U.S. patent application number 12/691200 was filed with the patent office on 2010-10-28 for peptides of il-2.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Pedro Alzari, Ralph Eckenberg, Michel Goldberg, Jean-Claude Mazie, Jean-Louis Moreau, Thierry Rose, Jacques Theze.
Application Number | 20100273723 12/691200 |
Document ID | / |
Family ID | 37804446 |
Filed Date | 2010-10-28 |
United States Patent
Application |
20100273723 |
Kind Code |
A1 |
Theze; Jacques ; et
al. |
October 28, 2010 |
PEPTIDES OF IL-2
Abstract
The present invention relates to new peptides of IL-2,
derivatives thereof, and their use as therapeutic agents.
Inventors: |
Theze; Jacques; (Paris,
FR) ; Eckenberg; Ralph; (Saint Germain en Laye,
FR) ; Moreau; Jean-Louis; (Paris, FR) ;
Goldberg; Michel; (Paris Cedex, FR) ; Rose;
Thierry; (Paris Cedex, FR) ; Alzari; Pedro;
(Paris, FR) ; Mazie; Jean-Claude; (Asnieres,
FR) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT PASTEUR
Paris Cedex
FR
|
Family ID: |
37804446 |
Appl. No.: |
12/691200 |
Filed: |
January 21, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11494583 |
Jul 28, 2006 |
7662368 |
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12691200 |
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10727514 |
Dec 5, 2003 |
7101965 |
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11494583 |
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09720828 |
Mar 1, 2001 |
6825334 |
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PCT/IB99/01424 |
Jul 16, 1999 |
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10727514 |
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Current U.S.
Class: |
514/21.3 |
Current CPC
Class: |
A61P 37/00 20180101;
C07K 2317/34 20130101; C07K 2317/73 20130101; Y10S 514/885
20130101; C07K 2317/76 20130101; C07K 16/246 20130101; C07K 2317/92
20130101 |
Class at
Publication: |
514/21.3 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 37/00 20060101 A61P037/00 |
Claims
1.-25. (canceled)
26. A method of inducing SHC phosphorylation or inducing the
SHC/MAPK pathway comprising administering to a subject in need
thereof an effective amount of a peptide consisting of SEQ ID NO: 2
or a peptide consisting of SEQ ID NO: 2 containing one conservative
mutation, wherein said peptide binds to the IL-2.beta. chain or the
monoclonal antibodies produced by H2-8 hybridoma.
27. The method of claim 26, wherein said peptide consists of SEQ ID
NO.: 2.
28. The method of claim 26, wherein said peptide is the peptide
consisting of SEQ ID NO: 2 containing one conservative
mutation.
29. The method of claim 28, wherein said conservative mutation is a
replacement of a non-polar R-group by another non-polar
R-group.
30. The method of claim 28, wherein said conservative mutation is a
replacement of a uncharged non-polar R-group by another uncharged
non-polar R-group.
31. The method of claim 28, wherein said conservative mutation is a
replacement of a charged polar R-group by another charged polar
R-group.
32. The method of claim 28, wherein Lys is substituted for Arg, or
vice versa so that a positive charge is maintained.
33. The method of claim 28, wherein Glu is substituted for Asp, or
vice versa so that a negative charge is maintained.
34. The method of claim 28, wherein Asp is substituted for Glu.
35. The method of claim 28, wherein one or more Ser is substituted
for Thr.
36. The method of claim 28, wherein one or more Gln is substituted
for Asn.
37. The method of claim 26, wherein said method is a method of
inducing SHC phosphorylation.
38. The method of claim 26, wherein said method is a method of
inducing the SHC/MAPK pathway.
Description
[0001] The present invention relates to new peptides of IL-2, and
derivatives thereof and their use as therapeutic agents.
[0002] Interleukin-2 (IL-2) is the main growth factor of T
lymphocytes (TH ZE et al. 1996, Immunol. Today 17:481-486). By
regulating T helper lymphocyte activity IL-2 increases the humoral
and cellular immune responses. By stimulating cytotoxic CD8 T cells
and NK cells this cytokine participates to the defense mechanisms
against tumors and viral infections. IL-2 is used in therapy
against metastatic melanoma and renal adenocarcinoma. IL-2 is used
in clinical trials in many forms of cancer (LOTZE and ROSENBERG
1988, Interleukin 2 as a Pharmaclologic Reagent. in Interleukin 2,
K. A. Smith, Academic Press: p. 237-89). It is also used in HIV
infected patients and leads to a significant increase in CD4 counts
(KOVACS et al. 1996, New Engl. J. of Medicine 1350-6).
[0003] Human IL-2 is a protein of 133 amino acids (aa) composed of
four .alpha. helices connected by loops of various length; its
tridimensional structure has been established. IL-2R is composed of
three chains .alpha., .beta. and .gamma.. IL-2R.alpha. controls the
affinity of the receptor. IL-2R.beta. and IL-2R.gamma. are
responsible for IL-2 signal transduction. The different molecular
areas of IL-2 interacting with the three chains of the IL-2R have
been defined. More specifically it has been determined that .alpha.
helix A as well as the NH.sub.2 terminal area of IL-2 (residues 1
to 30) control the interactions IL-2/IL-2R.beta. (ECKENBERG et al.
1997, Cytokine 9:488-98): IL-2R.beta. chain is the most important
in IL-2 signaling (TH ZE et al. 1990).
[0004] The effects of human interleukin-2 (IL-2) on its target
cells are mediated through specific cell surface receptors (IL-2R)
(TANIGUCHI et al. (1983) Nature 302:305-310; ROBB et al. (1984)
Proc. Natl. Acad. Sci. USA 81:6486-6490; SMITH K A. 1988a.
Interleukin-2; SMITH K A (1988b) Science 240:1169-1176). IL-2R
comprises at least three subunits encoded by different genes
(MINAMI et al. (1993) Annu. Rev. Immunol. 11:245-267; TANIGUCHI et
al. (1993) Cell 73:5-8). The first component to be identified,
IL-2R.alpha., is a 55 kDa protein that binds IL-2 with a Kd of
.apprxeq.10 nM (UCHIYAMA et al. (1981) J. Immunol. 126:1293-1297;
LEONARD et al. (1984) Nature 311:626-631). The role of IL-2R.alpha.
(KUMAR et al. (1987) J. Immunol. 139:3680-3684) and the influence
of IL-2 on IL-2R.alpha. gene expression have been studied (BISMUTH
et al. (1985) Eur. J. Immunol. 15:723-727; FROUSSARD et al. (1991)
Mol. Immunol. 28:87-93). The second IL-2R component, IL-2R.beta. is
a 75 kDa protein with a large intracytoplasmic domain (286 aa)
(TESHIGA WARA et al. (1987) J. Exp. Med. 165:223-238; HATAKEY AMA
et al. (1989) Science 244:551-556; TSUDO et al. (1989) Proc. Natl.
Acad. Sci. USA 86:1982-1986). The last component to be identified,
11--2R.gamma., is a 64 kDa protein (TAKESHITA et al. (1992) Science
257:379-382; ISHII et al. (1994) Int. Immunol. 6:1273-1277).
IL-2R.beta. and IL-2R.gamma. belong to the hematopoietin receptor
family whereas IL-2R.alpha. belongs to another family of molecules
(TH ZE J (1994) Eur. Cytokine Netw. 5:353-368). In the mouse system
all three chains are required to form a functional receptor (MOREAU
et al. (1995a) J. Immunol. 155:3401-3 4 08; CHASTAGNER et al.
(1996) Eur. J. Immunol. 26:201-206). In the human system two
receptors are functional. When associated, human IL-2R.beta. plus
IL-2R.gamma. form an intermediate affinity receptor with a Kd of
.apprxeq.1 nM, whereas expression of all three chains leads to the
formation of a high affinity IL-2R (Kd.apprxeq.10 .mu.M).
[0005] The structure of IL-2 (MACKAY D (1992) Science 257:410-413)
is composed of a compact core bundle of four antiparallel a helices
connected by three loops (FIG. 1). Some of the interactions between
IL-2 and IL-2R.alpha. (SAUV et al. (1991) Proc. Natl. Acad. Sci.
USA 88:4636-4640; WANG et al. (1995) Eur. J. Immunol. 25:1212-1216)
and IL-2R.gamma. subunits (Voss et al. (1993) Proc. Natl. Acad.
Sci. USA 90:2428-2432; Buchli et al. (1993) Arch. Biochrn. Biophys.
307:411-415) have been elucidated, but less is known concerning
IL-2/IL-2R.beta. interaction, despite the fact that IL-2R.beta.
chain plays a very critical role in signal transduction (TANIGUCHI
T (1995) Science 268:251-255).
[0006] It has been shown that one substitution Asp20 by Lys (mutant
D20K) prevents binding to IL-2R.beta. (COLLINS et al. (1988) Proc.
Natl. Acad. Sci. USA 85:7709-7713). In a recent report the role of
the sequence (Leu17, Leu18, Leu19, Asp20, Leu21) from IL-2 .alpha.
helix A, in IL-2/IL-2R.beta. interactions was analyzed by cassette
mutagenesis (BERNDT et al. (1994) Biochemistry 33:6571-6577).
However the data were difficult to interpret since most of the
proteins produced have multiple mutations inside and outside of the
sequence of interest. Only one analog with a single mutation was
studied (121V). More surprisingly it was reported in this study
that deletion of the segment spanning residues 17-31 (Dell) gives a
protein with full agonist activity.
[0007] IL-2 peptides and derivatives were described in Cytokine
(1997) 7:488-498, but were not tested in an in vitro system for
biochemical activity such as cytokine activity, and in particular
for IL-2-like activity.
[0008] In view of the aforementioned deficiencies attendant with
the prior art analysis of IL-2 agonists and antagonists, as well as
with methods of modulating IL-2 activity therewith, it is clear
that there exists a need in the art for the same.
[0009] Accordingly, one object of this invention is to provide
compositions having an IL-2-like activity and methods for their use
as therapeutic agents. The applications of such recombinant,
synthetic or hybrid peptides are thus one object of the invention.
These compositions are defined as having the following
characteristics: a) containing one or more peptides at least five
amino acids in length; and b) inhibiting or mimicking the binding
of helix A of interleukin-2 (IL-2) to a subunit of an IL-2 receptor
(IL-2R).
[0010] Another object of the invention is the use of a purified
peptide having the following characteristics: a) the peptide is at
least five amino acids in length; b) the peptide binds to a subunit
of an IL-2 receptor (IL-2R); and c) the peptide induces
phosphorylation of the subunit of the IL-2R.
[0011] A further object of the invention concerns the preparation
of the antibodies which recognize the peptides of the invention,
and the therapeutic use of these antibodies.
[0012] A further object of the invention is the use of DNA
sequences encoding the peptides of the invention and their
derivatives. Such DNA fragments are useful for gene therapy among
other applications. The use of a DNA of the following sequence ATG
GCT CCG ACG AGC AGC TCC ACC AAG AAA ACC CAG CTC CAG CTC GAA CAC CTG
CTG CTG GAC CTG CAG ATG ATC CTG AAC GGT ATC AAC AAC (SEQ ID NO.: 1)
or said SEQ ID NO.: 1 without the first codon ATG (SEQ ID NO.: 3)
is one particular object of the invention.
[0013] Yet another object of the invention is to provide a method
for detecting the activity of an IL-2-like peptide, wherein the
IL-2 activity is measured by the binding of the IL-2R to the
peptide having the IL-2 agonist or antagonist activity. A still
further object of the invention is the use of compounds which
inhibit the activity of an IL-2R by contacting the IL-2R with an
amount of the selected antagonist peptide sufficient to inhibit
binding of IL-2 to the IL-2R under conditions that allow binding of
the peptide to the IL-2R to occur.
[0014] Another object of the invention is to provide a method for
the selection of antibodies specific for the purified peptide with
IL-2-like activity as defined herein. These monoclonal or
polyclonal antibodies can inhibit binding of IL-2 to the IL-2R
under conditions that allow binding of the peptide to the IL-2R to
occur. The therapeutic use of these antibodies is also a part of
the present invention. In particular, these antibodies specific for
the purified peptides are useful for treating or preventing
undesirable immune reactions such as graft rejection or autoimmune
disorders, for example, rheumatoid arthritis.
[0015] A still further object of the invention is to provide a
method for inducing in a patient the biological effects of IL-2 by
administering to the patient an amount of the agonist peptide of
the invention sufficient to induce those biological effects, or by
administering a combination of various cytokines and purified
peptide. By various cytokines is meant for example IL-4, IL-9,
IL-15 or IL-2.
[0016] Another object of the invention relates to the nucleic acid
sequences corresponding to the amino acid sequence of the purified
peptide and its derivatives according to the invention. A preferred
embodiment is the nucleotide sequence encoding the purified peptide
IP130 having the following sequence (SEQ ID NO.: 2): Met Ala Pro
Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gln Leu Glu His Leu Leu Leu
Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn or a sequence which
does not comprise the first Met (SEQ ID NO.: 4).
[0017] This sequence or a sequence derived therefrom can be
inserted in an appropriate vector capable of expressing the product
in vivo, in a bacterium or in a eukaryotic cell, particularly in
yeast or a mammalian Cell. These constructs are useful for gene
therapy among other uses.
[0018] With the foregoing and other objects, advantages and
features of the invention that will become hereinafter apparent,
the nature of the invention may be more clearly understood by
reference to the following detailed description of the preferred
embodiments of the invention and to the appended claims.
[0019] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0020] FIG. 1 shows a schematic representation of the human IL-2
structure. The protein contains 133 amino-acid residues (molecular
weight: 15-18 kDa, depending on the degree of glycosylation). Four
.alpha.-helices, denoted A (residue positions 6-29), B--B'
(positions 53-72), C (positions 81-97), and D (positions 113-133)
surround a central hydrophobic core. Residues Leu 17 and Asp20 (see
text) occur in the N-terminal helix A. The structure of the loop
between a helix C and a helix D was undetermined. Atomic
coordinates were obtained from the Brookhaven Protein Data Bank
(BERNSTEIN et al. (1977) J. Mol. Biol. 112:535-542) entry link,
deposited by D. B. MCKAY (MACKAY D (1992) Science 257:410-413). The
figure was drawn with the program Molscript (KRAULIS (1991) J.
Appl. Crystallogr. 24:946-950).
[0021] FIG. 2 shows the binding and binding inhibition of mAb H2-8.
Binding experiments: plates were coated with IL-2, peptide 1-22 or
peptide 1-30. Control is represented by non coated plates. Binding
of mAb was revealed with alkaline phosphatase goat anti-mouse
polyvalent Ig conjugate. Inhibition experiments: concentrations of
mAbs H2-8 or 19B11 giving 1/2 maximal binding on IL-2 coated plates
were used. These dilutions were mixed for 1 hr at 37.degree. C.
with the indicated concentration of inhibitors before addition to
wells coated by IL-2.
[0022] FIG. 3 shows the binding of mAb H2-8 on peptide 1-30. Plates
were coated with mAb H2-8 and were incubated with peptide 1-30 as
described.
[0023] FIG. 4 shows the biological effects of mAb H2-8 on IL-2
Pro.sup.125. Different concentrations of IL-2 Pro.sup.125 were
tested on the proliferation of TS1.beta. cells (IL-2R.alpha.-,
human IL-2R.beta.+, mouse IL-2R.gamma.+). The proliferation was
measured by [.sup.3HTdR] incorporation as indicated.
[0024] Various concentrations (indicated in parenthesis-.mu.g/ml)
of mAb H2-8 were tested. As control the absence of effects of mAb
H2-8 on IL-2 dependent proliferation of TS1.beta. cells was
verified.
[0025] FIG. 5 shows the model of IL-2/IL-2R interactions. FIG. 8(A)
Position of residues Leu17 and Asp20 in the IL-2 structure with
respect to helices A, C and D, in a view perpendicular to the axis
of helix A. FIG. 8(B) Position of residue Leu17 with respect to
helices A and B--B'. The side chain of Leu17 is located in a
leucine-rich hydrophobic core of the molecule. The charged side
chain of Asp20 is partly exposed to solvent. The two orientations
of the molecule shown here are roughly perpendicular to that shown
in FIG. 1.
[0026] FIG. 8(C) The model of the IL-2/IL-2R complex (BAMBOROUGH et
al. (1994) Structure 2:839-851) is based on the structure of the
human growth hormone and its receptor (DE VOS et al. (1992) Science
255:306-312). For clarity, only .alpha.-helices A and D of IL-2 and
the .beta. and .gamma. chains of the receptor are shown. The IL-2
positions studied, Leu17, Asp20 and residues Arg15 and Tyr134 of
IL-2R.beta. are labelled. Positions Cys125 and Ser127 are also
shown. Secondary structural elements as defined by the program DSSP
(KABSCH et al. (1983) Biopolymers 22:2577-2637). Atomic coordinates
of the complex were obtained from the Brookhaven Protein Data Bank,
entry code film.
[0027] FIG. 6 is a schematic representation of IL-2/IL-2R
interactions and the IP130 sequence (SEQ ID NO.: 4). IL-2 receptor
is composed of three subunits (.alpha.,.beta., .gamma.). (See Imm.
Today, 1996).
[0028] FIG. 7 demonstrates that IP130 induces proliferation and
acts in synergy with IL-2. Proliferative activity was tested on
TS1.beta.2 (grown in IL-2). Background of 1.4.times.10.sup.3 cpm
was subtracted. Synergy with IL-2 is also observed. TS1.beta.
target cells are derived from TS1 murine cells (which only express
murine IL-2R.gamma.), after transfection with the human IL-2R.beta.
gene.
[0029] FIG. 8 shows that the proliferation induced by IP130 is not
due to synergy with residual growth factor coming from the culture
medium. TS1.beta. cells, used in this study, are cultured in IL-4.
TS1.beta. proliferates with IP130 and this proliferation is not
inhibited by 11B11 mAB, which neutralizes proliferation induced by
IL-4. FIG. 8A shows IP130 proliferative activity. FIG. 8B shows
IL-4 proliferative activity. FIG. 8C shows IP130+mAB 11B11
(anti-IL-4). FIG. 8D shows IL-4+mAB 11B11 (.quadrature.) and
IL-4+mAB 145 (control mAB) (). Proliferative activity was tested on
TS1.beta. (grown in IL-4).
[0030] FIG. 9 demonstrates that human IL-2R.beta. is essential for
the proliferation induced by IP130. FIG. 9A shows IP130 activity on
TS1 cells transfected with human IL-2R.beta.. FIG. 9B shows the
effect of anti-human IL-2R.beta. neutralizing antibody (A41) on
IP130 activity. TS1 cells only proliferate after transfection by
the human IL-2R.beta. gene. As for IL-2, murine IL-2R.beta. chain
does not allow proliferation in the presence of IP130. TS1.beta.
proliferation induced by IP130 is specifically neutralized by the
mAB A41 (anti human IL-2R.beta.).
[0031] FIG. 10 is a summary of IP130 (SEQ ID NO.: 4) and of
derivative molecule's structure-function studies. A family of
peptides was studied for helicity, oligomerization and biologic
activity (see also general presentation of the data).
[0032] FIG. 11 is a model of IP130/IL-2R.beta. interactions. IP130
is tetrameric and IL-2R.beta. forms a dimer in solution. Proposed
from the results obtained with the three dimensional structure of
IL-2 and growth hormone/receptor complex.
[0033] FIG. 12 shows the pattern of phosphorylation induced by
IP130 and involvement of SHC. IP130 induces SHC protein
phosphorylation. The two bands corresponding to SHC isoforms are
phosphorylated after a ten minute stimulation by IP130. Kinetics of
Shc phosphorylation is shown on the left. A Western blot of Shc
protein is shown on the right.
[0034] FIG. 13 is an electrophoretic mobility shift assay which
shows that IP130 does not induce STAT activity. STAT activation is
analyzed in KIT225 cells nuclear extracts after stimulation by
IL-2, IP130 or IL-2+IP130. Only cells stimulated by IL-2 or
IL-2+IP130 show a STAT activation. .beta.-casein probes were used
in the study. The same results were obtained with two other probes
(GAS and GIRE).
[0035] FIG. 14 depicts a model of signal transduction and IP130.
IL-2 uses three main pathways: 1.degree./JAK/STAT depending on
IL-2R.beta..gamma. complex; 2.degree./SHC/MAPK initiated on the
phosphorylated IL-2R.beta. chain and 3.degree./PI3K. In accordance
with the (IP130)/(IL-2R.beta.), model (FIG. 14), IP130 does not
induce the JAK/STAT pathway but induces the SHC/MAPK pathway. (JAK:
Janus activated kinase; PI3K: phosphatidyl inositol, 3-phosphate
kinase; STAT: signal transducers and activators of transcription;
MAPKK: mitogen activated protein kinase; MAPK: mitogen activated
protein kinase; : induction of transcription).
[0036] FIG. 15 shows the cell cycle entry (S+G2/M) of IP130
stimulated NK cells. PBMC are stimulated by IL-2, IP130 or IL-2+EP
130. Non-specific responses (J+1 in medium) are subtracted. NK
cells entry into S+G2/M phases are measured by propidium iodide and
analyzed with the ModFit 2.0 software (Becton Dickinson).
[0037] FIG. 16 shows that IP130 stimulates LAK activity. In this
experiment, the kinetics of LAK activity stimulation has been
studied. Histograms show the results with an effector/target ratio
of ten. .DELTA.% lysis=lysis induced by IL-2 and/or
IP130-spontaneous lysis.
[0038] The present invention relates to the use of IL-2 peptides
derived from interleukin-2 (IL-2), for their therapeutic use in
mammals, and particularly in humans. The peptides are selected from
fragments of IL-2 and derivatives of IL-2. The derivatives are
defined as containing an amino acid sequence capable of binding to
the IL-2R.beta. chain under the conditions described herein, or
capable of binding to the monoclonal antibodies produced by H2-8
hybridoma. The invention also relates to antibodies directed
against the peptides according to the invention which likewise
mimic and/or modulate IL-2 activity. The diagnostic and therapeutic
approaches involve the use of the purified peptides and the
antibodies for detecting and/or modulating IL-2 binding to IL-2R in
vitro and in vivo. In accordance with the present invention there
may be employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
SAMBROOK et al, "Molecular Coning: A Laboratory Manual" (1989);
"Current Protocols in Molecular Biology" Volumes I-Ill [Ausubel, R.
M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes [J.
E. Celis, ed. (1994)]; "Current Protocols in Immunology" Volumes
I-Ill [Coligan, J. E., ed. (1994)]; "Oligonucleotide Synthesis" (M.
J. Gait ed. 1984); "Nucleic Acid Hybridization" [B. D. Hames &
S. J. Higgins eds. (1985)]; "Transcription And Translation" [B. D.
Hames & S. J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.
I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL
Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning"
(1984).
[0039] Therefore, if appearing herein, the following terms shall
have the definitions set out below.
[0040] The terms "IL-2 peptide," "IL-2 agonist/antagonist,"
"IP130/IP130 derivatives," and any variants not specifically
listed, may be used herein interchangeably, and as used throughout
the present application. These terms refer to proteinaceous
material including single or multiple proteins or recombinant
product or peptides obtained by chemical synthesis, and extend to
those proteins having the amino acid sequence data described herein
and presented in FIG. 10 (SEQ ID NO.: 3). The profile of biological
activities set forth hereinafter is one of the aspects of the
present application. Accordingly, proteins displaying substantially
equivalent or altered activity are likewise contemplated. These
modifications may be deliberate, for example, such as modifications
obtained through site-directed mutagenesis, or may be accidental,
such as those obtained through mutations in hosts that are
producers of the complex or its named subunits. Also, the terms
"IL-2 peptide," "IL-2 agonist/antagonist" and "IP130/IP130
derivatives or purified peptide(s)" are intended to include within
their scope proteins specifically recited herein as well as all
substantially homologous analogs and allelic variations.
[0041] The amino acid residues described herein are preferred to be
in the "L" isomeric form. However, residues in the "D" isomeric
form can be substituted for any L-amino acid residue, as long as
the desired functional property or immunoglobulin-binding is
retained by the polypeptide. NH refers to the free amino group
present at the amino terminus of a polypeptide. COOH refers to the
free carboxy group present at the carboxy terminus of a
polypeptide. In keeping with standard polypeptide nomenclature, J.
Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid
residues are shown in the following Table of Correspondence:
TABLE-US-00001 TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter
AMINO ACID Y Tyr tyrosine G Gly glycine F Phe phenylalanine M Met
methionine A Ala alanine S Ser serine I Ile isoleucine L Leu
leucine T Thr threonine V Val valine P Pro proline K Lys lysine H
His histidine Q Gln glutamine E Glu glutamic acid W Trp tryptophan
R Arg arginine D Asp aspartic acid N Asn asparagine C Cys
cysteine
[0042] It should be noted that all amino-acid residue sequences are
represented herein by formulae whose left and right orientation is
in the conventional direction of amino-terminus to
carboxy-terminus. Furthermore, it should be noted that a dash at
the beginning or end of an amino acid residue sequence indicates a
peptide bond to a further sequence of one or more amino-acid
residues. The above Table is presented to correlate the
three-letter and one-letter notations which may appear alternately
herein.
[0043] A "replicon" is any genetic element (e.g., plasmid,
chromosome, virus) that functions as an autonomous unit of DNA
replication in vivo; i.e., capable of replication under its own
control.
[0044] A "vector" is a replicon, such as plasmid, phage or cosmid,
to which another DNA segment may be attached so as to bring about
the replication of the attached segment.
[0045] A "DNA molecule" refers to the polymeric form of
deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in
its either single stranded form, or a double-stranded helix. This
term refers only to the primary and secondary structure of the
molecule, and does not limit it to any particular tertiary forms.
Thus, this term includes double-stranded DNA found, inter alia, in
linear DNA molecules (e.g., restriction fragments), viruses,
plasmids, and chromosomes. In discussing the structure of
particular double-stranded DNA molecules, sequences may be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the nontranscribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA).
[0046] An "origin of replication" refers to those DNA sequences
that participate in DNA synthesis.
[0047] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in vivo when
placed under the control of appropriate regulatory sequences. The
boundaries of the coding sequence are determined by a start codon
at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl) terminus. A coding sequence can include, but is not
limited to, prokaryotic sequences, cDNA from eukaryotic mRNA,
genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. A polyadenylation signal and
transcription termination sequence will usually be located 3' to
the coding sequence.
[0048] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell.
[0049] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined by mapping with
nuclease S1), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase.
Eukaryotic promoters will often, but not always, contain "TATA"
boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno
sequences in addition to the -10 and -35 consensus sequences.
[0050] An "expression control sequence" is a DNA sequence that
controls and regulates the transcription and translation of another
DNA sequence. A coding sequence is "under the control" of
transcriptional and translational control sequences in a cell when
RNA polymerase transcribes the coding sequence into mRNA, which is
then translated into the protein encoded by the coding
sequence.
[0051] A "signal sequence" can be included before the coding
sequence. This sequence encodes a signal peptide, N-terminal to the
polypeptide, that communicates to the host cell to direct the
polypeptide to the cell surface or secrete the polypeptide into the
media, and this signal peptide is clipped off by the host cell
before the protein leaves the cell. Signal sequences can be found
associated with a variety of proteins native to prokaryotes and
eukaryotes.
[0052] The term "oligonucleotide," is defined as a molecule
comprised of two or more ribonucleotides, preferably more than
three. Its exact size will depend upon many factors which, in turn,
depend upon the ultimate function and use of the
oligonucleotide.
[0053] A cell has been "transformed" by exogenous or heterologous
DNA when such DNA has been introduced inside the cell. The
transforming DNA may or may not be integrated (covalently linked)
into chromosomal DNA making up the genome of the cell. In
prokaryotes, yeast, and mammalian cells for example, the
transforming DNA may be maintained on an episomal element such as a
plasmid. With respect to eukaryotic cells, a stably transformed
cell is one in which the transforming DNA has become integrated
into a chromosome so that it is inherited by daughter cells through
chromosome replication. This stability is demonstrated by the
ability of the eukaryotic cell to establish cell lines or clones
comprised of a population of daughter cells containing the
transforming DNA. A "clone" is a population of cells derived from a
single cell or common ancestor by mitcsis. A "cell line" is a clone
of a primary cell that is capable of stable growth in vitro for
many generations.
[0054] Two DNA sequences are "substantially homologous" when at
least about 75% (preferably at least about 80%, and most preferably
at least about 90 or 95%) of the nucleotides match over the defined
length of the DNA sequences. Sequences that are substantially
homologous can be identified by comparing the sequences using
standard software available in sequence data banks, or in a
Southern hybridization experiment under, for example, stringent
conditions as defined for that particular system. Defining
appropriate hybridization conditions is within the skill of the
art. See, e.g., MANIATIS et al., supra; DNA Cloning, Vols. I &
II, supra; Nucleic Acid Hybridization, supra.
[0055] It should be appreciated that also within the scope of the
present invention are the biological uses of the DNA sequences
encoding IL-2 peptides having the same amino acid sequence as IP130
(SEQ ID NO.: 2 or SEQ ID NO.:4), but which are degenerate to the
DNA encoding SEQ ID NO.: 2 or SEQ ID NO.: 4. By "degenerate to" is
meant that a different three-letter codon is used to specify a
particular amino acid. It is well known in the art that the
following codons can be used interchangeably to code for each
specific amino acid:
TABLE-US-00002 Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or
L) UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (Ile or I) AUU
or AUC or AUA Methionine (Met or M) AUG Valine (Val or V) GUU or
GUC of GUA or GUG Serine (Ser or S) UCU or UCC or UCA or UCG or AGU
or AGC Proline (Pro or P) CCU or CCC or CCA or CCG Threonine (Thr
or T) ACU or ACC or ACA or ACG Alanine (Ala or A) GCU or GCG or GCA
or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine (His or H) CAU or
CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn or N) AAU or
AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAU or
GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU or
UGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG
Glycine (Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W)
UGG Termination codon UAA (ochre) or UAG (amber) or UGA (opal)
[0056] It should be understood that the codons specified above are
for RNA sequences. The corresponding codons for DNA have a T
substituted for U.
[0057] Modifications of the peptides can be made in the DNA
encoding SEQ ID NO.: 2 or SEQ ID NO.: 4 such that a particular
codon is changed to a codon which codes for a different amino acid.
Such a mutation is generally made by making the fewest nucleotide
changes possible. A substitution mutation of this sort can be made
to change an amino acid in the resulting protein in a
non-conservative manner (i.e., by changing the codon from an amino
acid belonging to a grouping of amino acids having a particular
size or characteristic to an amino acid belonging to another
grouping) or in a conservative manner (i.e., by changing the codon
from an amino acid belonging to a grouping of amino acids having a
particular size or characteristic to an amino acid belonging to the
same grouping). Such a conservative change generally leads to less
change in the structure and function, of the resulting protein. A
non-conservative change is more likely to alter the structure,
activity or function of the resulting protein. The present
invention should be considered to include sequences containing
conservative changes which do not significantly alter the activity
or binding characteristics of the resulting protein.
[0058] The following is one example of various groupings of amino
acids:
Amino acids with nonpolar R groups [0059] Alanine [0060] Valine
[0061] Leucine [0062] Isoleucine [0063] Proline [0064]
Phenylalanine [0065] Tryptophan [0066] Methionine Amino acids with
uncharted polar R groups [0067] Glycine [0068] Serine [0069]
Threonine [0070] Cysteine [0071] Tyrosine [0072] Asparagine [0073]
Glutamine Amino acids with charged polar R groups (negatively
charged at Ph 6.0) [0074] Aspartic acid [0075] Glutamic acid Basic
amino acids (positively charged at pH 6.0) [0076] Lysine [0077]
Arginine [0078] Histidine (at pH 6.0) Another grouping may be those
amino acids with phenyl groups: [0079] Phenylalanine [0080]
Tryptophan [0081] Tyrosine Another grouping may be according to
molecular weight (i.e., size of R groups):
TABLE-US-00003 [0081] Glycine 75 Alanine 89 Serine 105 Proline 115
Valine 117 Threonine 119 Cysteine 121 Leucine 131 Isoleucine 131
Asparagine 132 Aspartic acid 133 Glutamine 146 Lysine 146 Glutamic
acid 147 Methionine 149 Histidine (at pH 6.0) 155 Phenylalanine 165
Arginine 174 Tyrosine 181 Tryptophan 204
Particularly preferred conservative substitutions are: [0082] Lys
for Arg and vice versa such that a positive charge may be
maintained; [0083] Glu for Asp and vice versa such that a negative
charge may be maintained; [0084] Ser for Thr such that a free --OH
can be maintained; and [0085] Gln for Asn such that a free NH, can
be maintained.
[0086] Amino acid substitutions may also be introduced in IL-2 or
peptides thereof to substitute an amino acid with a particularly
preferable property. For example, a Cys may be introduced a
potential site for disulfide bridges with another Cys. A His may be
introduced as a particularly "catalytic" site (i.e., His can act as
an acid or base and is the most common amino acid in biochemical
catalysis). Pro may be introduced because of its particularly
planar structure, which induces .beta.-turns in the protein's
structure.
[0087] The biologically active peptides of the invention preferably
encompass a region of the IL-2 sequence which includes amino acids
17-20, although one or more of these amino acid residues may be
substituted with another amino acid, or a modified amino acid. The
use of the preferred peptide containing at least 5 amino acids in
length, more preferably 8-12 amino acids, and most preferably at
least 15 amino acids in length is one aspect of the invention, as
well as the use of the peptide of the invention based on amino
acids 1-30 of IL-2.
[0088] Biological or physiological activity of IL-2 may be
considered to include the stimulation of CD4, CD8 and NK cells, and
may include antiviral and antitumor activities. Biological or
physiological activity of IP130 and other peptides of the invention
may include the foregoing activities of IL-2, as well as induction
of SHC phosphorylation and induction of the SHC/MAPK pathway.
[0089] A "heterologous" region of the DNA construct is an
identifiable segment of DNA within a larger DNA molecule that is
not found in association with the larger molecule in nature. Thus,
when the heterologous region encodes a mammalian gene, the gene
will usually be flanked by DNA that does not flank the mammalian
genomic DNA in the genome of the source organism. Another example
of a heterologous coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., a cDNA where the
genomic coding sequence contains introns, or synthetic sequences
having codons different than the native gene). Allelic variations
or naturally-occurring mutational events do not give rise to a
heterologous region of DNA as defined herein.
[0090] An "antibody" is any immunoglobulin, including antibodies
and fragments thereof, that binds a specific epitope. The term
encompasses polyclonal, monoclonal, and chimeric antibodies, the
last mentioned described in further detail in U.S. Pat. Nos.
4,816,397 and 4,816,567.
[0091] An "antibody combining site" is that structural portion of
an antibody molecule comprised of heavy and light chain variable
and hypervariable regions that specifically binds antigen.
[0092] The phrase "antibody molecule" in its various grammatical
forms as used herein contemplates both an intact immunoglobulin
molecule and an immunologically active portion of an immunoglobulin
molecule.
[0093] Exemplary antibody molecules are intact immunoglobulin
molecules, substantially intact immunoglobulin molecules and those
portions of an immunoglobulin molecule that contains the paratope,
including those portions known in the art as Fab, Fab',
F(ab').sub.2 and F(v), which portions are preferred for use in the
therapeutic methods described herein.
[0094] Fab and F(ab').sub.2 portions of antibody molecules are
prepared by the proteolytic reaction of papain and pepsin,
respectively, on substantially intact antibody molecules by methods
that are well-known. See for example, U.S. Pat. No. 4,342,566 to
Theofilopolous et al. Fab' antibody molecule portions are also
well-known and are produced from F(ab').sub.2 portions followed by
reduction of the disulfide bonds linking the two heavy chain
portions as with mercaptoethanol, and followed by alkylation of the
resulting protein mercaptan with a reagent such as iodoacetamide.
An antibody containing intact antibody molecules is preferred
herein.
[0095] The phrase "monoclonal antibody" in its various grammatical
forms refers to an antibody having only one species of antibody
combining site capable of immunoreacting with a particular antigen.
A monoclonal antibody thus typically displays a single binding
affinity for any antigen with which it immunoreacts. A monoclonal
antibody may therefore contain an antibody molecule having a
plurality of antibody combining sites, each immunospecific for a
different antigen; e.g., a bispecific (chimeric) monoclonal
antibody.
[0096] Preferred antibodies of the present invention bind to the
peptides of the invention, described above. The antibodies which
bind the peptides of the invention may be used as diagnostic agents
for analyzing IL-2 binding to its receptor, and may also be used as
therapeutic agents, to enhance or inhibit the binding of IL-2 to
its receptor. In a preferred embodiment, the antibody of the
invention inhibits the binding of IL-2 and/or IP130 to the
IL-2R.
[0097] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human.
[0098] The phrase "therapeutically effective amount" is used herein
to mean an amount sufficient to prevent, and preferably reduce by
at least about 30 percent, more preferably by at least 50 percent,
most preferably by at least 90 percent, a clinically significant
change in a feature of pathology such as for example, elevated
blood pressure, fever or white cell count as may attend its
presence and activity.
[0099] A DNA sequence is "operatively linked" to an expression
control sequence when the expression control sequence controls and
regulates the transcription and translation of that DNA sequence.
The term "operatively linked" includes having an appropriate start
signal (e.g., ATG) in front of the DNA sequence to be expressed and
maintaining the correct reading frame to permit expression of the
DNA sequence under the control of the expression control sequence
and production of the desired product encoded by the DNA sequence.
If a gene that one desires to insert into a recombinant DNA
molecule does not contain an appropriate start signal, such a start
signal can be inserted in front of the gene.
[0100] The term "standard hybridization conditions" refers to salt
and temperature conditions substantially equivalent to 5.times.SSC
and 65.degree. C. for both hybridization and wash. However, one
skilled in the art will appreciate that such "standard
hybridization conditions" are dependent on particular conditions
including the concentration of sodium and magnesium in the buffer,
nucleotide sequence length and concentration, percent mismatch,
percent formamide, and the like. Also important in the
determination of "standard hybridization conditions" is whether the
two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such
standard hybridization conditions are easily determined by one
skilled in the art according to well known formulae, wherein
hybridization is typically 10-20.degree. C. below the predicted or
determined T.sub.m with washes of higher stringency, if
desired.
[0101] In its primary aspect, the present invention concerns the
use of IL-2 peptides which modulate IL-2 activity. By "modulate" is
meant either agonist or antagonist activity which either increases
or suppresses the physiological effects of IL-2 such as the
proliferation of cells, as described below.
As stated above, the present invention also relates to a
recombinant DNA molecule or cloned gene, or a degenerate variant
thereof, which encodes an IL-2 peptide, or a fragment thereof, that
possesses a molecular weight preferably of about 2-5 kD and an
amino acid sequence set forth in FIG. 10 (SEQ ID NO.: 4) or a
sequence wherein SEQ ID NO.: 4 is modified by insertion, deletion
and/or substitution; preferably a nucleic acid molecule, in
particular a recombinant DNA molecule or cloned gene, encoding the
peptide has a nucleotide sequence, is complementary to, or
hybridizes under standard hybridization conditions to a DNA
sequence encoding SEQ ID NO:2 or SEQ ID NO.: 4.
[0102] The possibilities both diagnostic and therapeutic that are
raised by the existence of the IL-2 peptides, derive from the fact
that the peptides appear to participate in direct and causal
protein-protein interaction between the IL-2 peptide and the IL-2
receptor, specifically IL-2R.beta., and those factors that
thereafter mediate cellular events. In particular the IP130 peptide
has been shown to induce phosphorylation of IL-2R.beta., to induce
c-myc; and to induce natural killer (NK) cells to enter the cell
cycle. As suggested earlier and elaborated further on herein, the
present invention contemplates pharmaceutical intervention in the
cascade of reactions in which the IL-2R is implicated, to modulate
the activity initiated by IL-2 and peptides thereof.
[0103] Thus, in instances where it is desired to reduce or inhibit
the IL-2 induced activity, an appropriate IL-2 peptide inhibitor of
IL-2 could be introduced to block the interaction of the IL-2 with
the IL-2R. Correspondingly, instances where insufficient IL-2
induced activity is taking place could be remedied by the
introduction of additional quantities of the appropriate IL-2
peptide agonist, such as IP130, or its chemical or pharmaceutical
cognates, analogs, fragments and the like.
[0104] As discussed earlier, the IL-2 peptides or their binding
partners or other ligands or agents exhibiting either mimicry or
antagonism to IL-2 or control over its production, may be prepared
in pharmaceutical compositions, with a suitable carrier and at a
strength effective for administration by various means to a patient
experiencing an adverse medical condition associated with
undesirable levels of IL-2 for the treatment thereof. A variety of
administrative techniques may be utilized, among them parenteral
techniques such as subcutaneous, intravenous and intraperitoneal
injections, catheterizations and the like. Average quantities of
the IL-2 peptides or their subunits may vary and in particular
should be based upon the recommendations and prescription of a
qualified physician or veterinarian.
[0105] Also, antibodies including both polyclonal and monoclonal
antibodies, and drugs that modulate the production or activity of
IL-2 and/or peptides thereof may possess certain diagnostic
applications and may for example, be utilized for the purpose of
detecting and/or measuring IL-2 receptor activity or the like. For
example, IL-2 or peptides thereof may be used to produce both
polyclonal and monoclonal antibodies to themselves in a variety of
cellular media, by known techniques such as the hybridoma technique
utilizing, for example, fused mouse spleen lymphocytes and myeloma
cells. Likewise, small molecules that mimic or antagonize the
activity(ies) of the IL-2 peptides of the invention may be
discovered or synthesized, and may be used in diagnostic and/or
therapeutic protocols.
[0106] The general methodology for making monoclonal antibodies by
hybridomas is well known. Immortal, antibody-producing cell lines
can also be created by techniques other than fusion, such as direct
transformation of B lymphocytes with oncogenic DNA, or transfection
with Epstein-Barr virus. See, e.g., M. SCHREIER et al., "Hybridoma
Techniques" (1980); HAMMERLING et al., "Monoclonal Antibodies And
T-cell Hybridomas" (1981); KENNETT et al., "Monoclonal Antibodies"
(1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783;
4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632;
4,493,890.
[0107] Panels of monoclonal antibodies produced against IL-2
peptides can be screened for various properties; i.e., isotype,
epitope, affinity, etc. Of particular interest are monoclonal
antibodies that modulate the activity of IL-2 or peptides thereof.
Such monoclonals can be readily identified in cellular
proliferation assays. High affinity antibodies are also useful when
immuncaffinity purification of native or recombinant IL-2 or IL-2
peptides is possible.
[0108] Preferably, the anti-IL-2 antibody used in the diagnostic
methods of this invention is an affinity purified polyclonal
antibody. More preferably, the antibody is a monoclonal antibody
(mAb). In addition, it is preferable for the anti-IL-2 antibody
molecules used herein be in the form of Fab, Fab', F(ab').sub.2 or
F(v) portions of whole antibody molecules.
[0109] A diagnostic method of the present invention comprises
examining a cellular sample or medium by means of an assay
including an effective amount of a labeled IL-2 peptide or an
antagonist thereof, such as an anti-IP130 antibody, preferably an
affinity-purified polyclonal antibody, and more preferably a mAb.
In addition, it is preferable for the anti-IL-2 antibody molecules
used herein be in the form of Fab, Fab', F(ab').sub.2 or F(v)
portions or whole antibody molecules. As previously discussed,
patients capable of benefitting from this method include those
suffering from cancer, a pre-cancerous lesion, a viral infection or
other like pathological derangement. Methods for isolating the IL-2
peptide and inducing anti-IL-2 antibodies and for determining and
optimizing the ability of anti-IL-2 antibodies to assist in the
examination of the target cells are all well-known in the art.
[0110] Methods for producing polyclonal anti-polypeptide antibodies
are well-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et
al. A monoclonal antibody, typically containing Fab and/or
F(ab').sub.2 portions of useful antibody molecules, can be prepared
using the hybridoma technology described in Antibodies-A Laboratory
Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, New
York (1988), which is incorporated herein by reference. Briefly, to
form the hybridoma from which the monoclonal antibody composition
is produced, a myeloma or other self-perpetuating cell line is
fused with lymphocytes obtained from the spleen of a mammal
hyperimmunized with an IL-2 peptide or IL-2 R-binding portion
thereof.
[0111] Splenocytes are typically fused with myeloma cells using
polyethylene glycol (PEG) 6000. Fused hybrids are selected by their
sensitivity to HAT. Hybridomas producing a monoclonal antibody
useful in practicing this invention are identified by their ability
to immunoreact with the present IL-2 mutant or peptide and their
ability to inhibit specified IL-2 activity in target cells.
[0112] A monoclonal antibody useful in practicing the present
invention can be produced by initiating a monoclonal hybridoma
culture comprising a nutrient medium containing a hybridoma that
secretes antibody molecules of the appropriate antigen specificity.
The culture is maintained under conditions and for a time period
sufficient for the hybridoma to secrete the antibody molecules into
the medium. The antibody-containing medium is then collected. The
antibody molecules can then be further isolated by well-known
techniques.
[0113] Media useful for the preparation of these compositions are
both well-known in the art and commercially available and include
synthetic culture media, inbred mice and the like. An exemplary
synthetic medium is Dulbecco's minimal essential medium (DMEM;
DULBECCO et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/l
glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary
inbred mouse strain is the Balb/c.
[0114] Methods for producing monoclonal anti-IL-2 antibodies are
also well-known in the art. See NIMAN et al., Proc. Natl. Acad.
Sci. USA, 80:4949-4953 (1983). Typically, the present IL-2 peptide
or a peptide analog is used either alone or conjugated to an
immunogenic carrier, as the immunogen in the before described
procedure for producing anti-IL-2 monoclonal antibodies. Thy
hybridomas are screened for the ability to produce an antibody that
immunoreacts with the IL-2 mutant or peptide analog.
[0115] The present invention further contemplates the use of
therapeutic compositions which are useful in practicing the
therapeutic methods of this invention. In one embodiment, the
therapeutic composition includes, in admixture, a pharmaceutically
acceptable excipient (carrier) and one or more of a IL-2 peptide, a
purified peptide or a derivative thereof or a polypeptide analog
thereof or fragment thereof, as described herein as an active
ingredient. In a preferred embodiment, the therapeutic composition
comprises an active compound containing a purified peptide capable
of modulating the specific binding of the present IL-2 with the
IL-2R.
[0116] The preparation of therapeutic compositions which contain
polypeptides, analogs or active fragments as active ingredients is
well understood in the art. Typically, such compositions are
prepared as injectables, either as liquid solutions or suspensions,
however, solid forms suitable for solution in, or suspension in,
liquid prior to injection can also be prepared. The preparation can
also be emulsified. The active therapeutic ingredient is often
mixed with excipients which are pharmaceutically acceptable and
compatible with active ingredient. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol, or the like
and combinations thereof. In addition, if desired, the composition
can contain minor amounts of auxiliary substances such as wetting
or emulsifying agents, pH buffering agents which enhance the
effectiveness of the active ingredient.
[0117] The use of the compositions may be by administration in a
manner compatible with the dosage formulation, and in a
therapeutically effective amount. The quantity to be administered
depends on the subject to be treated, capacity of the subject's
immune system to utilize the active ingredient, and degree of
modulation of IL-2 binding capacity desired. Precise amounts of
active ingredient required to be administered depends on the
judgment of the practitioner and are peculiar to each individual.
However, suitable dosages may range from about 0.1 to 20,
preferably about 0.5 to about 10, and more preferably one to
several, milligrams of active ingredient per kilogram body weight
of individual per day and depend on the route of administration.
Suitable regimes for initial administration and booster shots are
also variable, but are typified by an initial administration
followed by repeated doses at one or more hour intervals by a
subsequent injection or other administration. Alternatively,
continuous intravenous infusion sufficient to maintain
concentrations of ten nanomolar to ten micromolar in the blood are
contemplated.
[0118] The use of the therapeutic compositions may be by
administration in a composition which further includes an effective
amount of the IL-2 agonist/antagonist or analog thereof, and one or
more of the following active ingredients: an antibiotic, a
steroid.
[0119] As used herein, "pg" means picogram, "ng" means nanogram,
"ug" or ".mu.g" mean microgram, "mg" means milligram, "ul" or
".mu.l" mean microliter, "ml" means milliliter, "l" means
liter.
[0120] Another feature of this invention is the expression of the
DNA sequences disclosed herein. As is well known in the act, DNA
sequences may be expressed by operatively linking them to an
expression control sequence in an appropriate expression vector and
employing that expression vector to transform and appropriate
unicellular host.
[0121] Such operative linking of a DNA sequence of this invention
to an expression control sequence, of course, includes, if not
already part of the DNA sequence, the provision of an initiation
codon, ATG, in the correct reading frame upstream of the DNA
sequence.
[0122] A wide variety of host/expression vector combinations may be
employed in expressing the DNA sequences of this invention. Useful
expression vectors, for example, may consist of segments of
chromosomal, non-chromosomal and synthetic DNA sequences. Suitable
vectors include derivatives of SV40 and known bacterial plasmids,
e.g., E. coli plasmids col E1, pCR1, pBR322, pMB9 and their
derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous
derivatives of phage .lamda., e.g., NM989, and other phage DNA,
e.g., M13 and filamentous single stranded phage DNA; yeast plasmids
such as the 2.mu. plasmid or derivatives thereof; vectors useful in
eukaryotic cells, such as vectors useful in insect or mammalian
cells; vectors derived from combinations of plasmids and phage
DNAs, such as plasmids that have been modified to employ phage DNA
or other expression control sequences; and the like.
[0123] Any of a wide variety of expression control
sequences--sequences that control the expression of a DNA sequence
operatively linked to it--may be used in these vectors to express
the DNA sequences of this invention. Such useful expression control
sequences include, for example, the early or late promoters of
SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp
system, the TAC system, the TRC system, the LTR system, the major
operator and promoter regions of phage .lamda., the control regions
of fd coat protein, the promoter for 3-phosphoglycerate kinase or
other glycolytic enzymes, the promoters of acid phosphatase (e.g.,
Pho5), the promoters of the yeast .alpha.-mating factors, and other
sequences known to control the expression of genes of prokaryotic
or eukaryotic cells or their viruses, and various combinations
thereof.
[0124] A wide variety of unicellular host cells are also useful in
expressing the DNA sequences of this invention. These hosts may
include well known eukaryotic and prokaryotic hosts, such as
strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fund such
as yeasts, and animal cells, such as CHO, B--W and L-M cells,
African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40,
and BMT10), insect cells (e.g., Sf9), and human cells and plant
cells in tissue culture.
[0125] It will be understood that not all vectors, expression
control sequences and hosts will function equally well to express
the DNA sequences of this invention.
[0126] Neither will all hosts function equally well with the same
expression system. However, one skilled in the art will be able to
select the proper vectors, expression control sequences, and hosts
without undue experimentation to accomplish the desired expression
without departing from the scope of this invention. For example, in
selecting a vector, the host must be considered because the vector
must function in it. The vector's copy number, the ability to
control that copy number, and the expression of any other proteins
encoded by the vector, such as antibiotic markers, will also be
considered.
[0127] In selecting an expression control sequence, a variety of
factors will normally be considered. These include, for example,
the relative strength of the system, its controllability, and its
compatibility with the particular DNA sequence or gene to be
expressed, particularly as regards potential secondary structures.
Suitable unicellular hosts will be selected by consideration of,
e.g., their compatibility with the chosen vector, their secretion
characteristics, their ability to fold proteins correctly, and
their fermentation requirements, as well as the toxicity to the
host of the product encoded by the DNA sequences to be expressed,
and the ease of purification of the expression products.
[0128] Considering these and other factors a person skilled in the
art will be able to construct a variety of vector/expression
control sequence/host combinations that will express the DNA
sequences of this invention on fermentation or in large scale
animal culture.
[0129] It is further intended that for the therapeutic use of the
peptides according to the invention, the IL-2 peptide analogs may
be prepared from nucleotide sequences of the protein
complex/subunit derived within the scope of the present invention.
Analogs, such as fragments, may be produced, for example, by pepsin
digestion of IL-2 material. Other analogs, such as muteins, can be
produced by standard site-directed mutagenesis of IL-2 coding
sequences. Analogs exhibiting "IL-2 activity" such as small
molecules, whether functioning as promoters or inhibitors, may be
identified by known in vivo and/or in vitro assays.
[0130] As mentioned above, a DNA sequence encoding IL-2 peptides
can be prepared synthetically rather than cloned. The DNA sequence
can be designed with the appropriate codons for the IL-2 peptide
amino acid sequence. In general, one will select preferred codons
for the intended host if the sequence will be used for expression.
The complete sequence is assembled from overlapping
oligonucleotides prepared by standard methods and assembled into a
complete coding sequence. See, e.g., EDGE, Nature, 292:756 (1981);
NAMBAIR et al., Science, 223:1299 (1984); JAY et al., J. Biol.
Chem., 259:6311 (1984).
[0131] Synthetic DNA sequences allow convenient construction of
genes which will express IL-2 peptide analogs or "muteins".
Alternatively, DNA encoding muteins can be made by site-directed
mutagenesis of native IL-2 genes or cDNAs, and muteins can be made
directly using conventional polypeptide synthesis.
[0132] A general method for site-specific incorporation of
unnatural amino acids into proteins is described in Christopher J.
Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G.
Schultz, Science, 244:182-188 (April 1989). This method may be used
to create analogs with unnatural amino acids.
[0133] In accordance with the gene therapy applications of the
present invention, the preparation of antisense oligonucleotides
and ribozymes may be used to modulate the expression of IL-2 at the
translational level. This approach utilizes antisense nucleic acid
and ribozymes to block translation of a specific mRNA, either by
masking that mRNA with an antisense nucleic acid or cleaving it
with a ribozyme.
[0134] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule.
(See WEINTRAUB, 1990; MARCUS-SEKURA, 1988.) In the cell, they
hybridize to that mRNA, forming a double stranded molecule. The
cell does not translate an mRNA in this double-stranded form.
Therefore, antisense nucleic acids interfere with the expression of
mRNA into protein. Oligomers of about fifteen nucleotides and
molecules that hybridize to the AUG initiation codon will be
particularly efficient, since they are easy to synthesize and are
likely to pose fewer problems than larger molecules when
introducing them into cells. Antisense methods have been used to
inhibit the expression of many genes in vitro (MARCUS-SEKURA, 1988;
HAMBOR et al., 1988).
[0135] The DNA sequences described herein may thus be used to
prepare antisense molecules against, and ribozymes that cleave
mRNAs for IL-2 or molecules which stimulate or reduce IL-2
secretion and their ligands.
[0136] The present invention also relates to a variety of
diagnostic applications, including methods for detecting IL-2
presence and activity, by reference to their ability to elicit the
activities which are mediated by the present IL-2 peptides. As
mentioned earlier, the IL-2 peptide can be used to produce
antibodies to itself by a variety of known techniques, and such
antibodies could then be isolated and utilized as in tests for the
presence of particular IL-2 and/or IL-2 R activity in suspect
target cells.
[0137] As described in detail above, antibody(ies) to the IL-2
peptides can be produced and isolated by standard methods including
the well known hybridoma techniques. For convenience, the
antibody(ies) to the IL-2 peptide will be referred to herein as
Ab.sub.1 and antibody(ies) raised in another species as
Ab.sub.2.
[0138] The presence of IL-2 and IL-2 peptides in cells can be
ascertained by the usual immunological procedures applicable to
such determinations. A number of useful procedures are known. Three
such procedures which are especially useful utilize either the IL-2
peptide labeled with a detectable label, antibody Ab.sub.1 labeled
with a detectable label, or antibody Ab.sub.2 labeled with a
detectable label. The procedures may be summarized by the following
equations wherein the asterisk indicates that the particle is
labeled, and "ILP" stands for the IL-2 peptide: [0139] A.
ILP*+Ab.sub.1=ILP*Ab.sub.1 [0140] B. ILP+Ab*=ILPAb.sub.1* [0141] C.
ILP+Ab.sub.1+Ab.sub.2*=ILPAb.sub.1Ab.sub.2*
[0142] The procedures and their application are all familiar to
those skilled in the art and accordingly may be utilized within the
scope of the present invention. The "competitive" procedure,
Procedure A, is described in U.S. Pat. Nos. 3,654,090 and
3,850,752. Procedure C, the "sandwich" procedure, is described in
U.S. Pat. Nos. RE 31,006 and 4,016,043. Still other procedures are
known such as the "double antibody," or "DASP" procedure.
[0143] In each instance, the ILP forms complexes with one or more
antibody(ies) or binding partners and one member of the complex is
labeled with a detectable label. The fact that a complex has formed
and, if desired, the amount thereof, can be determined by known
methods applicable to the detection of labels.
[0144] It will be seen from the above, that a characteristic
property of Ab.sub.2 is that it will react with Ab.sub.1. This is
because Ab.sub.1 raised in one mammalian species has been used in
another species as an antigen to raise the antibody Ab.sub.2. For
example, Ab.sub.2 may be raised in goats using rabbit antibodies as
antigens. Ab.sub.2 therefore would be anti-rabbit antibody raised
in goats. For purposes of this description and claims, Ab.sub.1
will be referred to as a primary or anti-ILP antibody, and Ab.sub.2
will be referred to as a secondary or anti-Ab.sub.1 antibody.
[0145] The labels most commonly employed for these studies are
radioactive elements, enzymes, chemicals which fluoresce when
exposed to ultraviolet light, and others.
[0146] A number of fluorescent materials are known and can be
utilized as labels. These include, for example, fluorescein,
rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A
particular detecting material is anti-rabbit antibody prepared in
goats and conjugated with fluorescein through an
isothiocyanate.
[0147] The IL-2 peptide or its binding partner(s) can also be
labeled with a radioactive element or with an enzyme. The
radioactive label can be detected by any of the currently available
counting procedures. The preferred isotope may be selected from
.sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.36Cl, .sup.51Cr,
.sup.57Co, .sup.59Fe, .sup.90Y, .sup.125I, .sup.131I, and
.sup.186Re.
[0148] Enzyme labels are likewise useful, and can be detected by
any of the presently utilized colorimetric, spectrophotometric,
fluorospectrophotometric, amperometric or gasometric techniques.
The enzyme is conjugated to the selected particle by reaction with
bridging molecules such as carbodiimides, diisocyanates,
glutaraldehyde and the like. Many enzymes which can be used in
these procedures are known and can be utilized. The preferred are
peroxidase, .beta.-glucuronidase, .beta.-D-glucosidase,
.beta.-D-galactosidase, urease, glucose oxidase plus peroxidase and
alkaline phosphatase. U.S. Pat. Nos. 3,654,090; 3,850,752; and
4,016,043 are referred to by way of example for their disclosure of
alternate labeling material and methods.
[0149] A particular assay system which can be utilized in
accordance with the present invention, is known as a receptor
assay. In a receptor assay, the material to be assayed is
appropriately labeled and then certain cellular test colonies are
inoculated with a quantity of both the labeled and unlabeled
material after which binding studies are conducted to determine the
extent to which the labeled material binds to the cell receptors.
In this way, differences in affinity between materials can be
ascertained.
[0150] A further embodiment of this invention is the diagnostic
application of commercial test kits suitable for use by a medical
specialist. The kit may be prepared to determine the presence or
absence of predetermined IL-2R activity or predetermined IL-2
activity capability in suspected target cells. In accordance with
the testing techniques discussed above, one class of such kits will
contain at least the labeled IL-2 peptide or its binding partner,
for instance an antibody specific thereto, and directions, of
course, depending upon the method selected, e.g., "competitive,"
"sandwich," "DASP" and the like. The kits may also contain
peripheral reagents such as buffers, stabilizers, etc.
[0151] Accordingly, a test kit may be prepared for the
demonstration of the presence or capability of cells for
predetermined IL-2 R activity, comprising: [0152] (a) a
predetermined amount of at least one labeled immunochemically
reactive component obtained by the direct or indirect attachment of
the present IL-2 peptide factor or a specific binding partner
thereto, to a detectable label; [0153] (b) other reagents; and
[0154] (c) directions for use of said kit.
[0155] More specifically, the diagnostic test kit may comprise:
[0156] (a) a known amount of the IL-2 peptide as described above
(or a binding partner) generally bound to a solid phase to form an
immunosorbent, or in the alternative, bound to a suitable tag, or
plural such end products, etc. (or their binding partners) one of
each; [0157] (b) if necessary, other reagents; and [0158] (c)
directions for use of said test kit.
[0159] In a further variation, the test kit may be prepared and
used for the purposes stated above, which operates according to a
predetermined protocol (e.g. "competitive," "sandwich," "double
antibody," etc.), and comprises: [0160] (a) a labeled component
which has been obtained by coupling the IL-2 peptide to a
detectable label; [0161] (b) one or more additional immunochemical
reagents of which at least one reagent is a ligand or an
immobilized ligand, which ligand is selected from the group
consisting of: [0162] (i) a ligand capable of binding with the
labeled component (a); [0163] (ii) a ligand capable of binding with
a binding partner of the labeled component (a); [0164] (iii) a
ligand capable of binding with at least one of the component(s) to
be determined; and [0165] (iv) a ligand capable of binding with at
least one of the binding partners of at least one of the
component(s) to be determined; and [0166] (c) directions for the
performance of a protocol for the detection and/or determination of
one or more components of an immunochemical reaction between the
IL-2 peptide and a specific binding partner thereto.
[0167] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only and are not intended to be limiting unless otherwise
specified.
EXAMPLES
Example 1
Characterization of Mouse Monoclonal Antibody H2.8
[0168] Female BALB/c mice were repeatedly immunized with 25 to 50
.mu.g of peptide 1-30 per, injection. The peptide was coupled to
the KLH carrier and injected with Complete Freund's adjuvant (first
injection) or incomplete Freund's adjuvant (subsequent injections).
The titer of the anti-IL-2 activity was assessed in a group of five
animals. Spleen cells from the animal giving the best response were
used for fusion with cell line SP2-0. Four hybridomas with specific
anti-IL-2 activity were cloned. The mABs were purified from the
corresponding ascitic fluid by amonium sulfate precipitation. The
purity of the reagents (>80%) was verified by polyacrylamide
gels. The properties of the mAbs were characterized. The results
are reported only for mAb H2-8. The isotype (IgGl) and the Kd
(1.4.times.10.sup.-9M) of mAb H2-8 were determined.
[0169] Mouse mAbs 19B11 (IgGl) and 2C4 (IgGl) previously
characterized (MOREAU et al (1995b) Mol. Immunol. 32:1047-1056;
REBOLLO et al (1992) Mol. Immunol. 29:119-130) were used as
controls. mAbs 19B11 and 2C4 inhibit the binding of IL-2 to
IL-2R.beta. and recognize the peptides 1-10 (see below), 1-22 and
1-30. Rat monoclonal 11B11 (IgG, k) specific for murine IL-4 was
provided by Dr. W. PAUL (National Institute of Health, Bethesda
Md., USA and used as previously described (MOREAU et al. (1995b)
Mol. Immunol. 32:1047-1056).
[0170] The inhibitory effect of purified mAb H2-8 was first assayed
on the binding of .sup.125I-labeled IL-2. Its effects were measured
on two transfectants derived from a mouse cell line expressing only
mIL-2R.gamma.. Transfectant TS1.beta. and TS1.alpha. were obtained
after transfection with human IL-2R.beta. and human IL-2R.alpha.
cDNA, respectively. Both cell lines bind IL-2. mAb H2-8 inhibits
the binding of IL-2 to TS1.beta. without significantly affecting
IL-2 binding to TS1.alpha. (data not shown).
[0171] The binding properties of mAb H2-8 were studied by ELISA.
Plates were coated with either IL-2 or peptides 1-22 or 1-30. mAb
H2-8 binds to IL-2 and peptide 1-30, but does not recognize peptide
1-22. As control mAb 19B11 (previously characterized) recognized
both peptides (FIG. 2).
[0172] Binding inhibition experiments were performed to further
characterize the specificity of mAb H2-8. Plates were coated with
IL-2 and a concentration of mAb H2-8 giving approximately 50% of
maximum binding was used. H2-8 was preincubated with different
peptides including five decapeptides (1-10, 5-15, 10-20, 15-25,
20-30). Only IL-2 and peptide 1-30 were able to inhibit the binding
of mAb H2-8 to IL-2. Peptide 1-30 was the most efficient inhibitor
in these experiments (FIG. 2). This result is compatible with the
fact that isolated peptide 1-30 folds in an .alpha. helical
configuration (.alpha.-helix content of 50%.+-.7%) whereas peptide
1-22 does not (13%.+-.5%) as measured by circular dichroism.
Therefore peptide 1-30 may adopt some unique structural
conformation very close to that of native IL-2. As control the
binding of mAb 19B11 is inhibited by IL-2 but also by peptides
1-10, 1-22 and 1-30. This confirms that the epitope of mAb 19B11 is
near the NH.sub.2 terminal position of IL-2 as previously suggested
(MOREAU et al. (1995b) Mol. Immunol. 32:1047-1056).
Example 2
Peptide Synthesis
[0173] Peptides were synthesized by the stepwise solid-phase using
the Boc/trifluorcacetic acid method (MERRIFIELD (1963) J. Am. Chem.
Soc. 85:2149-2145), on a .rho.-methyl-benzhydrylamine resin
(Applied Biosystems) with an Applied Biosystems 430A peptide
synthesizer.
Example 3
Involvement of aa at Positions 17 and 20 in mAb H2-8
Recognition
[0174] The reactivity of mAb H2-8 to various IL-2 mutants including
one mutant at position 17 (Leu.fwdarw.Asp), four mutants at
position 20 (Asp.fwdarw.Asn; Asp.fwdarw.Lys; Asp.fwdarw.Arg and
Asp.fwdarw.Leu) and a double mutant 17-20 (Leu17.fwdarw.Asp and
Asp20.fwdarw.Leu) were tested by Western blot analysis (FIG.
3).
[0175] mAb H2-8 does not recognize mutations at position 20 or the
double mutant (TH ZE J (1994) Eur. Cytokine Netw. 5:353-368; MOREAU
et al. (1995a) J. Immunol. 155:3401-3408; CHASTAGNER et al. (1996)
Eur. J. Immunol. 26:201-206; MACKAY D (1992) Science 257:410-413).
Recognition of the mutation at position 17 is also affected. As
positive control the results obtained with mAb 2C4 that recognize
an epitope near the NH.sub.2 terminal area of IL-2 are shown. Since
this mAb (as 19B11) recognizes peptide 1-10 which bears no
mutation, its binding to IL-2 is not affected. Similarly mutations
at position 125 and/or 127 do not affect binding to mAbs H28 and
2C4, and serve as additional controls. ELISA experiments performed
with all the mutants support the data obtained with Western blots
(data not shown).
[0176] mAb H2-8 as characterized in Example 1 and 19B11 (described
in Molec. Immunol. (1995) 32:1047-1056) have similar properties:
both bind to the NH.sub.2 terminal end of IL-2 and specifically
inhibit the binding of IL-2 to IL-2R.beta. chain. Since both
antibodies recognize sequences located in peptide 1-30 it was of
interest to compare the relationship between the corresponding
epitopes. Plates coated with mAb H2-8 were used to bind peptide
1-30. The binding of mAb 19B11 to these plates was positive, thus
indicating that the epitopes of mAbs H2-8 and 19B11 do not overlap
significantly. Various controls performed to verify these results
are shown (FIG. 3). The binding of 19B11 is strictly dependent on
the presence of peptide 1-30 and on the coating by mAb H2-8.
Results obtained with mAb 3H9 recognizing the peptide 30-54 further
demonstrated the specificity of the data presented in FIG. 3.
Example 4
Cell Lines Culture Media and Proliferation Assay
[0177] TS1 cells express only mouse IL-2R.gamma.. TS1.beta. cells
were obtained after transfection of TS1 cells with human
IL-2R.beta. cDNA cloned in the pdKCR expression vector kindly
provided by Dr. T. TANIGUCHI (Institute for Molecular and Cellular
Biology, Tokyo University, Japan). TS1.alpha. cells were obtained
after transfection of TS1 cells with human IL-2R.alpha. cDNA cloned
in pCMV4 expression vector provided by Drs W. A. KUZIEL and W. C.
GREENE (Gladstone Institute Virol./Immunol., San Francisco Calif.,
USA). TS1.beta. and TS1.alpha. were previously characterized
(PITTON et al. (1993) Cytokine 5:362-371). CTLL2 and YT were also
used for IL-2 binding studies.
[0178] All cultures were performed in complete medium composed of
RPMI 1640 (BioProducts, Walkerville, Md.), 10% heat inactivated FCS
(Seroyial, Vogelgsun, France), 2 mM glutamine, 100 units/ml
penicillin, 100 .mu.g/ml streptomycin, 50 mM
2-.beta.-mercaptoethanol (2.beta.ME). TS1.beta. and TS1.alpha. cell
lines were grown as TS1 cells in complete medium supplemented with
supernatant of recombinant baculovirus expressing murine IL-9
proteins (DIB 349) (UYTTENHOVE et al. (1988) Proc. Natl. Acad. Sci.
USA 85:6934-6938).
[0179] TS1.beta. cells were cultured (10.sup.4 cells/well) in 96
wells in flat-bottomed microtiter plates with a final volume of 0.2
ml. Various concentrations of human rIL-2, IL-2 muteins or mouse
rIL-9 were assayed. In order to test the inhibitory effect of mAbs,
different concentrations of these reagents were mixed in the
culture wells with the respective lymphokines for 30 min at low
temperature before adding the cells. The inhibitory effects of
mutein 20 Leu (described in Cytokine (1997) 9:488-498, which is
incorporated herein by reference in its entirety) was studied by
preincubating the cells (30 min at 4.degree. C.) with the indicated
concentration of inhibitor before adding IL-2 or IL-9 to the wells.
Cultures were pulsed with 0.5 .mu.Ci/well of (.sup.3H) TdR after 36
h of incubation and harvested 15 h later.
Example 5
Biological Properties of mAb H2-8
[0180] The biological properties of mAb H2-8 were evaluated on the
proliferation of the IL-2- or IL-9-dependent TS1.beta. cell line
(FIG. 4). In these experiments the IL-2 mutant IL-2 Pro.sup.125
(Cys.fwdarw.Pro) was used. This mutation slightly reduces the
affinity of IL-2 for IL-2R without affecting the maximum
proliferation obtained when higher concentrations of mutant are
used.
[0181] FIG. 4 shows that different concentrations of mAb H2-8
reduce the IL-2 proliferation of TS1.beta.. A progressive shift of
the IL-2 titration curve is observed with increasing concentrations
of mAb H2-8. Inhibitory effects of mAb H2-8 are comparable to these
obtained with mAb 19B11, which was also found to inhibit the
proliferation of cells bearing high affinity IL-2R (MOREAU et al.
(1995b) Mol. Immunol. 32:1047-1056). Addition of both mAb H2-8 and
19B11 completely abolishes IL-2 proliferation even at a very high
dose of IL-2.
[0182] As controls FIG. 4 shows that mAb 11B11 (specific for mouse
IL-4) does not affect the IL-2-dependent proliferation of
TS1.beta.. IL-9-induced proliferation of TS1.beta. is also not
affected by either H2-8 or 19B11.
Example 6
IL-2 Binding Assay and Inhibition
[0183] The IL-2 binding assay was performed as already described
(MOREAU et al. (1995b) Mol. Immunol. 32:1047-1056).
.sup.125I-labelled IL-2 binding to different cell lines was first
studied. Inhibition experiments were performed at concentration of
.sup.125I-labelled IL-2 giving between 50 to 70% maximum binding.
The effects of the different muteins was analyzed after 1 hr
preincubation at 4.degree. C. followed by incubation with
.sup.125I-labelled IL-2 (3 hr at 4.degree. C.). In each experiments
non-specific binding was determined. The data were expressed as %
inhibitory capacity of the different mutein versus wild type
protein.
Example 7
Physico-Chemical Properties of Peptide IP130 (Cvtokine, 1997)
[0184] The amino terminal peptide of IL-2 including aa 1 to 30 has
a molecular weight of 3422.
[0185] The circular dichroism studies performed with IP130
indicates that at 20.degree. C., in phosphate buffer (20 mM, pH
7.2), 50% of the residues are in an a helix configuration.
[0186] The quaternary structure of peptide IP130 was also studied
by sedimentation--diffusion equilibrium. At concentration above
5.times.10.sup.6M, most of the molecules are in a tetrameric form
(in equilibrium with an octomeric complex).
[0187] The aminoacid sequence 1 to 30 shows 7 leucines and 2
isoleucines among the first 20 residues. The periodicity of these
aa as well as the above results suggest a structural model for
IP130 that would comprise 4 peptides organized in 4 .alpha.
helices. In this model leucines and isoleucines side chains appear
on the same face. This face is hydrophobic and four of these faces
would build an hydrophobic core inacessible to water. At high
concentration peptide 1-10 tend to dimerize and this would explain
the formation of octameric peptides.
[0188] The binding of IP130 to soluble IL-2R.beta. chain was
studied. Soluble IL-212.0 has been found to be dimeric in solution.
From the results, a structural model has been proposed: the complex
would include four IP130 peptides and two IL-2R.beta. chains
((IP130).sub.4/(IL-2R.beta.).sub.2).
Example 8
Biological Properties of IP130
[0189] Studies were performed either with a murine cell line
transfected by human IL-2R.beta. gene (TS1.beta.) or with an IL-2
dependant human leukemic cell line (Kit 225 from Dr. T. HORI).
[0190] IP130 stimulates the proliferation of TS1.beta. in the
absence of IL-2. In the presence of IL-2 a strong synergy is
observed with the peptide. Both activities are obtained at
comparable concentrations (IC-50.apprxeq..mu.M).
[0191] IP130 acts only on cell lines expressing human IL-2R.beta..
This is in agreement with previous studies showing that murine
IL-2R.beta. does not bind IL-2 (CHASTAGNER et al. 1996, Eur. J.
immunol. 26:201-6). Consequently, classical murine cell lines
(C30-1, CTLL, HT-2, . . . ) usually used to assay IL-2 activity
remains insensitive to IP130 effects. Furthermore anti-human
IL-2R.beta. blocking mAb neutralizes the effects of IP130.
[0192] Alone the peptide induces the phosphorylation of proteins on
Kit 225 cell line. On the pattern of phosphorylated proteins, the
kinase Shc is easily recognized. After specific immunoprecipitation
and blotting with mAb 4G10 (anti-Ptyr), phosphorylated IL-2R.beta.
is identified on lysates from kit 225 cell line stimulated by
IP130. c-myc induction which depends on IL-2R.beta. phosphorylation
is also observed after IP130 stimulation. STAT-3 and STAT-5 are not
activated after IP130 stimulation since IL-2R.gamma. is not
involved in IP130 interaction with Kit 225.
Example 9
Immunological Properties of IP130 and Use as Therapeutic Agent
[0193] IL-2R.beta. chain is constitutively expressed by human NK
cells from all donors studied (DAVID et al., Blood 91:165-172,
1998). Monocytes only express IL-2R.gamma. chain. Other lymphocytes
do not express neither IL-2R.beta. nor IL-2R.alpha. nor
IL-2R.gamma. (DAVID et al. 1998).
[0194] IP130 induces NK cells to enter into the cell cycle.
[0195] IP130 has been also tested on the generation of LAK in
vitro. IP130 induces LAK activity as tested on K562 targets.
[0196] mAb H2-8 was isolated after immunization with IL-2 peptide
1-30. It recognizes both IL-2 and peptide 1-30, but not the shorter
peptides covering the same region (FIG. 2). This result suggests
that mAb H2-8 recognizes a conformational epitope on the N-terminal
region of IL-2, and that this epitope is mimicked by the 1-30
peptide. Indeed, circular dichroism measurements reveal a
significant fraction of .alpha.-helical structure for the 1-30
peptide. Furthermore, H2-8 can bind to peptide 1-30 even in the
presence of mAb 19B11 (which recognizes a linear epitope within the
non-helical part of the peptide 1-30), but does not recognize IL-2
mutants at position 20 (in the center of .alpha.-helix A) as
determined by Western blot analysis or ELISA (FIG. 3 and other data
not shown). The antibody also inhibits the bioactivity of IL-2 on
TS1.beta. cells (FIG. 4), whose proliferation is strictly dependent
on the expression of the human IL-2R.beta. chain. Taken together,
these results demonstrate that mAb H2-8 recognizes an epitope
around Asp20 of IL-2 a region that directly influences the
interaction of the cytokine with IL-2R.beta..
[0197] Having now fully described the invention, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the invention as set forth herein.
Sequence CWU 1
1
4193DNAHomo sapiens 1atggctccga cgagcagctc caccaagaaa acccagctcc
agctcgaaca cctgctgctg 60gacctgcaga tgatcctgaa cggtatcaac aac
93231PRTHomo sapiens 2Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr
Gln Leu Gln Leu Glu1 5 10 15His Leu Leu Leu Asp Leu Gln Met Ile Leu
Asn Gly Ile Asn Asn 20 25 30390DNAHomo sapiens 3gctccgacga
gcagctccac caagaaaacc cagctccagc tcgaacacct gctgctggac 60ctgcagatga
tcctgaacgg tatcaacaac 90430PRTHomo sapiens 4Ala Pro Thr Ser Ser Ser
Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu
Gln Met Ile Leu Asn Gly Ile Asn Asn 20 25 30
* * * * *