers. The most common and simple
example is
a peptide linker that typically consists of several amino acids joined through
peptide
bonds to the termini of the native protein. The linkers may be joined to the
terminal
amino acids through their side groups (e.g., through a disulfide linkage to
cysteine). In
preferred embodiments, however, the linkers will be joined to the alpha carbon
amino and
carboxyl groups of the terminal amino acids.
Functional groups capable of forming covalent bonds with the amino and
carboxyl terminal amino acids are well known to those of skill in the art. For
example,
functional groups capable of binding the terminal amino group include
anhydrides,
carbodimides, acid chlorides, activated esters and the like. Similarly,
functional groups
capable of forming covalent linkages with the terminal carboxyl include
amines, alcohols,
and the like. In a preferred embodiment, the linker will itself be a peptide
and will be
joined to the protein termini by peptide bonds. A preferred linker for the
circular
permutation of IL-13 is Gly-Gly-Ser-Gly.
In a preferred embodiment, circular permutation of IL-13 involves creating
an opening such that the formation of new termini does not interrupt secondary
structure
crucial to the formation of a structure that specifically binds the IL-13
receptor. Even if
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the three-dimensional structure is compatible with joining the termini, it is
conceivable
that the kinetics and thermodynamics of folding would be greatly altered by
circular
permutation if the cleavage separates residues that participate in short range
interactions
that are crucial for the folding mechanism or the stability of the native
state. Goldenberg,
Protein Eng., 7: 493-495 (1989). Thus, the choice of a cleavage site can be
important to
the protein's binding specificity and/or avidity.
The selection of an opening site in IL-13 may be determined by a number
of factors. Preferred opening sites will be located in regions that do not
show a highly
regular three-dimensional structure. Thus, it is preferred that cleavage sites
be selected in
regions of the protein that do not show secondary structure such as alpha
helices, pleated
sheets, a13 barrel structures, and the like.
Methods of identifying regions of particular secondary structure of IL-13
based on amino acid sequence are widely known to those of skill in the art.
See, for
example, Cohen et al., Science, 263: 488-489 (1994). Numerous programs exist
that
predict protein folding based on sequence data. Some of the more widely known
software packages include MatchMaker (Tripos Associates, St. Louis, Missouri,
USA),
FASMAN from GCG (Genetics Computer Group), PHD (European Molecular Biology
Laboratory, Heidelburg, Germany) and the like. In addition, the amino acid
sequence of
IL-13 is well known and the protein has been extensively characterized (see,
e.g., WO
94/04680).
Alternatively, where the substitution of certain amino acids or the
modification of the side chains of certain amino acids does not change the
activity of a
protein, it is expected that the modified amino acids are not critical to the
protein's
activity. Thus, amino acids that are either known to be susceptible to
modification or are
actually modified in vivo are potentially good candidates for cleavage sites.
Where the protein is a member of a family of related proteins, one may
infer that the highly conserved sequences are critical for biological
activity, while the
variable regions are not. Preferred cleavage sites are then selected in
regions of the
protein that do not show highly conserved sequence identity between various
members of
the protein family. Alternatively, if a cleavage site is identified in a
conserved region of a
protein, that same region provides a good candidate for cleavage sites in a
homologous
protein.
Methods of determining sequence identity are well known to those of skill
in the art. Sequence comparisons between two (or more) polynucleotides or
polypeptides
are typically performed by comparing sequences of the two sequences over a
"comparison
window" to identify and compare local regions of sequence similarity. Since
the goal is
to identify very local sequence regions that are not conserved, the comparison
window
will be selected to be rather small. A "comparison window", as used herein,
refers to a
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segment of at least about S contiguous positions, usually about 10 to about
50, more
usually about 15 to about 40 in which a sequence may be compared to a
reference
sequence of the same number of contiguous positions after the two sequences
are
optimally aligned.
Optimal alignment of sequences for comparison may be conducted by the
local homology algorithm of Smith et al. Adv. Appl. Math. 2: 482 (1981), by
the
homology alignment algorithm of Needleman et al., J. Mol. Biol. 48:443 (1970),
by the
search for similarity method of Pearson et al., Proc. Natl. Acad. Sci. USA,
85: 2444
(1988), by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA,
~ and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer
Group
(GCG), 575 Science Dr., Madison, WI), or by inspection.
A preferred opening site in IL-13 is just prior to Met-44 of hIL-13, just at
the beginning of the putative second alpha-helix resulting in a circularly
permuted IL-13
having a methionine at position 44 of the native IL-13 at the amino terminus
of the cpIL-
13 and the glycine at position 43 of the native IL-13 at the new carboxyl
terminus of the
cpIL-13. This carboxyl terminus can be joined to a second protein directly or
though a
spacer.
Circularly permuted IL-13 may be made by a number of means known to
those of skill in the art. These include chemical synthesis, modification of
existing
proteins, and expression of circularly permuted proteins using recombinant DNA
methodology.
The circularly permuted IL-13 may be synthesized using standard
chemical peptide synthesis techniques as discussed below. If the linker is a
peptide it
may be incorporated during the synthesis. If the linker is not a peptide it
may be coupled
to the peptide after synthesis.
Alternatively, the circularly permuted IL-13 can be made by chemically
modifying a native IL-13 (e.g. a native human IL-13). Generally, this requires
reacting
the IL-13 in the presence of the linker to form covalent bonds between the
linker and the
carboxyl and amino termini of the protein, thus forming a circular protein.
New termini
are then formed by cleaving the peptide bond joining amino acids at another
location.
This may be accomplished chemically or enzymatically using, for example, a
peptidase.
If the cleavage reaction tends to hydrolyze more than one peptide bond,
the reaction may be run briefly. Those molecules having more than one peptide
bond
cleaved will be shorter than the full length circularly permuted molecule and
the latter
may be isolated by any protein purification technique that selects by size
(e.g., by size
exclusion chromatography or electrophoresis). Alternatively, various sites in
the circular
protein may be protected from hydrolysis by chemical modification of the amino
acid
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side chains which may interfere with enzyme binding, or by chemical blocking
of the
vulnerable groups participating in the peptide bond.
In a preferred embodiment, the circularly permuted IL-13, or fusion
proteins comprising the circularly permuted IL-13 is synthesized using
recombinant DNA
methodology. Generally this involves creating a DNA sequence that encodes the
circularly permuted IL-13 (or entire fusion protein containing the IL-13),
placing the
DNA in an expression cassette under the control of a particular promoter,
expressing the
protein in a host, isolating the expressed protein and, if required,
renaturing the protein.
Recombinant expression of the fusion proteins of this invention is discussed
in more
detail below.
DNA encoding circularly permuted IL-13 or fusion proteins comprising
circularly permuted IL-13 may be prepared by any suitable method, including,
for
example, cloning and restriction of appropriate sequences or direct chemical
synthesis by
methods as discussed below. Alternatively, subsequences may be cloned and the
appropriate subsequences cleaved using appropriate restriction enzymes. The
fragments
may then be ligated to produce the desired DNA sequence.
In a preferred embodiment, DNA encoding the circularly permuted IL-13
may be produced using DNA amplification methods, for example polymerase chain
reaction (PCR). First, the segments of the native DNA on either side of the
new terminus
are amplified separately. The S' end of the one amplified sequence encodes the
peptide
linker, while the 3' end of the other amplified sequence also encodes the
peptide linker.
Since the 5' end of the first fragment is complementary to the 3' end of the
second
fragment, the two fragments (after partial purification, e.g. on LMP agarose)
can be used
as an overlapping template in a third PCR reaction. The amplified sequence
will contain
codons the segment on the carboxy side of the opening site (now forming the
amino
sequence), the linker, and the sequence on the amino side of the opening site
(now
forming the carboxyl sequence). The circularly permuted molecule may then be
ligated
into a plasmid and expressed as discussed below.
C. ModiFed IL-13
One of skill in the art will appreciate that IL-13 can be modified in a
variety of ways that do not destroy binding specificity and/or avidity and, in
fact, may
increase binding properties. Some modifications may be made to facilitate the
cloning,
expression, or incorporation of the circularly permuted growth factor into a
fusion
protein. Such modifications are well known to those of skill in the art and
include, for
example, a methionine added at the amino terminus to provide an initiation
site, or
additional amino acids placed on either terminus to create conveniently
located restriction
sites or termination codons.
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One of skill will recognize that other modifications may be made. Thus,
for example, amino acid substitutions may be made that increase specificity or
binding
affinity of the circularly permuted protein, etc. Alternatively, non-essential
regions of the
molecule may be shortened or eliminated entirely. Thus, where there are
regions of the
molecule that are not themselves involved in the activity of the molecule,
they may be
eliminated or replaced with shorter segments that merely serve to maintain the
correct
spatial relationships between the active components of the molecule.
D. Derivatized Peptides and Peptidomimetics
The design of chemically modified peptides and peptide mimics which are
resistant to degradation by proteolytic enzymes or have improved solubility or
binding
properties is well known.
Modified amino acids may contain additional chemical moieties or
modified amino acids not normally a part of a protein. Covalent modifications
of the
peptide are thus included within the scope of the present invention. Such
modifications
may be introduced into an IL-13 receptor binding molecule by reacting targeted
amino
acid residues of the molecule with an organic derivatizing agent that is
capable of reacting
with selected side chains or terminal residues. The following examples of
chemical
derivatives are provided by way of illustration and not by way of limitation.
It should be
noted that to determine whether any derivatized or modified molecule is an IL-
13
receptor binding molecule of the present invention, the molecule can be tested
to
determine whether it binds to an IL-13R with an affinity at least about 3
times that of wild
type IL-13, more preferably about S times that of wild type IL-13, and even
more
preferably at least about 10 times that of wild type IL-13, or more.
The design of peptide mimics which are resistant to degradation by
proteolytic enzymes is well known, both for hormone agonist/antagonist and for
enzyme
inhibitor design. See e.g., Sawyer, in STRUCTURE-BASED DRUG DESIGN, P.
Verapandia, Ed., NY 1997; U.S. Patent No. 5,552,534; and U.S. Patent No.
5,550,251, all
of which are incorporated by reference.
Historically, the major focus of peptidomimetic design has evolved from
receptor-targeted drug discovery research and has not been directly impacted
by an
experimentally-determined three-dimensional structure of the target protein.
Nevertheless, a hierarchical approach of peptide~peptidomimetic molecular
design and
chemical modification has evolved over the past two decades, based on
systematic
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transformation of a peptide ligand and iterative analysis of the structure-
activity and
structure-conformation relationships of "second generation" analogs. Such work
has
typically integrated biophysical techniques (x-ray crystallography and/or NMR
spectroscopy) and computer-assisted molecular modeling with biological testing
to
advance peptidomimetic drug design.
The three-dimensional structural properties of peptides are defined in
terms of torsion angles ('f, cp, w, ~ between the backbone amine nitrogen
(N"), backbone
carbonyl carbon (C1), backbone methionine carbon (C"), and side chain
hydrocarbon
functionalization (e.g., Ca, C~', Cs, Ce of Lys) derived from the amino acid
sequence. A
Ramachandran plot ('h versus cp) may define the preferred combinations of
torsion angles
for ordered secondary structures (conformations), such as " helix, p turn, Y
turn, or p sheet.
Molecular flexibility is directly related to covalent and/or noncovalent
bonding
interactions within a particular peptide. Even modest chemical modifications
by N"-
methyl, C"-methyl or Cp-methyl can have significant consequences on the
resultant
conformation.
The N"-C"-C' scaffold may be transformed by introduction of olefin
substitution (e.g., C"-C~ --> C = C or dehydroamino acid or insertion (e.g.,
C"-C' -~ C"-
C=C-C' or vinylogous amino acid. Also the Cp carbon may be substituted to
advance the
design of so-called "chimeric" amino acids. Finally, with respect to N-
substituted amides
it is also noteworthy to mention the intriguing approach of replacing the
traditional
peptide scaffold by achiral N-substituted glycine building blocks. Overall,
such N"-C"-C
scaffold or C"-C~ side chain modifications expand peptide-based molecular
diversity (i.e.,
so-called "peptoid" libraries) as well as extend our 3-D structural knowledge
of
traditional cp-'IJ-x space.
In one approach, such as disclosed by Sherman and Spatola, J. Am. Chem.
Soc. 112: 433 (1990), one or more amide bonds are replaced in an essentially
isosteric
manner by a variety of chemical functional groups. For example, any amide
linkage in an
IL-13 receptor binding molecule can be replaced by a ketomethylene moiety,
e.g. (-
C(=O)-CHZ_) for (-(C=O)-NH-). A few of the known amide bond replacements
include:
aminomethylene or 'Y[CH2NH]; ketomethylene or'h[COCHz]; ethylene or
'f[CHZCHZ];
olefin or'Y[CH=CH]; ether or'F[CH20]; thioether or 'Y[CHZS]; tetrazole
or'h[CN4];
thiazole or 'Y[thz]; retroamide or'F[NHCO]; thioamide or 'Y[CSNH]; and ester
or
')l[C02]. These amide bond surrogates alter conformational and H-bonding
properties
that may be requisite for peptide molecular recognition and/or biological
activity at
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receptor targets. Furthermore, such backbone replacements can impart metabolic
stability
towards peptidase cleavage relative to the parent peptide. The discovery of
yet other
nonhydrolyzable amide bond isostere has particularly impacted the design of
protease
inhibitors, and these include: hydroxymethylene or 'f [CH(OH)];
hydroxyethylene or
'h[CH(OH)CH2] and 'Y[CH2CH(OH)]; dihydroxyethylene or ('Y[CH(OH)CH(OH)],
hydroxyethylamine or 'Y[CH(OH)CHZN], dihydroxyethylene and CZ_syrnmetric
hydroxymethylene. Such backbone modifications have been extremely effective,
as they
may represent transition state mimics or bioisosteres of the hypothetical
tetrahedral
intermediate (e.g., 'f [C(OH)2NH]) for this class of proteolytic enzymes. Such
derivatives
are expected to have the property of increased stability to degradation by
enzymes, and
therefore possess advantages for the formulation of compounds which may have
increased in vivo half lives, as administered by oral, intravenous,
intramuscular,
intraperitoneal, topical, rectal, intraocular, or other routes.
Both peptide backbone and side chain modifications may provide
prototypic leads for the design of secondary structure mimicry, as typically
suggested by
the fact that substitution of D-amino acids, N"-Me-amino acids, Ca-Me amino
acids,
and/or dehydroamino acids within a peptide lead may induce or stabilize
regiospecific
U-turn, T-turn, p-sheet, or °'-helix conformations. To date, a variety
of secondary structure
mimetics have been designed and incorporated in peptides or peptidomimetics.
The
a-turn has been of particular interest to the area of receptor-targeted
peptidomimetic drug
discovery. This secondary structural motif exists within a tetrapeptide
sequence in which
the first and fourth Ca atoms are <_ 7 A separated, and they are further
characterized as to
occur in a nonhelical region of the peptide sequence and to possess a ten-
membered
intramolecular H-bond between the i and i--~4 amino acid residues. One of the
initial
approaches of significance to the design of p-turn mimetics was the monocyclic
dipeptide-
based template which employs side chain to backbone constraint at the i+1 and
i+2 sites.
Over the past decade a variety of other monocyclic or bicyclic templates have
been
developed as p-turn mimetics. Monocyclic U-turn mimetic has been described
that
illustrate the potential opportunity to design scaffolds that may incorporate
each of the
side chains (i, i+1, i+2 and i+3 positions), as well as five of the eight NH
or C=O
functionalities, within the parent tetrapeptide sequence, tetrapeptide
sequence modeled in
type I-IV p-turn conformations. Similarly, a benzodiazepine template has shown
utility as
a U-turn mimetic scaffold which also may be multisubstituted to simulate side
chain
functionalization,; particularly at the i and i+3 positions of the
corresponding tetrapeptide
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sequence modeled in type I-VI p-turn conformations. A recently reported y-turn
mimetic,
illustrates an innovative approach to incorporate a retroamide surrogate
between the i and
i~l amino acid residues with an ethylene bridge between the N1 (i.e., nitrogen
replacing
the carbonyl C') and N atoms of the i and i+2 positions, and this template
allows the
possibility for all three side chains of the parent tripeptide sequence.
Finally, the design
of a p-sheet mimetic provides an attractive template to constrain the backbone
of a peptide
to that simulating an extended conformation. The p-sheet is of particular
interest to the
area of protease-targeted peptidomimetic drug discovery.
Aromatic amino acids may be replaced with D- or L-napthylalanine, D- or
L-phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or 4-
pyreneylalanine, D- or
L-3-thieneylalanine, D- or L-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-
alanine, D- or
L-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-
(trifluoromethyl)-
phenylglycine, D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D-
or L-p-
biphenylphenylalanine, D- or L-p-methoxybiphenylphenylalanine, D- or L-2-
indole(alkyl)alanines, and D- or L-alkylainines where alkyl may be substituted
or
unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-
butyl, sec-isotyl,
iso-pentyl, non-acidic amino acids, of C1-C20.
Acidic amino acids can be substituted with non-carboxylate amino acids
while maintaining a negative charge, and derivatives or analogs thereof, such
as the non-
limiting examples of (phosphono)alanine, glycine, leucine, isoleucine,
threonine, or
serine; or sulfated (e.g., -S03H) threonine, serine, tyrosine.
Other substitutions may include unnatural hyroxylated amino acids made
by combining "alkyl" (as defined and exemplified herein) with any natural
amino acid.
Basic amino acids may be substituted with alkyl groups at any position of the
naturally
occurring amino acids lysine, arginine, ornithine, citrulline, or (gua:nidino)-
acetic acid, or
other (guanidino)alkyl-acetic acids, where "alkyl" is define as above. Nitrile
derivatives
(e.g., containing the CN-moiety in place of COOH) may also be substituted for
asparagine or glutamine, and methionine sulfoxide may be substituted for
methionine.
Methods of preparation of such peptide derivatives are well known to one
skilled in the
art.
In addition, any amino acid can be replaced by the same amino acid but of
the opposite chirality. Thus, any amino acid naturally occurring in the L-
configuration
(which may also be referred to as the R or S, depending upon the structure of
the
chemical entity) may be replaced with an amino acid of the same chemical
structural
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type, but of the opposite caroled, generally referred to as the D- amino acid
but which can
additionally be referred to as the R- or the S-, depending upon its
composition and
chemical configuration. Such derivatives have the property of greatly
increased stability
co degradation by enzymes, and therefore are advantageous in the formulation
of
compounds which may have longer in vivo half lives, when administered by oral,
intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or
other routes.
Additional amino acid modifications may include the following: Cysteinyl
residues may be reacted with alpha-haloacetates (and corresponding amines),
such as 2-
chloroacetic acid or chloroacetamide, to give carboxyrnethyl or
carboxyamidomethyl
derivatives. Cysteinyl residues may also be derivatized by reaction with
compounds such
as bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid,
chloroacetyl
phosphate, N-alkylmaleimides, 3-vitro-2-pyridyl disulfide, methyl 2-pyridyl
disulfide, p-
chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-
oxa-1,3-
diazole.
Histidyl residues may be derivatized by reaction with compounds such as
diethylprocarbonate e.g., at pH S.5-7.0 because this agent is relatively
specific for the
histidyl side chain, and para-bromophenacyl bromide may also be used; e.g.,
where the
reaction is preferably performed in 0.1 M sodium cacodylate at pH 6Ø
Lysinyl and amino terminal residues may be reacted with compounds such
as succinic or other carboxylic acid anhydrides. Derivatization with these
agents is
expected to have the effect of reversing the charge of the lysinyl residues.
Other suitable
reagents for derivatizing alpha-amino-containing residues include compounds
such as
imidoesters/e.g., as methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4
pentanedione; and
transaminase-catalyzed reaction with glyoxylate.
Arginyl residues may be modified by reaction with one or several
conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-
cyclohexanedione, and ninhydrin according to known method steps.
Derivatization of
arginine residues requires that the reaction be performed in alkaline
conditions because of
the high pKa of the guanidine functional group. Furthermore, these reagents
may react
with the groups of lysine as well as the arginine epsilon-amino group.
Tyrosyl residues may be modified by reaction with aromatic diazonium
compounds or tetranitromethane. N-acetylimidizol and tetranitromethane may be
used to
form O-acetyl tyrosyl species and 3-vitro derivatives, respectively.
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Carboxyl side groups (aspartyl or glutamyl) may be selectively modified
by reaction with carbodiimides (R'-N-C-N-R') such as 1-cyclohexyl-3-(2-
morpholinyl-
(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)
carbodiimide.
Furthermore aspartyl and glutamyl residues may be converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
Glutaminyl and asparaginyl residues may be frequently deamidated to the
corresponding glutamyl and aspartyl residues. Alternatively, these residues
may be
deamidated under mildly acidic conditions. Either form of these residues falls
within the
scope of the present invention.
Derivatization with bifunctional agents is useful for cross-linking the
peptide to certain chemical moieties. Commonly used cross-linking agents
include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide
esters, for
example, esters with 4-azidosalicylic acid, homobifunctional imidoesters,
including
disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and
bifunctional
maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-
[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that
are
capable of forming crosslinks in the presence of light. Alternatively,
reactive water-
insoluble matrices such as cyanogen bromide-activated carbohydrates and the
reactive
substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128;
4,247,642;
4,229,537; and 4,330,440 (which are herein incorporated entirely by
reference), may be
employed for protein immobilization.
Other modifications may include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation
of the
alpha-amino groups of lysine, arginine, and histidine side chains (T. E.
Creighton,
Proteins: Structure and Molecule Properties, W. H. Freeman & Co., San
Francisco, pp.
79-86 (1983)), acetylation of the N-terminal amine, methylation of main chain
amide
residues (or substitution with N-methyl amino acids) and, in some instances,
amidation of
the C-terminal carboxyl groups, according to known method steps.
Such derivatized molecules may improve the solubility, absorption,
permeability across the blood brain barner biological half life, and the like.
Such
modifications may alternatively eliminate or attenuate any possible
undesirable side effect
of the protein and the like. Molecules capable of mediating such effects are
disclosed, for
example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing
Co.,
Easton, Pa. (1980).
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Such chemical derivatives also may provide attachment to solid supports,
including but not limited to, agarose, cellulose, hollow fibers, or other
polymeric
carbohydrates such as agarose, cellulose, such as for purification, generation
of antibodies
or cloning; or to provide altered physical properties, such as resistance to
enzymatic
degradation or increased binding affinity or modulation of IL-13, which is
desired for
therapeutic compositions comprising IL-13 receptor binding molecules,
antibodies thereto
or fragments thereof. Such peptide derivatives are well-known in the art, as
well as
method steps for making such derivatives using carbodiimides active esters of
N-hydroxy
succinimmide, or mixed anhydrides, as non-limiting examples.
Variation upon the sequences of IL-13 receptor binding molecules of the
present invention may also include: the addition of one or more (e.g., two,
three, four, or
five) lysine, arginine or other basic residues or one, or more (e.g., two,
three, four, or five)
glutamate or aspartate or other acidic residues at one end of the peptide,
where "acidic"
and "basic" are as defined herein. Negative charges can also be introduced by
the
addition of carboxyl, phosphate, borate, sulfonate or sulfate groups. Such
modifications
may increase the alpha-helical content of the peptide by the "helix dipole
effect". They
also can provide enhanced aqueous solubility of the peptide, and allow the
correct
insertion of peptides into a membrane structure.
In another approach, a variety of uncoded or modified amino acids such as
D-amino acids and N-methyl amino acids have been used to modify mammalian
peptides.
Alternatively, a presumed bioactive conformation has been stabilized by a
covalent
modification, such as cyclization or by incorporation of gamma-lactam or other
types of
bridges. See, e.g., Veber and Hirschmann, et al., Proc. Natl. Acad. Sci. USA,
1978 75
2636 and Thorsett, et al., Biochem Biophys. Res. Comm., 1983 111 166. The
primary
purpose of such manipulations has not been to avoid metabolism or to enhance
oral
bioavailability but rather to constrain a bioactive conformation to enhance
potency or to
induce greater specificity for a receptor subtype.
VII. The Effector Molecule.
As described above, the effector molecule component of'the chimeric
molecules of this invention may be any molecule whose activity it is desired
to deliver to
cells that overexpress IL-13 receptors. Particularly preferred effector
molecules include
cytotoxins such as Pseudomonas exotoxin or Diphtheria toxin, radionuclides,
ligands
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such as growth factors, antibodies, detectable labels such as fluorescent or
radioactive
labels, and therapeutic compositions such as liposomes and various drugs.
A) Cytotoxins.
Particularly preferred cytotoxins include Pseudomonas exotoxins,
Diphtheria toxins, ricin, saponin, gelonin, ribosome inactivating protein, and
abrin.
Pseudomonas exotoxin and Diphtheria toxin, modified to remove their capacity
for non-
specific binding, are the most preferred.
i) Pseudomonas exotoxin (PE).
Pseudomonas exotoxin A (PE) is an extremely active monomeric protein
(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, which inhibits
protein
synthesis in eukaryotic cells through the inactivation of elongation factor 2
(EF-2) by
catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP ribosyl
moiety of
oxidized NAD onto EF-2).
The toxin contains three structural domains that act in concert to cause
cytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding. Domain II
(amino
acids 253-364) is responsible for translocation into the cytosol and domain
III (amino
acids 400-613) mediates ADP ribosylation of elongation factor 2, which
inactivates the
protein and causes cell death. The function of domain Ib (amino acids 365-399)
remains
undefined, although a large part of it, amino acids 365-380, can be deleted
without loss of
cytotoxicity. See Siegall et al., J. Biol. Chem. 264: 14256-14261 (1989).
Where the targeting molecule (e.g. an IL-13R binding molecule) is fused
to PE, a preferred PE molecule is one in which domain Ia (amino acids 1
through 252) is
deleted and amino acids 365 to 380 have been deleted from domain Ib. However
all of
domain Ib and a portion of domain II (amino acids 350 to 394) can be deleted,
particularly if the deleted sequences are replaced with a linking peptide such
as GGGGS.
In addition, the PE molecules can be further modified using site-directed
mutagenesis or other techniques known in the art, to alter the molecule for a
particular
desired application. Means to alter the PE molecule in a manner that does not
substantially affect the functional advantages provided by the PE molecules
described
here can also be used and such resulting molecules are intended to be covered
herein.
For maximum cytotoxic properties of a preferred PE molecule, several
modifications to the molecule are recommended. An appropriate carboxyl
terminal
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sequence to the recombinant molecule is preferred to translocate the molecule
into the
cytosol of target cells. Amino acid sequences which have been found to be
effective
include, REDLK (as in native PE), REDL, RDEL, or KDEL, repeats of those, or
other
sequences that function to maintain or recycle proteins into the endoplasmic
reticulum,
referred to here as "endoplasmic retention sequences". See, for example,
Chaudhary et al,
Proc. Natl. Acad. Sci. USA 87:308-312 and Seetharam et al, J. Biol. Chem. 266:
17376-
17381 (1991).
Deletions of amino acids 365-380 of domain Ib can be made without loss
of activity. Further, a substitution of methionine at amino acid position 280
in place of
glycine to allow the synthesis of the protein to begin and of serine at amino
acid position
287 in place of cysteine to prevent formation of improper disulfide bonds is
beneficial.
In a preferred embodiment, the targeting molecule is inserted in
replacement for domain Ia. A similar insertion has been accomplished in what
is known
as the TGFa-PE40 molecule (also referred to as TP40) described in Heimbrook et
al.,
Proc. Natl. Acad. Sci., USA, 87: 4697-4701 (1990) and in U.S. Patent
5,458,878.
Preferred forms of PE contain amino acids 253-364 and 381-608, and are
followed by the native sequences REDLK or the mutant sequences KDEL or RDEL.
Lysines at positions 590 and 606 may or may not be mutated to glutamine.
In a particularly preferred embodiment, the IL-13 receptor targeted
cytotoxins of this invention comprise the PE molecule designated PE38. This PE
molecule is a truncated form of PE composed of amino acids 253-364 and 381-
608.
Moreover, PE38 can be further modified to create a variant known as PE38QQR by
replacing the lysine residues at positions 509 and 606 by glutamine and
replacing the
residue at 613 by arginine (Debinski et al. Bioconj. Chem., 5: 40 (1994)).
In another particularly preferred embodiment, the IL-13 receptor targeted
cytotoxins of this invention comprise the PE molecule designated PE4E. PE4E is
a "full
length" PE with a mutated and inactive native binding domain where amino acids
57,
246, 247, and 249 are all replaced by glutamates (see, e.g., Chaudhary et al.,
J. Biol.
Chem., 265: 16306 (1995)).
The targeting molecule (e.g. the IL-13R binding molecule) may also be
inserted at a point within domain III of the PE molecule. Most preferably the
targeting
molecule is fused between about amino acid positions 607 and 609 of the PE
molecule.
This means that the targeting molecule is inserted after about amino acid 607
of the
molecule and an appropriate carboxyl end of PE is recreated by placing amino
acids about
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604-613 of PE after the targeting molecule. Thus, the targeting molecule is
inserted
within the recombinant PE molecule after about amino acid 607 and is followed
by amino
acids 604-613 of domain III. The targeting molecule may also be inserted into
domain Ib
to replace sequences not necessary for toxicity. Debinski, et al. Mol. Cell.
Biol., 11:
1751-1753 (1991).
In a preferred embodiment, the PE molecules are fused to the targeting
molecule by recombinant means. The genes encoding protein chains may be cloned
in
cDNA or in genomic form by any cloning procedure known to those skilled in the
art
(see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring
Harbor Laboratory, (1989)). Methods of cloning genes encoding PE fused to
various
ligands are well known to those of skill in the art (see, e.g., Siegall et
al., FASEB J., 3:
2647-2652 (1989); and Chaudhary et al. Proc. Natl. Acad. Sci. USA, 84: 4538-
4542
(1987)).
Those skilled in the art will realize that additional modifications,
deletions,
insertions and the like may be made to the chimeric molecules of the present
invention or
to the nucleic acid sequences encoding IL-13 receptor-directed chimeric
molecules.
Especially, deletions or changes may be made in PE or in a linker connecting
an antibody
gene to PE, in order to increase cytotoxicity of the fusion protein toward
target cells or to
decrease nonspecific cytotoxicity toward cells without antigen for the
antibody. All such
constructions may be made by methods of genetic engineering well known to
those
skilled in the art (see, generally, Sambrook et al., supra) and may produce
proteins that
have differing properties of affinity, specificity, stability and toxicity
that make them
particularly suitable for various clinical or biological applications.
ii) Diphtheria toxin (DT).
Like PE, Diphtheria toxin (DT) kills cells by ADP-ribosylating elongation
factor 2 thereby inhibiting protein synthesis. Diphtheria toxin, however, is
divided into
two chains, A and B, linked by a disulfide bridge. In contrast to PE, chain B
of DT,
which is on the carboxyl end, is responsible for receptor binding and chain A,
which is
present on the amino end, contains the enzymatic activity (Uchida et al.,
Science, 175:
901-903 (1972); Uchida et al. .l. Biol. Chem., 248: 3838-3844 (1973)).
In a preferred embodiment, the targeting molecule-Diphtheria toxin fusion
proteins of this invention have the native receptor-binding domain removed by
truncation
of the Diphtheria toxin B chain. Particularly preferred is DT388, a DT in
which the
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carboxyl terminal sequence beginning at residue 389 is removed. Chaudhary, et
al.,
Bioch. Biophys. Res. Comm., 180: 545-551 (1991).
Like the PE chimeric cytotoxins, the DT molecules may be chemically
conjugated to the IL-13 receptor targeting molecule, but, in a preferred
embodiment, the
targeting molecule will be fused to the Diphtheria toxin by recombinant means.
The
genes encoding protein chains may be cloned in cDNA or in genomic form by any
cloning procedure known to those skilled in the art. Methods of cloning genes
encoding
DT fused to various ligands are also well known to those of skill in the art
(see, e.g.,
Williams et al. J. Biol. Chem. 265: 11885-11889 (1990)).
The term "Diphtheria toxin" (DT) as used herein refers to full length
native DT or to a DT that has been modified. Modifications typically include
removal of
the targeting domain in the B chain and, more specifically, involve
truncations of the
carboxyl region of the B chain.
B) Detectable labels.
Detectable labels suitable for use as the effector molecule component of
the chimeric molecules of this invention include any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical, electrical, optical
or
chemical means. Useful labels in the present invention include magnetic beads
(e.g.
DynabeadsrM), fluorescent dyes (e.g., fluorescein isothiocyanate, texas red,
rhodamine,
green fluorescent protein, and the like), radiolabels (e.g., 3H, l2sh 3sS,
laC, or 32P),
enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others
commonly used
in an ELISA), and colorimetric labels such as colloidal gold or colored glass
or plastic
(e.g. polystyrene, polypropylene, latex, etc.) beads.
Means of detecting such labels are well known to those of skill in the art.
Thus, for example, radiolabels may be detected using photographic film or
scintillation
counters, fluorescent markers may be detected using a photodetector to detect
emitted
illumination. Enzymatic labels are typically detected by providing the enzyme
with a
substrate and detecting the reaction product produced by the action of the
enzyme on the
substrate, and colorimetric labels are detected by simply visualizing the
colored label.
C) Ligands.
As explained above, the effector molecule may also be a ligand or an
antibody. Particularly preferred ligand and antibodies are those that bind to
surface
markers on immune cells. Chimeric molecules utilizing such antibodies as
effector
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molecules act as bifunctional linkers establishing an association between the
immune
cells bearing binding partner for the ligand or antibody and the tumor cells
overexpressing the IL-13 receptor. Suitable antibodies and growth factors are
known to
those of skill in the art and include, but are not limited to, IL-2, IL-4, IL-
6, IL-7, tumor
S necrosis factor (TNF), anti-Tac, TGFa, and the like.
D) Other therapeutic moieties.
Other suitable effector molecules include pharmacological agents or
encapsulation systems containing various pharmacological agents. Thus, the
targeting
molecule of the chimeric molecule may be attached directly to a drug that is
to be
delivered directly to the tumor. Such drugs are well known to those of skill
in the art and
include, but are not limited to, doxirubicin, vinblastine, genistein, an
antisense molecule,
and the like.
Alternatively, the effector molecule may be an encapsulation system, such
as a liposome or micelle that contains a therapeutic composition such as a
drug, a nucleic
acid (e.g. an antisense nucleic acid), or another therapeutic moiety that is
preferably
shielded from direct exposure to the circulatory system. Means of preparing
liposomes
attached to proteins are well known to those of skill in the art. See, for
example, U.S.
Patent No. 4,957,735, Connor et al., Pharm. Ther., 28: 341-365 (1985)
VIII. Attachment of the Targeting Molecule to the Effector Molecule.
One of skill will appreciate that the targeting molecule and effector
molecules may be joined together in any order. Thus, where the targeting
molecule is a
polypeptide, the effector molecule may be joined to either the amino or
carboxy termini
of the targeting molecule. The targeting molecule may also be joined to an
internal
region of the effector molecule, or conversely, the effector molecule may be
joined to an
internal location of the targeting molecule, as long as the attachment does
not interfere
with the respective activities of the molecules.
The targeting molecule and the effector molecule may be attached by any
of a number of means well known to those of skill in the art. Typically the
effector
molecule is conjugated, either directly or through a linker (spacer), to the
targeting
molecule. However, where both the effector molecule and the targeting molecule
are
polypeptides it is preferable to recombinantly express the chimeric molecule
as a single-
chain fusion protein.
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A) Conjugation of the effector molecule to the targeting molecule.
In one embodiment, the targeting molecule (e.g., IL-13R binding
molecule) is chemically conjugated to the effector molecule (e.g., a
cytotoxin, a label, a
ligand, or a drug or liposome). Means of chemically conjugating molecules are
well
S known to those of skill.
The procedure for attaching an agent to an antibody or other polypeptide
targeting molecule will vary according to the chemical structure of the agent.
Polypeptides typically contain variety of fimctional groups; e.g., carboxylic
acid (COOH)
or free amine (-NHZ) groups, which are available for reaction with a suitable
functional
group on an effector molecule to bind the effector thereto.
Alternatively, the targeting molecule and/or effector molecule may be
derivatized to expose or attach additional reactive functional groups. The
derivatization
may involve attachment of any of a number of linker molecules such as those
available
from Pierce Chemical Company, Rockford Illinois.
A "linker", as used herein, is a molecule that is used to join the targeting
molecule to the effector molecule. The linker is capable of forming covalent
bonds to
both the targeting molecule and to the effector molecule. Suitable linkers are
well known
to those of skill in the art and include, but are not limited to, straight or
branched-chain
carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the
targeting
molecule and the effector molecule are polypeptides, the linkers may be joined
to the
constituent amino acids through their side groups (e.g., through a disulfide
linkage to
cysteine). In a preferred embodiment, however, the linkers will be joined to
the alpha
carbon amino and carboxyl groups of the terminal amino acids.
A bifunctional linker having one functional group reactive with a group on
a particular agent, and another group reactive with an antibody, may be used
to form a
desired immunoconjugate. Alternatively, derivatization may involve chemical
treatment
of the targeting molecule, e.g., glycol cleavage of the sugar moiety of a the
glycoprotein
antibody with periodate to generate free aldehyde groups. The free aldehyde
groups on
the antibody may be reacted with free amine or hydrazine groups on an agent to
bind the
agent thereto. (See U.S. Patent No. 4,671,958). Procedures for generation of
free
sulthydryl groups on polypeptide, such as antibodies or antibody fragments,
are also
known (See U.S. Pat. No. 4,659,839).
Many procedure and linker molecules for attachment of various
compounds including radionuclide metal chelates, toxins and drugs to proteins
are
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known. See, for example, European Patent Application No. 188,256; U.S. Patent
Nos.
4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and
4,589,071; and
Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987).
In some circumstances, it is desirable to free the effector molecule from
the targeting molecule when the chimeric molecule has reached its target site.
Therefore,
chimeric conjugates comprising linkages which are cleavable in the vicinity of
the target
site may be used when the effector is to be released at the target site.
Cleaving of the
linkage to release the agent may be prompted by enzymatic activity or
conditions to
which the chimeric molecule is subjected either inside the target cell or in
the vicinity of
the target site. When the target site is a tumor, a linker which is cleavable
under
conditions present at the tumor site (e.g. when exposed to tumor-associated
enzymes or
acidic pH) may be used.
A number of different cleavable linkers are known to those of skill in the
art. See, U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. The mechanisms
for
release of an agent from these linker groups include, for example, irradiation
of a
photolabile bond and acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for
example,
includes a description of immunoconjugates comprising linkers which are
cleaved at the
target site in vivo by the proteolytic enzymes of the patient's complement
system. In
view of the large number of methods that have been reported for attaching a
variety of
radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and
other agents
to antibodies, one skilled in the art will be able to determine a suitable
method for
attaching a given agent to a polypeptide.
B) Production of fusion proteins.
Where the targeting molecule and/or the effector molecule is relatively
short (i.e., less than about SO amino acids) they may be synthesized using
standard
chemical peptide synthesis techniques. Where both molecules are relatively
short the
chimeric .molecule may be synthesized as a single contiguous polypeptide.
Alternatively
the targeting molecule and the effector molecule may be synthesized separately
and then
fused by condensation of the amino terminus of one molecule with the carboxyl
terminus
of the other molecule thereby forming a peptide bond. Alternatively, the
targeting and
effector molecules may each be condensed with one end of a peptide spacer
molecule
thereby forming a contiguous fusion protein.
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Solid phase synthesis in which the C-terminal amino acid of the sequence
is attached to an insoluble support followed by sequential addition of the
remaining
amino acids in the sequence is the preferred method for the chemical synthesis
of the
polypeptides of this invention. Techniques for solid phase synthesis are
described by
Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The
Peptides:
Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis,
Part A.,
Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et
al., Solid
Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984).
In a preferred embodiment, the chimeric fusion proteins of the present
invention are synthesized using recombinant DNA methodology. Generally this
involves
creating a DNA sequence that encodes the fusion protein, placing the DNA in an
expression cassette under the control of a particular promoter, expressing the
protein in a
host, isolating the expressed protein and, if required, renaturing the
protein.
DNA encoding the fusion proteins (e.g. IL-13-PE38QQR) of this invention
may be prepared by any suitable method, including, for example, cloning and
restriction
of appropriate sequences or direct chemical synthesis by methods such as the
phosphotriester method of Narang et al. Meth. Enzymol. 68: 90-99 (1979); the
phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151 (1979); the
diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22: 1859-1862
(1981);
and the solid support method of U.S. Patent No. 4,458,066.
Chemical synthesis produces a single stranded oligonucleotide. This may
be converted into double stranded DNA by hybridization with a complementary
sequence, or by polymerization with a DNA polymerase using the single strand
as a
template. One of skill would recognize that while chemical synthesis of DNA is
limited
to sequences of about 100 bases, longer sequences may be obtained by the
ligation of
shorter sequences.
Alternatively, subsequences may be cloned and the appropriate
subsequences cleaved using appropriate restriction enzymes. The fragments may
then be
ligated to produce the desired DNA sequence.
In a preferred embodiment, DNA encoding fusion proteins of the present
invention may be cloned using DNA amplification methods such as polymerase
chain
reaction (PCR). Thus, in a preferred embodiment, the gene for IL-13 is PCR
amplified,
using a sense primer containing the restriction site for NdeI and an antisense
primer
containing the restriction site for HindIII. In a particularly preferred
embodiment, the
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primers are selected to amplify the nucleic acid starting at position 19, as
described by
McKenzie et al. (1987), supra. This produces a nucleic acid encoding the
mature IL-13
sequence and having terminal restriction sites. A PE38QQR fragment may be cut
out of
the plasmid pWDMH4-38QQR or plasmid pSGC242FdN1 described by Debinski et al.
Int. J. Cancer, 58: 744-748 (1994), and by Debinski et al. Clin. Res., 42:
251A (abstract
(1994) respectively. Ligation of the IL-13 and PE38QQR sequences and insertion
into a
vector produces a vector encoding IL-13 joined to the amino terminus of
PE38QQR
(position 253 of PE). The two molecules are joined by a three amino acid
junction
consisting of glutamic acid, alanine, and phenylalanine introduced by the
restriction site.
While the two molecules are preferably essentially directly joined together,
one of skill will appreciate that the molecules may be separated by a peptide
spacer
consisting of one or more amino acids. Generally the spacer will have no
specific
biological activity other than to join the proteins or to preserve some
minimum distance
or other spatial relationship between them. However, the constituent amino
acids of the
spacer may be selected to influence some property of the molecule such as the
folding,
net charge, or hydrophobicity.
The nucleic acid sequences encoding the fission proteins may be expressed
in a variety of host cells, including E. coli, other bacterial hosts, yeast,
and various higher
eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell
lines. The
recombinant protein gene will be operably linked to appropriate expression
control
sequences for each host. For E. coli this includes a promoter such as the T7,
trp, or
lambda promoters, a ribosome binding site and preferably a transcription
termination
signal. For eukaryotic cells, the control sequences will include a promoter
and preferably
an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc.,
and a
polyadenylation sequence, and may include splice donor and acceptor sequences.
The plasmids of the invention can be transferred into the chosen host cell
by well-known methods such as calcium chloride transformation for E. coli and
calcium
phosphate treatment or electroporation for mammalian cells. Cells transformed
by the
plasmids can be selected by resistance to antibiotics conferred by genes
contained on the
plasmids, such as the amp, gpt, neo and hyg genes.
Once expressed, the recombinant fusion proteins can be purified according
to standard procedures of the art, including ammonium sulfate precipitation,
affinity
columns, column chromatography, gel electrophoresis and the like (see,
generally, R.
Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher, Methods
in
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Erczymology Vol. 182: Guide to Protein Purification., Academic Press, Inc.
N.Y.
(1990)). Substantially pure compositions of at least about 90 to 95%
homogeneity are
preferred, and 98 to 99% or more homogeneity are most preferred for
pharmaceutical
uses. Once purified, partially or to homogeneity as desired, the polypeptides
may then be
used therapeutically.
One of skill in the art would recognize that after chemical synthesis,
biological expression, or purification, the IL-13 receptor targeted fusion
protein may
possess a conformation substantially different than the native conformations
of the
constituent polypeptides. In this case, it may be necessary to denature and
reduce the
polypeptide and then to cause the polypeptide to re-fold into the preferred
conformation.
Methods of reducing and denaturing proteins and inducing re-folding are well
known to
those of skill in the art (See, Debinski et al. J. Biol. Chem., 268: 14065-
14070 (1993);
Kreitman and Pastan, Bioconjug. Chem., 4: 581-585 (1993); and Buchner, et al.,
Anal.
Biochem., 205: 263-270 (1992)). Debinski et al., for example, describe the
denaturation
and reduction of inclusion body proteins in guanidine-DTE. The protein is then
refolded
in a redox buffer containing oxidized glutathione and L-arginine.
One of skill would recognize that modifications can be made to the IL-13
receptor targeted fusion proteins without diminishing their biological
activity. Some
modifications may be made to facilitate the cloning, expression, or
incorporation of the
targeting molecule into a fusion protein. Such modifications are well known to
those of
skill in the art and include, for example, a methionine added at the amino
terminus to
provide an initiation site, or additional amino acids placed on either
terminus to create
conveniently located restriction sites or termination codons.
IX. Identification of Target Cells.
Tumor cells overexpress IL-13 receptors. In particular, carcinoma tumor
cells (e.g. renal carcinoma cells) overexpress IL-13 receptors at levels
ranging from about
2100 sites/cell to greater than 150,000 sites per cell. Similarly, gliomas and
Kaposi's
sarcoma also overexpress IL-13 receptors (IL-13R). Moreover, at least some
cells of
virtually every cancer type tested to date appear to overexpress IL-13
receptors. For
example, overexpression of IL-13R has been found in some pancreatic cancers
and about
one-quarter of the head and neck cancers studied. Thus it appears that IL-13
receptor
overexpression is a general characteristic of many solid tumor neoplastic
cells.
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Thus, the methods of this invention can be used to target an effector
molecule to IL-13R-overexpressing neoplastic cells. Neoplasias are well known
to those
of skill in the art and include, but are not limited to, cancers of the skin
(e.g., basal or
squamous cell carcinoma, melanoma, Kaposi's sarcoma, etc.), cancers of the
reproductive
system (e.g., testicular, ovarian; cervical), cancers of the gastrointestinal
tract (e.g.,
stomach, small intestine, large intestine, colorectal, etc.), cancers of the
mouth and throat
(e.g. esophageal, larynx, oropharynx, nasopharynx, oral, etc.), cancers of the
head and
neck, bone cancers, breast cancers, liver cancers, prostate cancers (e.g.,
prostate
carcinoma), thyroid cancers, heart cancers, retinal cancers (e.g., melanoma),
kidney
cancers, lung cancers (e.g., mesothelioma), pancreatic cancers, brain cancers
(e.g.
gliomas, medulloblastomas, pituitary adenomas, etc.) and cancers of the lymph
system
(e.g. lymphoma).
In a particularly preferred embodiment, the methods of this invention are
used to target effector molecules to kidney cancers, colorectal cancers
(especially
colorectal carcinomas), to skin cancers (especially Kaposi's sarcoma), and to
brain
cancers (especially gliomas, and medulloblastomas).
One of skill in the art will appreciate that identification and confirmation
of IL-13 overexpression by cells requires only routine screening using well-
known
methods. Typically this involves providing a labeled molecule that
specifically binds to
the IL-13 receptor. The cells in question are then contacted with this
molecule and
washed. Quantification of the amount of label remaining associated with the
test cell
provides a measure of the amount of IL-13 receptor (IL-13R) present on the
surface of
that cell.
In a preferred embodiment, the IL-13 receptor present may be quantified
by measuring the binding of lzsl-labeled IL-13 (l2sl-IL-13) to the cell in
question. Details
of such a binding assay are provided in the Examples.
X. Pharmaceutical Compositions
The antagonists, agonists, and chimeric molecules of the invention are
useful for parenteral, topical, oral, or local administration, such as by
aerosol or
transdermally, for prophylactic and/or therapeutic treatment. Compositions of
these
molecules can be administered in a pharmaceutically acceptable Garner in a
variety of
unit dosage forms depending upon the method of administration. For example,
unit
dosage forms suitable for oral administration include powder, tablets, pills,
capsules and
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lozenges. It is recognized that the antagonists, agonists, and fusion proteins
of this
invention are peptides or proteins and, when administered orally, must be
protected from
digestion. This is typically accomplished either by complexing the peptide or
protein
with a composition to render it resistant to acidic and enzymatic hydrolysis
or by
packaging the molecule in an appropriately resistant carrier such as a
liposome. Means of
protecting peptides or proteins from digestion are well known in the art.
The pharmaceutical compositions of this invention are particularly useful
for parenteral administration, such as intravenous administration or
administration into a
body cavity or lumen of an organ. The compositions for administration will
commonly
comprise a solution of the antagonist, agonist, or chimeric molecule dissolved
in a
pharmaceutically acceptable carrier, preferably an aqueous Garner. A variety
of aqueous
carriers can be used, e.g., buffered saline and the like. These solutions are
sterile and
generally free of undesirable matter. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions may
contain
pharmaceutically acceptable auxiliary substances as required to approximate
physiological conditions such as pH adjusting and buffering agents, toxicity
adjusting
agents and the like, for example, sodium acetate, sodium chloride, potassium
chloride,
calcium chloride, sodium lactate and the like. The concentration of
antagonists, agonists,
or chimeric molecule in these formulations can vary widely, and will be
selected
primarily based on fluid volumes, viscosities, body weight and the like in
accordance
with the particular mode of administration selected and the patient's needs.
Thus, a typical pharmaceutical composition for intravenous administration
would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about
100 mg
per patient per day may be used, particularly when the drug is administered to
a secluded
site and not into the blood stream, such as into a body cavity or into a lumen
of an organ.
Actual methods for preparing parenterally administrable compositions will be
known or
apparent to those skilled in the art and are described in more detail in such
publications as
Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton,
Pennsylvania (1980).
The compositions containing the peptides or proteins of the invention or a
cocktail thereof (i.e., with other proteins) can be administered for
therapeutic treatments.
In therapeutic applications, compositions are administered to a patient
suffering from a
disease, in an amount sufficient to cure or at least partially arrest the
disease and its
complications. An amount adequate to accomplish this is defined as a
"therapeutically
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effective dose." Amounts effective for this use will depend upon the severity
of the
disease and the general state of the patient's health.
Single or multiple administrations of the compositions may be
administered depending on the dosage and frequency as required and tolerated
by the
patient. In any event, the composition should provide a sufficient quantity of
the proteins
of this invention to effectively treat the patient.
Among various uses of the cytotoxic chimeric molecules, such as fusion
proteins, of the present invention are included inhibiting a variety of
disease conditions
caused by specific human cells that may be eliminated by the toxic action of
the protein.
One preferred application is the inhibition of the growth of cancer cells,
such as by the
use of an IL-13 receptor binding molecule of the invention attached to a
cytotoxin which
kills or inhibits growth of cells which are contacted by or, especially, bound
by the
chimeric molecule.
Where the chimeric molecule comprises an IL-13 receptor targeting
molecule attached to a ligand, the ligand portion of the molecule is chosen
according to
the intended use. Proteins on the membranes of T cells that may serve as
targets for the
ligand includes CD2 (T'11), CD3, CD4 and CDB. Proteins found predominantly on
B
cells that might serve as targets include CD 10 (CALLA antigen), CD 19 and
CD20.
CD45 is a possible target that occurs broadly on lymphoid cells. These and
other possible
target lymphocyte target molecules for the chimeric molecules bearing a ligand
effector
are described in Leukocyte Typing IIl, A.J. McMichael, ed., Oxford University
Press
(1987). Those skilled in the art will realize ligand effectors may be chosen
that bind to
receptors expressed on still other types of cells as described above, for
example,
membrane glycoproteins or ligand or hormone receptors such as epidermal growth
factor
receptor and the like.
It will be appreciated by one of skill in the art that there are some regions
that are not heavily vascularized or that are protected by cells joined by
tight junctions
and/or active transport mechanisms which reduce or prevent the entry of
macromolecules
present in the blood stream. Thus, for example, systemic administration of
therapeutics to
treat gliomas, or other brain cancers, is constrained by the blood-brain
barner which
resists the entry of macromolecules into the subarachnoid space.
One of skill in the art will appreciate that in these instances, the
therapeutic
compositions of this invention can be administered directly to the tumor site.
Thus, for
example, brain tumors (e.g., gliomas) can be treated by administering the
therapeutic
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composition directly to the tumor site (e.g., through a surgically implanted
catheter).
Where the fluid delivery through the catheter is pressurized, small molecules
(e.g. the
therapeutic molecules of this invention) will typically infiltrate as much as
two to three
centimeters beyond the tumor margin.
Alternatively, the therapeutic composition can be placed at the target site
in a slow release formulation. Such formulations can include, for example, a
biocompatible sponge or other inert or resorbable matrix material impregnated
with the
therapeutic composition, slow dissolving time release capsules or
microcapsules, and the
like.
Typically the catheter or time release formulation will be placed at the
tumor site as part of a surgical procedure. Thus, for example, where major
tumor mass is
surgically removed, the perfusing catheter or time release formulation can be
emplaced at
the tumor site as an adjunct therapy. Of course, surgical removal of the tumor
mass may
be undesired, not required, or impossible, in which case, the delivery of the
therapeutic
compositions of this invention may comprise the primary therapeutic modality.
XI. Dia~~nostic Kits.
In another embodiment, this invention provides for kits for inhibiting the
growth of tumors or for the detection of cells overexpressing IL-13 receptors.
Kits will
typically comprise a chimeric molecule of the present invention (e.g. IL-13R
binding
molecule-label, IL-13R binding molecule -cytotoxin, IL-13 R binding molecule -
ligand,
etc.). In addition the kits will typically include instructional materials
disclosing means
of use of chimeric molecule (e.g. as a cytotoxin, for detection of tumor
cells, to augment
an immune response, etc. ). The kits may also include additional components to
facilitate
the particular application for which the kit is designed. Thus, for example,
where a kit
contains a chimeric molecule in which the effector molecule is a detectable
label, the kit
may additionally contain means of detecting the label (e.g. enzyme substrates
for
enzymatic labels, filter sets to detect fluorescent labels, appropriate
secondary labels such
as a sheep anti- mouse-HRP, or the like). The kits may additionally include
buffers and
other reagents routinely used for the practice of a particular method. Such
kits and
appropriate contents are well known to those of skill in the art.
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EXAMPLES
The following examples are offered to illustrate, but not to limit the
claimed invention.
Example 1 : Materials
Restriction endonucleases and DNA ligase were obtained from New
England Biolabs (Beverly, MA), BRL (Gaithersburg, MD), PANVERA (Madison, WI)
and Boehringer Mannheim (Indianapolis, III. Fast Protein Liquid
Chromatographic
(FPLC) columns and media were purchased from Pharmacia (Piscataway, NJ).
Oligonucleotide primers were synthesized at Bioserve Biotechnologies (Laurel,
MD).
Advantage-HF Polymerase chain reaction (PCR) kit was from Clontech (Palo Alto,
CA).
All plasmids carrying the genomes encoding the IL13 proteins were under
a T7 bacteriophage late promoter, a T7 transcription terminator at the end of
the open
reading frame and a fl origin of replication and gene for ampicillin
resistance. Plasmids
were amplified in E. coli (DHSalpha high efficiency transformation) (BRL) and
DNA
was extracted using Qiagen kits (Chatsworth, CA). TF-1 human erythroleukemia
cell line
was obtained from the ATCC (Manassas, VA) and were grown in human GM-CSF. B9
mouse plasmacytoma cell line was a kind gift of Dr. Lucine Aaden and were
grown in
human hIL-6. PM-RCC renal cell carcinoma cell line was established in our
laboratory.
(Puri, R.K. et al., Cellular Immunology 171:80-86 (1996); Puri, R.K. et al.,
Blood
87:4333-4339 (1996); Obiri, N.I. et al., JClin Investig 91:88-93 (1993).
Primary
monocytes and THP-1 cells were kindly provided by Dr. Subhash Dhawan (CBER
FDA)
and Dr. Ray Donnelley (CBER FDA), respectively. Buffycoat from peripheral
blood
were obtained from healthy volunteers who donated blood at NIH Blood Bank.
Mononuclear cells were isolated by centrifugation over Ficoll-Paque Plus
(Pharmacia
Biotech).
Example 2: Protein Homology Search and Prediction of Secondary Structure
Homology search and secondary structure analysis of IL-13: A computer
program, GCG (Genetics Computer Group, Inc., Madison, WI) was used for
homology
search, data base search, and prediction of secondary structure of IL-13 on
Silicon
Graphics Workstation in Human Genome Center, the Institutes of Medical
Science, the
University of Tokyo (Tokyo, Japan) and the Center for Information Technology,
National
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Institutes of Health (Bethesda, MD). The protein sequence of mouse
(i113_mouse.swissprot), rat (126913.gb ro), human (Cafl743334-l.gp_main) and
bos
taurus (bta132441-l.gp main ) were obtained from Swiss Prot and GenBank. Since
126913.gb ro is not the protein sequence but a cDNA sequence, it was used for
homology
searching after first being translated into the corresponding protein
sequence. The IL13
sequence is shown after deletion of predicted signal sequence of Homo Sapiens
or
equivalent [Fig. 1]. Hydrophobicity and secondary structure of IL13 was
predicted by
Kyte-Doolittle method and Chou-Fasman method respectively. (Chou, P. et al.,
Biochemistry 13:222-245 (1974)).
Example 3: Construction of plasmids encoding IL-13R112D
The mutagenesis of IL13 gene was performed using a cDNA as a template
using sense primer 5'- taa ttt gcc cat atg tcc cca ggc cct gtg cct -3', anti-
sense primer 5'-
taa ttt gcc cga att cag ttg aag tct ccc tcg cg -3' to mutate Arg112 to Asp112
and to
incorporate NdeI and EcoRI restriction enzyme sites at the 5'- and 3'-
termini,
respectively. After subcloning the PCR products into pCR2.1 (Invitrogen), the
plasmid
was digested with NdeI and EcoRI. The fragment was inserted into a prokaryotic
.
expression vector, pG420, digested with same restriction enzymes. We confirmed
the
existence of mutation and restriction sites by sequencing of the plasmid.
Example 4: Expression and purification of recombinant proteins
Expression and purification of IL-13R112D and wtIL-13 was carned out
by essentially the same techniques as previously reported for IL4. (Kreitman,
R.J. et al.,
Cytokine 7:311-318 (1995)). However, instead of BL21(lambdaDE3) E. coli, we
used
BL21(lambdaDE3)pLys E. coli genome that contains T7 RNA polymerase under the
lac
promoter and lac operator. The protein expression was induced by adding 1mM
IPTG.
WtILl3 and IL-13R112D were produced in inclusion bodies, which contained a
major,
rather pure protein of 13 kDa. After washing, inclusion bodies were
solubilized, refolded
and purified by FPLC ion-exchange chromatography. The resulting protein was
highly
purified (> 95% pure) and showed one thick band at 13 kDa in Coomassie Blue
stained
SDS- polyacrylamide-gels after electrophoresis. To confirm the identity, the
protein was
shown to react with anti-human IL-13 antibodies on Western blots. We also
observed a
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minor 26 kDa protein in SDS-PAGE gels that we regarded as dimerized IL13R112D
or
wtILl3.
Example 5: Cell Proliferation Assays
Proliferation assays were performed as described previously. (Leland, P.
et al., Oncology Research 7:227-235 (1995)). TF-1 and B9 cells were washed 2-3
times
to remove GM-CSF and IL-6 and then 1X103 to 5X103 cells were cultured in 96-
well
plates in RPMI complete medium containing 10 % fetal bovine serum. Varying
concentration of wtIL-13 and IL-13R112D were added to the wells, and the cells
were
cultured for 1-2 days. Tritiated thymidine (0.5 pCi) was added to each well 6-
9 hours
before the plates were harvested in a Skatron cell harvester (Skatron, Inc.,
Sterling, VA).
Filter mats were counted in a beta plate counter (Wallac).
Example 6: Protein Synthesis Inhibition Assay
Protein synthesis inhibition assay was performed as previously described.
(Puri, R.K., Cancer Research 51:6209-6209 (1991); Puri, R.K. et al., Cancer
Research
51 :3011-3017 (1991)). In brief, 1X103 PM-RCC cells were cultured in leucine-
free
medium (Biofluids, Rockville, MD) for 4 h to allow adherence to flat-bottomed
microtiter
plates. These cells then received various concentrations of IL-13-PE38,
incubated for 20-
24 h at 37 °C and then 1 ~Ci of 3H-leucine (NEN, Boston, MA) was added
to each well
and cultured for an additional 4 h. For blocking experiments, 2000 ng/ml of wt-
IL-13 or
IL-13R112D was added prior to the addition of IL13-PE38. Finally, cells were
washed
and harvested on fiberglass filtermat and cell associated radioactivity was
measured in a
Beta Plate Counter (Wallac, Gaithersburg, MD). The concentration of IL-13-PE38
at
which 50 % inhibition of protein synthesis (IC50) occurred was calculated.
Example 7: IL-13 Receptor Binding Studies
Recombinant human IL-4 and rhIL-13 were labeled with 125I
(Amersham) by using IODO-GEN reagent (Pierce, Rockford, IL) according to the
manufacturer's instructions. The specific activity of radiolabeled IL-4 and IL-
13 ranged
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from 21.0 to 22.0 pCi/~g and 17.6 to 18.0 pCi/wg, respectively. The
equilibrium binding
studies were carned out as described elsewhere. (Obiri, N.I. et al., J Clin
Investig 91:88-
93 (1993); Husain, S.R. et al., Molecular Medicine 3, no. 5:327-338 (1997);
Obiri, N.I. et
al., JBiol Chem 270:8797-8804 (1995); Obiri, N.I. et al., Jlmmunol 158:756-764
(1997)). Briefly, 1X10~6 cells in 100 ~1 of binding buffer were incubated at 4
°C for 2 hr
with lzsl-IL-4 (500 pM) or'zsI-IL-13 (500 pM) in the absence or presence of
increasing
concentrations (10 pM - 200 nM) of unlabeled wt IL-13 or IL-13R112D. The
duplicate
samples of the cells associated with 125I-IL-4 or 125I-IL-13 were separated
from free
izsl-IL-4 or lzsl-IL-13 by centrifugation through cushion of phthalate oils.
The cell pellets
were counted in a Gamma-counter (Wallac, Gaithersburg, MD).
Example 8: Flow Cytometry
Flow cytometric analysis of monocytes were performed as described
elsewhere. (Cosentino, G. et al., J Immunol 155:3145-3151 (1995)). Primary
monocytes
were cultured at 1X10' cells /ml in polypropylene tubes for 72 h with various
concentration of wt-IL-13 or IL-13R112D. Cells were washed and incubated at
4°C for
60 min in FACS staining buffer (HBSS containing FBS, 0.1 % sodium azide)
containing
FITC-conjugated anti-human CD14 (Becton Dickinson, San Jose, CA) antibodies at
a
concentration as recommended by manufacturer. For controls, cells were either
incubated
in FACS staining buffer alone or with isotype control antibody IgG2a, and
antimouse Ig
FITC-conjugate was then used as a secondary antibody for staining. The cells
were
subsequently washed, and fluorescence data were collected on a FACScan/C32
(Becton
Dickinson, San Jose, CA). The data were analyzed with a WinList software
program, and
fluorescence intensity was expressed as mean cannel number (MCN) on 256
channel/104
log scale.
Example 9: Electrophoretic mobility-shift assay (EMSA)
EMSA was performed as described before. (Murata, T. et al., Intl J
Cancer 70:230-240 (1997);Murata, T. et al., Blood 91, no. 10:3884-3891
(1998)). After
incubation with various concentrations of wt IL-13 or IL-13R112D for 10
minutes, THP-
1 cells, primary monocytes and Tory EBV immortalized B cells (a gift from Dr.
Giovanna
Tosato, CBER/FDA) were washed with cold PBS and solubilized with cold whole-
cell
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extraction buffer ( 1 mM MgCl2 , 20 mM HEPES, pH 7.0, 10 mM KCI, 300 mM NaCI,
0.5
mM dithiothreitol, 0.1 % NP-40, 1 mM PMSF, 1 mM Na3 V04 and 20% glycerol). DNA-
protein interactions were assessed by EMSA using the Bandshift kit from
Pharmacia.
Briefly, 40 p.g of sample proteins were incubated for 20 min. at room
temperature with 1
ng of 32 P-labeled double stranded oligonucleotide probe SBE-1 (sense oligo 5'-
gAT CgC
TCT TCT TCC CAg gAA CTC AAT g-3, anti sense oligo 5'-TCg ACA TTg AgT TCC
Tgg gAA gAA gAg C-3') in binding buffer [10 mM Tris-HCI, pH 7.5, 50 mM NaCI,
0.5
mM DTT, 10% glycerol, 0.05% NP-40, 0.05 mg/ml poly (dI-dC)2 ]. In some
experiment,
a 200-fold excess of cold SBE-1 probe was added as a competitor, and 2 w1 of
loading
dye was added to samples which were then electrophoresed in a 5% non-reducing
polyacrylamide gel at 150 V for 2 hr. Gels were dried for 2 hr and
autoradiographed
overnight at room temperature.
Example 10~ Proliferation activity of wtIL-13 and IL-13R112D on hematopoietic
cell lines
After purification of IL13 and mutated IL13, the goal was to compare their
biological activities on various cell types that express different types of
IL13R. First, we
tested the mitogenic activity of these two forms of IL-13. IL13 has been shown
to induce
proliferation of TF-1 human erythroleukemia cell line. (Kitamura, T. et al.,
JCellular
Physiol 140:323-334 (1989); Kitamura, T. et al., Blood 73:375-380 (1989)). We
tested
the proliferative activity of IL-13R112D on the TF-1 cell line. The
proliferative activity
of IL13R112D was more than 10 times better than that wtIL-13. The
concentration of
wtILl3 that produced half maximal proliferation (ED50) was about 2 ng/ml,
compared to
less than 0.2 ng/ml for IL13R112D. Similarly, IL-13R112D stimulated mouse
plasmacytoma cell line B9 much more strongly than did wtIL-13. IL-13R112D was
5.7-
19 fold better than wtIL-13 in proliferation assays. Thus, the proliferation
activity of IL-
13R112D on hematopoietic cells which express the Type III IL13 receptor
complex is
about one log better than wtIL-13.
Example 11: IL13R112D suppresses CD14 expression on monocytes more strongly
than does wtIL-13:
Since IL-13 has been shown to downregulate CD14 expression on
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monocytes (Cosentino, G. et al., Jlmmunol, 155:3145-3151 (1995)), we
investigated
whether the IL13R112D mutant has stronger activity than does wtILl3. We found
that
IL-13R112D and wtIL-13 suppressed CD14 expression on monocytes in a dose
dependent manner. IL13R112D was superior than wtILl3 in downregulation of
CD14.
For example, 1 ng/ml IL-13R112D induced downregulation of CD14 which was
similar
to that induced by 10 ng/ml wtILl3. In addition 10 ng/ml IL13R112D produced
similar
downregulation as did 50 ng/ml wtILl3.
Example 12: STAT 6 activation in THP-1 cells, monocytes and EBV-immortalized
B cells
IL13 has been shown to phosphorylate and activate STAT6 protein for
signal transduction in various cell types. (Murata, T. et al., Jlmmunol 156,
no. 8:2972-
2978 (1996); Obiri, N.I. et al., JBiol Chem 272, no. 32:20251-20258 (1997);
Murata, T.
et al., Cellular Immunology 175:33-40 (1997); Murata, T. et al., International
J Cancer
70, no. 2:230-240 (1997); Murata, T. et al., Internationallmmunology 10:1103-
1110
(1998); Murata, T. et al., Blood 91:3884-3891 (1998)). Therefore, we compared
the
ability of wtIL-13 and of IL-13R112D to stimulate STAT6 in a monocytic cell
line, THP-
1, which expresses the Type II receptor complex, and in primary monocytes
which
express Type III receptor complex. (Murata, T. et al., Intl JMoI Med 1:551-557
(1998)).
The concentration at which IL-13R112D and wtIL-13 stimulated maximal
activation of
STAT6 of primary monocytes was 10 ng/ml and 50 ng/ml, respectively. However, 1
ng/ml of IL-13R112D stimulated maximal activation of STAT6 in THP-1 cells
compared
to 10 ng/ml wtIL-13 that stimulated a slightly less than maximal activation.
These studies
demonstrate that IL13R112D has approximately 5-10 times better activity than
wtILl3 on
human monocytes.
Example 13: Inhibition of f~ZSIlILI3 and ~~25I1IL4 binding by IL13 mutant
We tested the ability of the IL13 mutant to replace [lzsl]IL-13 and [IZSI]IL-
4 binding in PM-RCC cells which express the Type I IL13 receptor complex. The
concentration of IL-13R112D that inhibited [lzsl]IL13 binding by 50% (ED50)
was 150
pM, compared to 650 pM by wtILl3. On the other hand, the ED50 of wtIL-13 and
IL-
13R112D to replace [l2sl]IL-4 binding was 800 pM and 100 pM, respectively.
Thus, IL-
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13R112D interacted more strongly with the IL4R and the IL13R than did wtIL-13.
Example 14: Inhibition of IL13-toxin-mediated cytotoxicity by IL13 mutant:
We have previously demonstrated that an IL13-cytotoxin (IL13-PE38) is
specifically and highly cytotoxic to a PM-RCC cell line. (Puri, R.K. et al.,
Blood
87:4333-4339 (1996)). To determine the relative ability of the IL13 mutant and
wtILl3
to act as the targeting moiety of a chimeric molecule bearing a cytotoxin, we
compared
the ability of wtIL-13 and of IL-13R112D to displace cytotoxicity mediated by
IL-13-
PE38 in PM-RCC cells. IL-13R112D appeared to be better than wtILl3 in blocking
cytotoxicity of IL13-toxin. IL13PE38 was highly cytotoxic to these cells with
a
concentration that inhibited protein synthesis by 50% (IC50) of less than 0.1
ng/ml. In
the presence of 2000 ng/ml wtILl3, the IC50 increased to 60 ng/ml while in the
presence
of IL13R112D, the IC50 reached 105 ng/ml. To carefully determine the extent of
superiority of IL13 mutant in blocking cytotoxicity of IL13PE38, we used
varying
concentration of cytotoxins in the presence of a fixed concentration of
IL13PE38. In this
assay, the IL13 mutant was approximately 10 times better than wtILl3 in
blocking the
cytotoxicity.
Example 15: Loadin~of Antigen Presenting Cells with Antigen
Peripheral blood mononuclear cells (PBMCs) are isolated from a patient
and cultured in RPMI-1640 medium. The cells are incubated with GM-CSF and IL-
13 in
which the arginine at position 112 has been mutated to an aspartic acid. Tumor
cells from
the patient are lysed and lysate representing approximately 106 tumor cells is
added to the
medium to pulse the cells with tumor antigen. The cells are incubated with the
lysate for
4 hours. The cells are then washed with fresh medium and an aliquot is tested
to ensure
presentation of the antigen by the cells. Testing is conducted using a mixed
leukocyte
reaction. The aliquot of pulsed antigen presenting cells (APCs) are placed in
a well of a
standard 96-well plate, to which are added T cells from a second donor. The T
cells are
incubated with the pulsed APCs for 4 hours and then tested to see if they
become lytic to
cells expressing the antigen with which the APCs were pulsed. Testing of the T
cells is
performed by standard SlChromium release or cytokine release assays.
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Example 16: Materials and Methods for Example 17
This Example sets forth materials and methods used in Example 17.
Materials
S Restriction endonucleases and DNA ligase were obtained from New
England Biolabs (Beverly, MA), BRL (Gaithersburg, MD), PANVERA (Madison, WI)
and Boehringer Mannheim (Indianapolis, Ilk. Fast Protein Liquid
Chromatographic
(FPLC) columns and media were purchased from Pharmacia (Piscataway, NJ).
Sequence
specific oligonucleotide primers were synthesized at Bioserve Biotechnologies
(Laurel,
MD). Advantage-HF Polymerase chain reaction (PCR) kit was from Clontech (Palo
Alto,
CA).
The pET based expression vector with ampR gene was used for
construction of mutein clone. Plasmids were amplified in Escherichia coli (DHS
a high
efficiency transformation) (GIBCO BRL Life Technology, Grand Island, NY) and
DNA
was extracted using Qiagen kits (Chatsworth, CA). TF-1 human erythroleukemia
cell line
was obtained from ATCC (Manassas, VA) and were grown in human granulocyte
macrophage colony-stimulating factor. The PM-RCC renal cell carcinoma cell
line was
previously established (Obiri, N. I. et al., J. Clin. Invest. 91:88-93
(1993)). THP-1 cells
were kindly provided by Dr. Ray Donnelly (CBER FDA, Bethesda, MD). AIDS
related
Kaposi's sarcoma cell line KSY-1 cells was kindly provided by P. Gill
(University of
Southern California, Los Angeles, CA) (Husain, S. R et al., Nat. Med. 5:817-
822 (1999)).
Construction of plasmids encoding IL-13R112D and IL-13E13KR112D
The mutagenesis of IL-13 gene was performed using a cDNA of wild type
IL-13 (Minty, A. et al., Nature 362:248-250 (1993)) as a template using sense
primer S'-
taa ttt gcc cat atg tcc cca ggc cct gtg cct -3', anti-sense primer 5'- taa ttt
gcc cga att cag
ttg aag tct ccc tcg cg -3' in order to mutate Arg112 to Asp (R112D) and
incorporate NdeI
and EcoRI restriction enzyme sites at 5'- and 3'- termini, respectively.
Similarly, sense
primer 5 '- agg aga tat aca tat gtc ccc agg ccc tgt gcc tcc ctc tac agc cct
cag gaa get cat
tga gga -3 ' , and anti-sense primer primer 5'- taa ttt gcc cga att cag ttg
aag tct ccc tcg cg -
3' were used to construct expression vector for IL-13E13KR112D. After
subcloning the
PCR products into pCR2.1 ~ (Invitrogen, Carlsbad, CA), the plasmid was
digested with
NdeI and EcoRI. The fragment was inserted into an prokaryotic pET based
expression
vector digested with same restriction enzymes. The existence of mutation and
restriction
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sites was confirmed by sequencing of the plasmid.
Expression and purification of recombinant proteins
Expression and purification of wild type IL-13 and IL-13 mutants were
earned out by essentially similar techniques as previously reported (Kreitman,
R J. et al.,
Cytokine 7:311-318 (1995); Oshima, Y. et al., J. Biol. Chem. 275:14375-14380
(2000)).
The protein expression was induced by adding 1mM IPTG. WtIL-13 and all IL-13
mutants were produced in inclusion bodies. After washing, inclusion bodies
were
solubilized, refolded and purified by FPLC ion-exchange chromatography. The
purified
protein showed a prominent single band at approximately 13 kDa in Coomassie
Blue
stained SDS polyacrylamide gel (Fig. 1). IL-13PE38QQR fusion protein was
expressed
in Escherichia coli and purified as previously described (Debinski, W. et al.,
J. Biol.
Chem. 270:16775-16780 (1995)).
Cell proliferation assays:
Proliferation assays were performed as described previously (Oshima, Y.
et al., .l. Biol. Chem. 275:14375-14380 (2000); Leland, P. et al., Oncol. Res.
7:227-235
(1995)). Briefly, TF-1 cells were washed 2-3 times to remove GM-CSF and then
2X104
cells per well were cultured in 96-well plates in RPMI medium containing 5 %
fetal
bovine serum. Varying concentration of wtIL-13, IL-13 mutants or both were
added to
the wells, and the cells were cultured for approximately 2 days. Tritiated
thymidine (0.5
pCi) was added to each well 6-12 h before the plates were harvested in a
Skatron cell
harvester (Skatron, Inc., Sterling, VA). Glass fiber filter mats were counted
in a beta
plate counter (Wallac, Gaithersburg, MD).
Protein Synthesis Inhibition Assay
Protein synthesis inhibition assay was performed as previously described
(Oshima, Y. et al., J. Biol. Chem. 275:14375-14380 (2000); Puri, R K. et al.,
Cancer Res.
51:3011-3017 (1991)). In brief, 1X103 PM-RCC cells were cultured with various
concentrations of IL-13PE38QQR incubated for 20-24 h at 37 °C and then
1 pCi of 3H-
leucine (NEN, Boston, MA) was added to each well and cultured for an
additional 4 h.
For blocking experiments, 2 ~g/ml of wild type IL-13 or IL-13 mutants was
added prior
to the addition of IL-13PE38QQR Finally, cells were washed and harvested on
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fiberglass filtermat and cell associated radioactivity was measured in a Beta
Plate Counter
(Wallac, Gaithersburg, MD). The concentration of IL-13PE38QQR at which 50
inhibition of protein synthesis (ICSO) occurred was calculated.
IL-13 receptor binding studies
Recombinant human IL-13 was labeled with lzsl (Amersham) by using
IODO-GEN reagent (Pierce, Rockford, IL) according to the manufacture's
instructions.
The specific activity of radiolabeled IL-13 was 18.0 - 26.4 wCi/wg. The
equilibrium
binding studies were carned out as described elsewhere (Obiri, N. I. et al.,
J. Biol. Chem.
270:8797-8804 (1995); Obiri, N. I. et al., J. Clin. Invest. 91:88-93 (1993),
Oshima, Y. et
al., J. Biol. Chem. 275:14375-14380 (2000)). Briefly, 1X106 cells in 100 ~,1
binding
buffer were incubated at 4 °C for 2 hr with lzsl-IL-13 (500 pM) in the
absence or presence
of various concentration of unlabeled wild type IL-13 or IL-13 mutein.
Duplicate
samples of cells associated with lzsl-IL-13 were separated from unbound lzsl-
IL-13 by
centrifugation through cushion of phthalate oils. The cell pellets were
counted in a
Gamma-counter (Wallac, Gaithersburg, MD).
Electrophoretic mobility-shift assay (EMSA)
EMSA was performed as described before (Murata, T. et al., Int. J. Cancer
70:230-240 (1997); Murata, T. et al., Blood 91:3884-3891 (1998); Oshima, Y. et
al., J.
Biol. Chem. 275:14375-14380 (2000)). After incubation with various
concentrations of
wild type IL-13 or IL-13 mutants for 15 minutes, THP-1 cells, Tory Epstein-
Barr virus
(EBV) immortalized B cells (a gift from Dr. Giovanna Tosato, CBER/FDA), or KSY-
1
cells were washed with cold PBS and solubilized with cold whole-cell
extraction buffer
(1 mM MgClz , 20 mM HEPES, pH 7.0, 10 mM KCI, 300 mM NaCI, 0.5 mM
dithiothreitol, 0.1% NP-40, 1 mM PMSF, 1 mM Na3V04 and 20% glycerol). DNA-
protein interactions were assessed by EMSA using the Bandshift kit from
Pharmacia.
Briefly, 80 ~g of sample proteins were incubated for 20 min. at room
temperature with 1
ng of 3zP-labeled double stranded oligonucleotide probe ( 4.2 x 109 CPM/pg
)SBE-1
(sense oligo 5'-gat cgc tct tct tcc cag gaa ctc aat g-3', anti sense oligo S'-
tcg aca ttg agt
tcc tgg gaa gaa gag c-3 ~ in binding buffer [ 10 mM Tris-HCI, pH 7.5, 50 mM
NaCI, 0.5
mM DTT, 10% glycerol, 0.05% NP-40, 0.05 mg/ml poly (dI-dC)z ], and 2 ~l of
loading
dye was added to samples which were then electrophoresed in a 5% non-reducing
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polyacrylamide gel at 150 V for 2.5 hr. Gels were dried for 2 hr and
autoradiographed
overnight at room temperature.
CDl4 regulation by IL-13
Flow cytometric analysis of human primary monocytes was performed as
described elsewhere (Cosentino, G. et al., J. Immunol. 155:3145-31 S 1 (1995);
Oshima, Y.
et al., J. Biol. Chem. 275:14375-14380 (2000)). Primary monocytes were
cultured at
1X10' cells /ml in polypropylene tubes for 48 h with lng/ml wtlL-13 with or
without
1 ~g/ml IL-13E13KR112D. Cells were washed and incubated at 4 °C for 60
min in FACS
staining buffer (HBSS containing 0.5% FBS, 0.1 % sodium azide) containing FITC-
conjugated anti-human CD14 (Becton Dickinson, San Jose, CA) antibodies as per
the
manufacture's recommendations. For controls, cells were either incubated in
FACS
staining buffer alone or with isotype control antibody, mouse IgG2a and then
antimouse
Ig FITC-conjugated was used as secondary antibody for staining. The cells were
subsequently washed, and fluorescence data were collected on a FACScan/C32
equipment (Becton Dickinson, San Jose, CA). The results were analyzed with a
CELLQuest (Becton Dickinson, San Jose, CA) program, and fluorescence intensity
was
expressed as mean channel number (MCN) on 2S6 channel/104 log scale.
Example 17' Comparison of Activity of IL-13R112D and IL-13E13KR112D
Recombinant protein isolation and purification
Recombinant wtIL-13, IL-13R112D and IL-13 double mutein, IL-
13E13KR112D were expressed in Escherichia coli and purified from inclusion
bodies.
After purification, each recombinant protein was analyzed using SDS-PAGE and
stained
with Coomassie blue. Visual inspection of bands suggested that purity of all
preparation
was more than 95 %.
IL-13E13KR112D competes for the binding of radiolabeled IL-13
Binding studies were performed on the PM-RCC renal cell carcinoma cell
line and the U251 glioblastoma cell line. Both cell lines express type I IL-13
receptor
(Obiri, N. I. et al., J. Immunol. 158:756-764 (1997); Murata, T. et al., Int.
J. Mol. Med.
1:551-557 (1998)). Both cell lines showed similar results. As expected, wtIL-
13
displaced specific binding of radiolabeled IL-13. Double mutein IL-13 also
inhibited
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binding of l2sl-IL-13. Double mutein IL-13 showed 1.3-3.0 times better
affinity than
wtIL-13. Introducing mutations did not alter the binding affinity
dramatically.
IL-13 double mutein blocks proliferative activity of IL-13
Proliferative responses of wtIL-13, IL-13R112D and double mutein, IL-
13E13KRI12D was measured alone or in combinations of wtIL-13 and IL-
13E13KR112D
in TF-1 cell line which express type III IL-13R (composed of IL-13Ra1, IL-4Ra
and IL-
2Ry chains). As expected, IL-13 stimulated the growth of TF-1 cells in a
concentration-
dependent manner. IL-13R112D was superior to wtIL-13 in stimulating TF-1 cell
proliferation. In contrast, IL-13E13KR112D did not show any proliferative
activity. To
determine the effect of IL-13EI3KR112D on IL-13 induced proliferation of TF-1
cells,
cells were cultured in the presence of various concentration of IL-13E13KR112D
and half
maximal growth stimulatory concentration of wtIL-13. Interestingly, double
mutein IL-
13 significantly blocked the mitogenic activity of wtIL-13 in a concentration-
dependent
manner.
IL-13 double mutein can neutralize the downregulation of CDl4 expression by
wtlL-13
on monocytes:
Since IL-13 has been shown to downregulate CD14 expression on
monocytes (Cosentino, G. et al., J. Immunol. 155:3145-3151 (1995); Oshima, Y.
et al., J.
Biol. Chem. 275:14375-14380 (2000)), we investigated whether IL-13 double
mutein can
nullify the downregulating activity induced by wtIL-13. wtIL-13 suppressed
CD14
expression on monocytes and IL-13E13KR112D neutralized the effect of wtIL-13.
IL-13 double mutein blocks signal transduction induced by wtlL-13
STATE activation is early cellular response induced by IL-13 and is
responsible for biological effect mediated by IL-13 including upregulation of
MHC class
II expression, CD23 expression, and switching of immunoglobulin class in B
cells
(Shimoda, K. et al., Nature 380:630-633 (1996)). We therefore tested whether
IL-13
double mutein can block the IL-13 induced signaling. Tory Epstein-Barr virus
immortalized B cells and THP-1 monocytic cells express Type III IL-13 receptor
while
KSY-1 AIDS associated Kaposi's sarcoma cells express Type I IL-13 receptor. In
all cell
types both wtIL-13 and IL-13R112D induced STATE activation in a concentration
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dependent manner, but IL-13R112D was 5-10 fold better than wtIL-13 in inducing
STATE activation. In sharp contrast, double mutein IL-13 IL-13E13KR112D did
not
stimulate STATE activation even in the presence of very high concentrations.
In addition,
double mutein blocked the wtIL-13 induced STATE activation in THP-1 cell line.
10
S ng/ml wtIL-13 stimulated maximal activation of STATE, however in the
presence of 10
fold excess of IL-13E13KR112D STATE activation was slightly suppressed.
However,
50-fold excess of double mutein significantly blocked STATE activation.
IL-13E13KR112D blocks cytotoxicity mediated by IL-13PE38QQR.
A chimeric fusion protein composed of wtIL-13 and a mutated form of
Pseudomonas exotoxin (PE38QQR) termed IL-13PE38QQR has previously been
produced (e.g., Debinski, W. et al., Clin. Cancer Res. 1:1253-1258 (1995);
Debinski, W.
et al., J. Biol. Chem. 270:16775-16780 (1995); Puri, R K. et al., Blood
87:4333-4339
(1996); Debinski, W. et al., J. Biol. Chem. 271:22428-22433 (1996); Husain, S.
R et al.,
Clin. Cancer Res. 3:151-156 (1997); Maini, A. et al., J. Urol. 158:948-953
(1997);
Debinski, W. et al., Nat. Biotechnol. 16:449-453 (1998); Husain, S. R et al.,
Blood
95:3506-3513 (2000)). This cytotoxin is highly cytotoxic to IL-13 receptor
positive
tumor cells in vivo and in vitro. IL-13PE38QQR mediates cytotoxicity through
binding to
IL-13 receptors and receptor internalization, therefore IL-13 receptor
agonists and
antagonists must be able to neutralize the cytotoxicity of the chimeric fusion
toxin. To
demonstrate interaction between IL-13 double mutein to IL-13 receptor, we
tested
whether IL-13E13KR112D can block the cytotoxicity mediated by IL-13PE38QQR..
IL-
13PE38QQR mediated cytotoxicity in a concentration dependent manner, and IL-
13, IL-
13R112D or double mutein blocked this cytotoxicity in a concentration
dependent manner
in both cell types studied. IL-13E13KR112D seemed slightly superior than wtIL-
13,
however, it seemed inferior than IL-13R112D in the neutralization of IL-
13PE38QQR
induced cytotoxicity.
Example 18: Materials and Methods for Example 19
Materials
Sequence specific oligonucleotide primers were synthesized at Bioserve
Biotechnologies (Laurel, MD). The pET based expression vector (Novagen,
Madison,
WI) was used for construction of mutein clone. Plasmids were amplified in
Escherichia
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coli, DHSa (GIBCO BRL Life Technology, Grand Island, NY) and DNA was extracted
using plasmid purification kits (Qiagen, Chatsworth, CA). Restriction
endonucleases and
DNA ligase were obtained from New England Biolabs (Beverly, MA), BRL
(Gaithersburg, MD), PANVERA (Madison, WI) and Boehringer Mannheim
(Indianapolis, IN). The TF-1 human erythroleukemia cell line was purchased
from
ATCC (Manassas, VA). The PM-RCC renal cell carcinoma cell line was established
in
our laboratory (Obiri, N. I. et al., J Clin Investig 91:88 (1993)). THP-1 ,
Ebstein-Barr
Virus (EBV) immortalized B cell and AIDS-related Kaposi's sarcoma cell line
KSY-1
were obtained and maintained as previously described (Oshima, Y. et al., JBiol
Chem
275:14375 (2000)).
Construction of plasmids encoding IL-13R112D and IL-13E13K
The mutagenesis of IL-13 gene was performed using cDNA of wtIL-13
(Minty, A. et al., Nature 362:248 (1993)) as a template. Sense primer 5'- agg
aga tat aca
1 S tat gtc ccc agg ccc tgt gcc tcc ctc tac agc cct cag gaa get cat tga gga -
3' , and anti-sense
primer primer S'- taa ttt gcc cga att cag ttg aac cgt ccc tcg cg -3' were used
to mutate Glu
13 (E13) to Lys (K13) and incorporate NdeI and EcoRI restriction enzyme sites
at the 5'-
and 3'- termini, respectively. Construction of the expression vector for IL-
13R112D was
described before (Oshima, Y. et al., JBiol Chem 275:14375 (2000)). After
subcloning
the PCR products, the fragment was restricted by NdeI and EcoRI and inserted
into an
expression vector. Existence of mutation and restriction sites was confirmed
by
sequencing of the plasmid.
Expression and purification of recombinant proteins
Expression and purification of wild type IL-13 and IL-13 mutants were
carned out by techniques similar to those previously reported (Oshima, Y. et
al., JBiol
Chem 275:14375 (2000); Kreitman, R. J. et al., Cytokine 7:311 (1995)). WtIL-13
and IL-
13 mutants were produced in inclusion bodies.
Cell proliferation assays
Proliferation assays were performed as described previously (Oshima, Y.
et al., JBiol Chem 275:14375 (2000); Leland, P. et al., Oncology Research
7:227 (1995)).
Briefly, 1X104 TF-1 cells per well were cultured in 96-well plates in RPMI
with 5% fetal
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bovine serum. Varying concentrations of wtIL-13, IL-13 mutant or both were
added to
the wells, and the cells were cultured for approximately 2 days. Tritiated
thymidine (0.5
pCi) was added to each well 6-12 h before the plates were harvested in a
Skatron cell
harvester (Skatron, Inc., Sterling, VA). Glass fiber filter mats were counted
in a beta
plate counter (Wallac, Gaithersburg, MD).
IL-13 receptor binding studies
WtIL-13 was labeled as previously described (Obiri, N. I. et al., JBiol
Chem 270:8797 (1995); Oshima, Y. et al., JBiol Chem 275:14375 (2000)). The
specific
activity of radiolabeled IL-13 was 26 ~Ci/~g. The equilibrium binding studies
were
performed as described (Obiri, N. I. et al., JBiol Chem 270:8797 (1995);
Oshima, Y. et
al., JBiol Chem 275:14375 (2000)). Briefly, SXlOs cells in 100 w1 binding
buffer were
incubated at 4 °C for 2 hr with lzsl-IL-13 (200 or 500 pM) in the
absence or presence of
various concentrations of unlabeled wtIL-13 or IL-13 mutant. Receptor-bound
lzsl-IL-13
was separated from unbound'zsI-IL-13. The cell pellets were counted in a Gamma-
counter (Wallac, Gaithersburg, MD).
Electrophoretic mobility-shift assay (EMSA)
EMSA was performed as described before (Murata, T. et al., International
J Cancer 70:230 (1997); Murata, T. et al., Blood 91:3884 (1998); Oshima, Y. et
al., JBiol
Chem 275:14375 (2000)). After incubation with various concentrations of wtIL-
13 or IL-
13 mutants for 15 minutes, THP-1 cells, EBV immortalized B cells, or KSY-1
cells were
washed with cold PBS and solubilized with cold whole-cell extraction buffer (1
mM
MgClz, 20 mM HEPES, pH 7.0, 10 mM KCI, 300 mM NaCI, 0.5 mM dithiothreitol,
0.1%
NP-40, 1 mM PMSF, 1 mM Na3V04 and 20% glycerol). DNA-protein interactions were
assessed by EMSA using the Bandshift kit (Pharmacia, Piscataway, N~ using the
3zP-
labeled double stranded oligonucleotide probe ( 4.2 x 109 CPM/~g ) SBE-1.
CD14 regulation by IL-13
Primary monocytes were cultured at 1X10' cells /ml for 48 h with 1 p,g/ml
wtIL-13 with or without 1 ~glml lL-13E13K. Staining of the cells was performed
as
described elsewhere (Oshima, Y. et al., JBiological Chemistry 275:14375
(2000)). The
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fluorescence data were collected on a FACScan/C32 (Becton Dickinson, San Jose,
CA).
The results were analyzed with the CELLQuest (Becton Dickinson) program.
Sequence alignment and Molecular Modeling of IL-l3Ra' CRH domain and IL-13
The sequences of CRH domains were aligned by the Bestfit program of
GCG software (Genetics Computer Group, Inc., Madison, WI). Percent similarity
and
identity of extracellular domains between IL-l3Ra' and IL-2Rg chains were
40.5% and
31.9 %, respectively. These numbers indicate reasonable sequence similarity
justifying
the use of IL-2Ry chain as a template for modeling IL-l3Ra' chain. Conserved
sequence
patterns such as "WSXWS" motif and disulfide bonds between (3-strands of IL-
l3Ra' and
IL-2Ry were perfectly aligned. The alignment of sequences between hIL-4 and
hIL-13
was also performed as previously reported (Minty, A. et al., Nature 362:248
(1993);
Bamborough, P. et al., Protein Engineering 7:1077 (1994)). The similarity and
identity
of a-helix A and D of IL-13 to known structure of hIL-4 was in a similar range
as
1 S observed for IL-2Ry and IL-l3Ra' chains. However, the similarity of a-
helix B and C
could not be reasonably aligned (Bamborough, P. et al., Protein Engineering
7:1077
( 1994)).
The coordinate of the CRH domain of the IL-4Ra chain was also used in
our model and obtained from protein data bank entry IILL. The model building
and
refinement procedures in general followed the procedure previously described
in detail
(Greer, J. Proteins 7:317 (1990)). An initial model was built using the
Homology module
of InsightII (Molecular Simulations Inc., San Diego, CA). Small loops and
splices were
created and handled such that the energy was kept at minimum for best model.
The
structures were finally refined using the Discover program (Molecular
Simulations Inc.,
San Diego, CA).
Example 19: IL-13E13K is an Antagonist of IL-13 Activity
Recombinant protein isolation and purification
Recombinant wtIL-13, IL-13E13K and IL-13R112D in which 1 12th
arginine (R) residue of IL-13 molecule was substituted for aspartic acid (D)
were
expressed in Escherichia coli and purified from inclusion bodies as previously
described
(Oshima, Y. et al., JBiol Chem 275:14375 (2000)). After purification, each
recombinant
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protein was analyzed using SDS-PAGE and stained with Coomassie blue. Each
protein
showed a prominent single band at approximately 13 kDa with purity of at least
95%.
IL-13E13K competes for the binding of radiolabeled IL-13
Binding studies were performed on U251 glioblastoma and PM-RCC renal
cell carcinoma cell lines, both of which express type I IL-13 receptors
(Obiri, N. I. et al.,
Jlmmunol 158:756 (1997)). As expected, wtIL-13 displaced specific binding of
radiolabeled IL-13. Interestingly, IL-13E13K also inhibited binding of l2sl-IL-
13. IL-
13E13K was better in displacing l2sl-IL-13 binding that wtIL-13. The ECso
(concentration causing 50% inhibition of lzsl-IL-13 binding) of wtIL-13 and IL-
13E13K
on U251 cells was approximately 20 nM and 2.5 nM, respectively. On PM-RCC, it
was
approximately 100 nM and 25 nM, respectively. Thus, IL-13E13K appeared to show
approximately 4.0 to 8 fold better binding avidity than wtIL-13 in displacing
~25I-IL-13
binding.
IL-13E13K blocks proliferative activity of IL-13
TF-1 erythroleukemia cells proliferate in response to IL-13 (Oshima, Y. et
al., JBiol Chem 275:14375 (2000)). Proliferative activity of wtIL-13 and IL-
13E13K
was measured either alone or in combination of both. As expected, wtIL-13
stimulated
the growth of TF-1 cells in a concentration-dependent manner (Oshima, Y. et
al., JofBiol
Chem 275:14375 (2000)). In contrast, IL-13E13K did not show any proliferative
activity.
This result indicated that inserting a mutation at position 13 completely
suppressed its
agonistic activity and that the amino acid residue at position 13 seemed
essential for the
IL-13-induced proliferation of TF-1 cells. To determine the effect of IL-
13E13K on
wtIL-13 induced proliferation of TF-1 cells, we cultured cells in the presence
of 1 pg/ml
IL-13E13K and various concentrations of wtIL-13. Interestingly, IL-13E13K
blocked the
mitogenic activity of wtIL-13. This block of IL-13 mitogenic activity was
concentration-
dependent. A 100 - 333 fold excess of IL-13E13K completely neutralized wtIL-13
induced mitogenic activity.
IL-13E13K can neutralize the downregulation of CD14 expression by wtlL-13 on
human
primary monocytes:
IL-13 has been shown to downregulate CD14 expression on monocytes
(Cosentino, G. et al., Jlmmunol 155:3145 (1995); Oshima, Y. et al., JBiol Chem
87
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275:14375 (2000)). Therefore, we investigated whether IL-13E13K can nullify
the
downregulating activity induced by wtIL-13. wtIL-13 suppressed CD14 expression
on
monocytes and IL-13E13K completely neutralized the effect of wtIL-13. For
example,
IL-13 decreased the mean channel number (MCN or mean fluorescence intensity,
MFI) in
the gated region from 591 to 492 (p < 0.01). IL-13E13K reversed this effect
and MCN
recovered to 600.
IL-13E13K blocks signal transduction induced by wtlL-13
IL-13 has been shown to transduce signal through the Janus kinase (Jak)
and signal transducer and activator of transcription (STAT) pathways (e.g.,
Murata, T. et
al., Jlmmunology 156:2972 (1996); Murata, T. et al., Intl JCancer 70:230
(1997);
Murata, T. et al., Intl Immunol 10:1103 (1998); Obiri, N. I. et al., JBiol
Chem 272:20251
(1997)). STAT6 is phosphorylated and activated after IL-13 stimulation, which
in turn
regulates gene transcription. We, therefore, tested whether IL-13E13K can
block the IL-
13 induced signaling. THP-1 and EBV immortalized B cells express Type II and
Type III
IL-13 receptors, respectively, while KSY-1 AIDS-associated Kaposi's sarcoma
cells
express Type I IL-13 receptors. In all cell types, both wtIL-13 and IL-13RI12D
induced
STAT6 activation in a concentration dependent manner. IL-13R112D, a potent IL-
13
agonist, also induced STAT6 activation and it was 5-10 fold better than wtlL-
13 (Oshima,
Y. et al., JBiol Chem 275:14375 (2000)). In sharp contrast, IL-13E13K did not
stimulate
STAT6 activation even at very high concentrations (50 ng/ml). In addition, IL-
13E13K
blocked wtIL-13 induced STAT6 activation in the THP-1 cell line. 10 ng/ml wtIL-
13
stimulated maximal activation of STAT6, however in the presence of 10-fold
excess IL-
13E13K, STAT6 activation was significantly suppressed and 50-fold excess of IL-
13E13K almost completely blocked STAT6 activation.
Homology modeling oflL-13, IL-l3Ra'and IL-4Ra
We created a model of interaction between the cytokine receptor
homology (CRH) domains of IL-l3Ra', IL-4Ra chains and IL-13 based on homology
of
CRH domains between IL-2Ry and IL-l3Ra' chains and between IL-4 and IL-13.
This
model was created based on our hypothesis that IL-13 interacts with IL-l3Ra'
and IL-
4Ra chains simultaneously in the type II IL-13R complex. This model suggests
that the
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receptor binding interface of the IL-13 molecule is located in a-helix A and
D, and that
a-helix D and A may interact with IL-13a' and IL-4Ra chains, respectively.
Example 20: Materials and Methods Used in Example 21
Cells
L1236, L591 and L428 human Hodgkin's disease (HD)-derived cell lines
have been established and well characterized (Diehl et al., Cancer Surv.,
4:399-419
(1985); Schaadt et al., Intl J Cancer; 26:723-31 (1980); Wolf et al., Blood;
87:3418-28
(1996)). TF-1 human erythroleukemia cells, A172 cells, and U251 human
glioblastoma
cells were obtained from the American Type Culture Collection (Manassas, VA).
THP-1
human monocytic cells were kindly provided by Dr. Ray Donnelly (CBER, FDA,
Bethesda, MD).
Antagonist, IL -13-toxin and IL-13 binding protein
IL-13 antagonist, IL-13E13K was produced as previously reported. IL-13-
PE3SQQR was expressed and purified as previously reported (Puri et al., Blood,
87:4333-
4339 (1996); Debinski et al., Clin Cancer Research; 1:1253-1258 (1995);
Debinski et al.,
JBiol Chem, 270:16775-16780 (1995)). Recombinant human IL-13Ra2/Fc chimera,
termed IL-13 binding protein (IL-13-BP) was purchased from R&D systems
(Minneapolis, MN).
Rt-PCR
Rt-PCR analysis was performed essentially as previously reported (Murata
et al., Biochem Biophys Res Commun; 238:90-94 (1997)). Total RNA was isolated
with
Sv-Total RNA Isolation System (Promega, Madison, WI). Rt-PCR was performed
using
Access RT-PCR System (Promega) according to manufacture's instruction. The
primers
used were: sense 5'-tcaacatcacccagaaccag-3' and antisense 5'-
taagagcaggtcctttacaaac-3'
for IL-13, sense 5'-ccatcattaccattcacatccc-3' and 5'-tctgttgttccagttcagttc-3'
for IL-2Ry,
sense 5'-tggctttcgtttgcttgg -3' and antisense 5'-gcgtgtgtatcttcgcttc-3' for IL-
13Ra2, sense
5'-accaatgagagtgagaagcc-3' and antisense S'-tttcctgcattatccttgacc-3' for
IL13Ra1, sense
5'-gctcttgccctgttttctg -3' and antisense 5'-tccctttttcttcctctacctc-3' for IL-
4Ra and sense
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S'-gtgggccgctctaggcacca-3' and antisense 5'-cggttggccttagggttcaggggg-3' for
actin.
Determination of secreted IL-13 by HD cell line
L1236 and L428 cells were washed and 1.0 X 106 cells/ml were cultured
for 72 hr. Culture supernatant was collected after centrifugation at 1,500 RPM
for 5
minutes and stored at 70°C until assays. Dot blot analysis was
performed using S&S
Minifold~I (Schleicher & Schuell, Keene, NH) according to manufacturer's
instructions.
ELISA for IL-13 was performed using CYTELISA Human IL-13 kit (Cytimmune
Sciences Inc., College Park, MD). Briefly, 1x106/ml cells were cultured for 3
days and
the supernatant was harvested. 5 ml of supernatant was loaded on nytran~ plus
membrane (Schleicher & Schuell, Keene, NH) per well. The proteins were
immobilized
by incubating in 5% skim milk and then IL-13 was detected by incubating with
500 times
diluted goat anti-human IL-13 polyclonal IgG (Santa Cruz Biotechnology, Inc.,
Santa
Cruz, CA) as a primary antibody and then incubated with 1000 x diluted anti-
goat Ig-
HRP conjugated (Santa Cruz Biotechnology) as a secondary antibody. The HRP
activity
was visualized by ECLTM (Amersham Life Science, Piscataway, NJ).
Cell proliferation and cytotoxicity assays
Cell proliferation (Leland et al., Oncology Res; 7:227-235 (1995)) and
cytotoxicity assays
(Puri et al., Cancer Res, 51:3011-3017 (1991)) were done essentially following
the same
techniques as previously reported. Studies of cell growth in the absence and
in the
presence of IL-13 (Figure 19, panel C) were performed as follows. 1x105/ml
L1236,
L591 or L428 cells were incubated with or without various concentrations of IL-
13 for 54
hr in 37°C humidified 5% C02 incubator. After lp,Ci per well 3H-
thymidine pulse per
well, cells were incubated for an additional 12 hrs and then harvested by
Skatron Cell
Harvester (Skatron, Inc., Sterling, VA). Glass fiber filter mats were counted
in a beta
plate counter (Wallac, Gaithersburg, MD). Data in Figure 19, panel C is shown
as mean
+ standard deviation of percent CPM. Standard deviation is shown when bigger
than
symbol.
Studies of the cytotoxicity of an IL-13-targeted immunotoxin (Figure 19,
panel D) were performed as follows. L1236 or L428 cells (1x104/ml)were
incubated with
or without various concentrations of IL-13-PE38QQR for 22 hr at 37°C in
humidified 5%
C02 incubator. Cells were pulsed with 1 ~Ci per well 3H-Leucine and incubated
for an
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additional 4 hr and then harvested and counted. Data is shown as mean +
standard
deviation of percent CPM. Standard deviation is shown when bigger than symbol.
Example 21: IL-13 Antagonists Suppress Proliferation of Hodgkin's Disease
Cells
Suppression of HD cell proliferation by IL-13 antagonist
The IL-13 antagonist, IL-1 3E13K discussed in Examples 18 and 19 was
examined to determine whether this antagonist can suppress Hodgkin's
disease/Reed-
Sternberg ("H/RS" or "HD/RS") cell proliferation. The IL-13 antagonist
significantly
inhibited the proliferation of L1236 cells as determined by viable cell number
counting or
3H-thymidine uptake. In contrast, the growth of L428 cells was not affected by
the
antagonist, ruling out nonspecific toxicity of the antagonist.
Because equilibrated or secreted IL-13 in the culture media was washed
out at the beginning of the assay, the effects of the antagonist were not so
robust.
Therefore, in order to show better dose response, HD/RS cells were cultured in
the
presence of wild-type IL-13 (0.1 ng/ml). This concentration was similar to the
EDso
(concentration for half maximum proliferation) of IL-13 on L1236 cells.
Interestingly,
this concentration was also similar to the IL-13 concentration in the culture
media of
L1236 cells determined by ELISA. IL-13 induced the proliferation of L1236
cells, which
was also suppressed by IL-13 antagonist in a dose-dependent manner. The dose
that
induced half maximal inhibition of cell growth (ICSO) by the antagonist was
approximately 20 ng/ml. For a positive control, we utilized an IL-13 binding
protein
("BP") that suppresses IL-13 proliferative activity by absorbing IL-13. The
ICso of IL-
13-BP (Zhang et al., JBiol Chem; 272:9474-9480 (1997)) on L1236 cells was also
approximately 20 ng/ml. L428 cells were not affected by the antagonist or IL-
13-BP in
either assay.
Expression of IL-13 mRNA and functional receptor for IL-13
To determine the mechanisms) of different activity of IL-13 and IL-
13E13K on various HD/RS cell lines, we examined the expression of mRNA for IL-
13R
chains. All 3 HD/RS cell lines expressed mRNA for IL-13 and IL-13Ra1, IL-4Ra
and
IL-2Ry chains. For a control, brain tumor cell lines U251 and A172 were
examined.
These cell lines were noted not to express IL-13 nor the IL-2Ry chain, but to
express IL-
4Ra, IL-13Ra1 and IL-13Ra2 chains. These data show that brain tumor cell lines
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express type I IL-13R. Hematopoietic cell lines TF-1 and THP-1 did not express
IL-13
nor the IL-13Ra2 chain, but did express IL-4Ra, IL-13Ra1 and IL-2Ry chains.
Thus,
these cell lines expressed type III IL-13R. These results also confirm our
previous studies
(Murata et al., Biochem Biophys Res Commun, 238:90-94 (1997)). Thus, all HD/RS
cell
lines tested expressed type III IL-13R, although a faint band for IL-13Ra2
chain was seen
in L428 cells. All three HD/RS cell lines also expressed mRNA for IL-13 and
all tested
cell lines expressed protein for IL-13 as determined by dot blot analysis. The
concentration of IL-13 determined by ELISA was 20 pg/ml and 187 pg/ml in L1236
and
L428 cells, respectively.
The functional status of IL-13R was next investigated by two ways:
1) stimulation of proliferation by wild-type IL-13 and 2) internalization of
IL-13 ligand-
receptor complex. In the proliferation assay, L1236 cells responded to
stimulation by IL-
13, however, L428 and L591 cells did not. The EDS° of IL-13 in L1236
cells was
approximately 0.1 ng/ml. Interestingly, the B9 marine B-cell line has a
similar EDS° of
IL-13. For internalization assay, a PE based recombinant toxin termed IL-13-
PE3SQQR
comprised of IL-13 and a mutated form of Pseudomonas exotoxin was utilized.
This
cytotoxin generally shows cytotoxicity via internalization of ligand-receptor
complex
(Puri et al., Blood, 87:4333-4339 (1996); Pastan et al., Annual Rev Biochem,
61:331-354
(1992)), therefore the cytotoxicity can be regarded as internalization.
Interestingly, IL-
13-PE38QQR was cytotoxic to L1236 cells but had no effect on L428 cells.
Example 22: Use of IL-13 A~onists in the Maturation of Dendritic Cells
Typically, peripheral blood derived monocytes are collected from normal
donor or cancer patients. Monocytes are generally purified by standard
elutriation density
gradient technique or by plastic adherence for 2 hour at 37°C.
Elutriated or adherent cells
are washed two times with HBSS (Life Technologies, Inc. Gaithersburg, MD) and
resuspended in XVIVO-15 medium (BioWhittaker, Gaithersburg, MD) at a
concentration
of 1 x106 cell/ml. Three to five ml of this cell suspension is plated in each
well of 6-well
plate (Costar Corp., Cambridge, MA). The medium contains 100 ng/ml granulocyte-
macrophage colony stimulating factor (GM-CSF) and 50 ng/ml of an IL-13R112D.
After
7 days of culture at 37°C in S% C02, the culture medium is exchanged
with fresh medium
containing GM-CSF/IL-13R112D with TNF-alpha (10 ng/ml) and the cells are
cultured
for an additional 7 days. After 14-day of culturing, adherent cells (DCs) are
loosened
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gently using sterile cell scrapers. The cells are then transferred to sterile
50-ml centrifuge
tubes and washed once and counted before phenotyping. These cells are now
differentiated into dendritic cells, which acquire unique surface phenotypes.
For example
DCs begin to express CD 11 c, CD80, CD83 and high levels of HLA-DR but not CD
14.
The DCs are then pulsed as desired with an antigen, typically a tumor cell
lysate, peptide
antigen, apoptotic body fused to whole tumor cells, or a gene modified to
express
antigens and co-stimulatory molecules. Cells (one million to 10 million) are
then washed
with lactate ringer and injected to patients at multiple time points and
schedules to boost
their immune response to the antigen. The DCs migrate to lymph nodes and
educate
cytotoxic T cells in the context of MHC class I molecules. The T cells then
circulate and
seek and kill cells that express the antigen that was used to pulse DCs.
As little as 10 ng/ml IL-13, when combined with 10 or 100 ng/ml GM-
CSF in the above protocol, can generate dendritic cells. Since IL-13R112D is
up to 10
times more potent than wild type IL-13 in promoting the maturation and
activation of
potent DCs, as little as 0.1 ng/ml of IL-13R112D can be used in the above-
described
protocol to activate DCs. Thus, the agonists of the invention obviate the need
for a high
concentration of activator in the in vitro maturation of dendritic cells.
Examele 23: Use of Antagonists of the Invention in Asthma, Allergic IRhinitis,
and
Atopic Dermatitis
Recent studies have demonstrated that IL-13 is a necessary and sufficient
factor for the expression of allergic asthma. IL-13 also plays a key role in
allergic rhinitis
and atopic dermatitis. IL-13 induces pathophysiological features of asthma in
animals in
a manner that is independent of IgE, IL-4, and eosinophils (Wills-Karp et al.,
Science
282, 2258, 1998). Further studies in patients with asthma have demonstrated
that IL-13 is
locally produced in bronchoalveolar lavage (BAL) cells when these cells are
challenged
with allergen (Huang, et al. J. Immunol. 155:2688, (1995)). Up to 3 ng/ml of
IL-13
protein/ml BAL was detected in allergen challenged patients. Therefore,
neutralization of
locally produced IL-13 will mitigate symptoms of this life-threatening
disease.
The antagonists of IL-13 of the invention neutralize the effect of IL-13 and
thus are useful in the treatment of asthma. To demonstrate this in an animal
model of
asthma, the following protocol may be utilized. Mice are typically sensitized
to soluble
antigens from the fungus Aspergillus fumigatus and subsequently challenged
with an
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intranasal bolus of spores from this fungus. The spore challenge in sensitized
mice then
sets in motion a chronic allergic lung disease characterized by airway
hyperreactivity,
mucus hypersecretion, smooth muscle hypertrophy and peribronchial fibrosis,
all of
which are features of clinical asthma (Kunkel et al., American J. Pathology
156:723-732
(2000)). The mice are first challenged intraperitoneally and subcutaneously
with
aspergillus antigen and then with fungus antigen given intratracheally. IL-13
antagonists
(1 to SO ng/ml diluted in 0.1% human serum albumin) are administered
intratracheally
several days after the aspergillus antigen challenge. IL-13 antagonists (1 to
50
microgram/kg, three times per week for two weeks) may also be given
intraperitoneally
or intravenously, for a total of 100 microliters of drug.
For the prevention of asthma in humans, IL-13 antagonists (1 to 50
micrograms/Kg) are administered three times per week for two weeks by
injection or
intranasally. Conveniently, the administration may be by inhaler or nebulizer.
Example 24~ Prevention of Hepatic Fibrosis Induced by Scbistosomiasis
Infection
In Schistosomiasis, chronic parasite-induced granuloma formation can lead
to tissue destruction and fibrosis, which causes much of the morbidity and
mortality in
humans. IL-13 has been shown to play a central role in the pathogenesis of
schistosomiasis and appears to be a profibrotic agent (Chiarmonte et al., J.
Clin. Invest.
104:777 (1999); Fallon et al., J. Immunol. 164:2585 (2000)). Moreover,
fibrosis is a
major pathological manifestation of a number of allergic, autoimmune and
infectious
diseases..
To demonstrate the activity of IL-13 antagonists in animal models, mice
are infected with Schistosoma mansoni by percutaneous injection. Typically,
these mice
are acutely infected with this pathogen for 8 weeks (Smithers et al.,
Parasitology 55:695
(1965)). IL-13 antagonists are administered intraperitoneally or intravenously
at a dose
of 1 to 50 microgram/kg every alternate day for two weeks, and degree of
fibrosis or
resolution of fibrosis induced by infection is studied.
In clinical use, IL-13 antagonists can be administered i.p. or i.v. for the
treatment of infection induced fibrosis. The starting dose of IL-13 antagonist
is 1
microgram/kg, and can be escalated to 50 micrograms/kg. The doses will be
given every
alternate day for two weeks. In more intractable cases, a continuous infusion
can be used,
with a higher dose (100 microgram/Kg) of an IL-13 antagonist. For severe
hepatic
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fibrosis, portal infusion of an IL-13 antagonist (doses from 1 to 200
microgram/kg) may
also be explored.
Example 25: Use of IL-13 Antagonists in the Slowing the Proliferation of
Cancer
Cells
IL-13 has been shown to be an autocrine growth factor for Hodgkin's'
disease (HD) derived Reed Sternberg (RS) cells and renal cell carcinoma cell
lines in
vitro. IL-13 is also an autocrine growth factor for AIDS-associated Kaposi's
sarcoma
cells. It is expected that other cancer cell types also produce IL-13 and that
IL-13 serves
as an autocrine growth factor for these cancer cells as well. IL-13
antagonists will have a
significant role in the management of cancers in which IL-13 acts as an
autocrine growth
factor.
The use of IL-13 antagonists in slowing or stopping the growth of cancer
cells for which IL-13 acts as an autocrine growth factor can be demonstrated
in animal
models. In a typical protocol, up to 5 million HD/RS cells in 100-microliters
of
phosphate buffered saline are injected subcutaneously into nude Balb/C mice
(male and
female). After 4-6 days, when the mice have developed palpable tumors, they
are
injected with the IL-13 antagonist under study. Separate cohorts of mice are
injected,
with each cohort receiving a different dose of antagonist, with the doses
ranging from 1 to
100 microgram/kg). The administrations can be intraperitoneal, intravenous, or
intratumoral, and are given every alternate day for one to two weeks. Tumor
response is
determined by the measurement of tumor volume using Vernier calipers. Survival
of the
animals is followed. Additional cohorts of mice have the antagonist
administered in a
continuous infixsion, at a higher dose (SO to 200 microgram/kg).
In the clinic, antagonists are administered in the same manner. If desired,
and if the cancer is not of a type in which all cells are known to secrete IL-
13, a test can
be performed to determine whether proliferation of cells of the cancer will be
slowed by
administration of an IL-13 antagonist. For this test, a needle biopsy of tumor
cells is
obtained and the cells are tested for the production of IL-13 by standard
ELISA tests, with
the production of IL-13 being indicative that the patient will benefit from
the
administration of an IL-13 antagonist. This test can be confirmed by culturing
the cells in
the presence of an IL-13 antagonist and comparing their growth to a like
culture of cells
cultured in the absence of the antagonist.
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Once it is confirmed that the tumor is an IL-13 secreting tumor or
otherwise confirmed that IL-13 is an autocrine growth factor for the cancer,
an IL-13
antagonist is administered. Typically, the starting dose of IL-13 antagonist
is based on
the amount of IL-13 produced by the cancer cells, with the goal being to
contact the cells
with a ten-fold or greater excess of inhibitor. Generally, one million cells
produce up to 1
n8 of IL-13 in three days. Assuming a tumor burden of 5x108 tumor cells as an
example,
S microgram/kg would be administered as a starting dose every alternate day
for two
weeks. The doses can be escalated by half logs to 100 microgram/kg. Severe
cases can
also be treated by continuous infusion, commencing at a higher dose (100
microgramlkg)
of antagonist.
Example 26~ Use of IL-13 Anta~~,onists in Leishmania maior Infection
Leishmaniasis is an important disease affecting millions of people
worldwide. Various mouse models are available that can be utilized for the
study of
immune response to disease. Using these model systems, it has been determined
that IL-
13 is a susceptibility factor for Leishmania major infection (Matthews et al.,
J. Immunol.
164:1458 (2000)). Blocking IL-13 can help shift the immune response to a Thl
type, that
effects a resolution of the disease. High affinity-IL-13 antagonists can
neutralize the
effect of IL-13 in Leishmaniasis by systemic administration at the time of
infection or
after an infection has established.
Animal models are used to demonstrate the effect of IL-13 antagonists on
this disease. IL-13 antagonists are administered to mice intraperitoneally or
intravenously
at the doses of 1 to 50 microgram/kg every alternate day for two weeks and the
infection
of the mice by Leishmania major induced infection studies.
In the clinic, patients are treated with an IL-13 antagonist in the clinic at
1
microgramlkg every alternate day for two weeks. If the response is not
satisfactory in the
judgment of the clinician, the dose of the antagonist is escalated by half
logs to ~50
microgram/kg. Again, the doses are given every alternate day for two weeks. In
severe
cases, a continuous infusion can be administered, at a higher dose (100
microgram/Kg) of
antagonist.
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It is understood that the examples and embodiments described herein are
for illustrative purposes only and that various modifications or changes in
light thereof
will be suggested to persons skilled in the art and are to be included within
the spirit and
purview of this application and scope of the appended claims. All
publications, patents,
and patent applications cited herein are hereby incorporated by reference for
all purposes.
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