U.S. patent number 5,599,905 [Application Number 08/094,669] was granted by the patent office on 1997-02-04 for interleukin-4 receptors.
This patent grant is currently assigned to Immunex Corporation. Invention is credited to M. Patricia Beckmann, David J. Cosman, Rejean Idzerda, Carl J. March, Bruce Mosley, Linda Park.
United States Patent |
5,599,905 |
Mosley , et al. |
February 4, 1997 |
Interleukin-4 receptors
Abstract
Mammalian Interleukin-4 receptor proteins, DNAs and expression
vectors encoding mammalian IL-4 receptors, and processes for
producing mammalian IL-4 receptors as products of cell culture, are
disclosed. A method for suppressing an IL-4-dependent immune or
inflammatory response in a mammal, including a human, by
administering an effective amount of soluble IL-4 receptor (sIL-4R)
and a suitable diluent or carrier.
Inventors: |
Mosley; Bruce (Seattle, WA),
Cosman; David J. (Seattle, WA), Park; Linda (Seattle,
WA), Beckmann; M. Patricia (Pouslbo, WA), March; Carl
J. (Seattle, WA), Idzerda; Rejean (Seattle, WA) |
Assignee: |
Immunex Corporation (Seattle,
WA)
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Family
ID: |
27500837 |
Appl.
No.: |
08/094,669 |
Filed: |
July 20, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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480694 |
Feb 14, 1990 |
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370924 |
Jun 23, 1989 |
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326156 |
Mar 20, 1989 |
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319438 |
Mar 2, 1989 |
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265047 |
Oct 19, 1988 |
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Current U.S.
Class: |
530/350;
435/69.1; 536/23.5; 424/85.2; 530/351 |
Current CPC
Class: |
C07K
16/2866 (20130101); A61P 37/00 (20180101); A61P
37/08 (20180101); G01N 33/6869 (20130101); C07K
14/7155 (20130101); Y10S 514/886 (20130101); A61K
38/00 (20130101); Y10S 514/885 (20130101); Y10S
514/826 (20130101) |
Current International
Class: |
C07K
16/28 (20060101); C07K 16/18 (20060101); G01N
33/68 (20060101); C07K 14/435 (20060101); C07K
14/715 (20060101); A61K 38/00 (20060101); C07K
014/705 (); C12N 015/12 () |
Field of
Search: |
;530/350,351 ;435/69.1
;536/23.5 ;424/85.2 ;514/2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO87/02990 |
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May 1987 |
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WO |
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WO89/09621 |
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Oct 1989 |
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WO |
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WO91/03555 |
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Mar 1991 |
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WO |
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WO93/11234 |
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Jun 1993 |
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WO |
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Other References
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Ohara et al, Nature, 325, 1987, pp. 537-540. .
Cabrillat et al, Biochem. Biophys. Res. Comm 149, 1987, pp.
995-1001. .
Nakajima et al, J. Immunol 139, 1987, pp. 774-779. .
Lowenthal et al, J Immunol 140, 1988, pp. 456-464. .
Sims et al, Science 241, 1988, pp. 585-589. .
Yamasaki et al Science 241, 1988, pp. 825-828. .
Chester and Hawkins, "Clinical issues in antibody design", Tibtech
13:294-300, 1995. .
Paul, William E., Ed., Fundamental Immunology, Raven Press, New
York, 1993, p. 826. .
Ohara and Paul, "Receptors for B-cell stimulatory factor-1
expressed on cells of haematopoietic lineage", Nature 325:537-540,
1987. .
Park et al., "Characterization of the high-affinity cell-surface
receptor for murine B-cell-stimulating factor 1", Proc. Nat. Acad.
Sci. USA, 84:1669-1673, 1987. .
Park et al., "Characterization of the Human B Cell Stimulatory
Factor 1 Receptor", J. Exp. Med., 166:476-488, 1987. .
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Stimulatory Factor 1 (BSFI): Presence of Functional Receptors on
CBA/N Splenic B Cells", J. Immunol. 139:774-779, 1987. .
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Interleukin 4 (BSF1)", J. Immunol. 140:456-464, 1988. .
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Expression on Immature (Lyt-2.sup.- /L3T4.sup.-) Thymocytes", J.
Immunol. 140:474-478, 1988. .
Park et al., "Interleukin-4 Binds to Murine Fibroblasts: Receptor
Characterization and Induction of Colony Stimulating Activity
Production", UCLA Symposia Abstracts, J. Cell Biochem. Suppl. 12A,
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Dower et al., "Interleukin Receptors", ISI Atlas of Science:
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cDNA Inserts in Mammalian Cells", Mol. Cell. Biol. 3:280-289, 1983.
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Aruffo and Seed, "Molecular cloning of a CD28 cDNA by a
high-effciency COS cell expression system", Proc. Natl. Acad. Sci.
USA 84:8573-8577, 1987. .
Yamasaki et al., "Cloning and Expression of the Human Interleukin-6
(BSF-2/IFN.beta. 2) Receptor", Science 241:825-828, 1988. .
Sims et al., "cDNA Expression Cloning of the IL-1 Receptor, a
Member of the Immunoglobulin Superfamily", Science 241:585-589,
1988. .
De Maagd et al., "The human thymus microenvironment: heterogeneity
detected by monoclonal anti-epithelial cell antibodies", Immunology
54:745-754, 1985. .
Larche et al., "A novel T-lymphocyte molecule that may function in
the induction of self-tolerance and MHC-restriction within the
human thymic microenviroment", Immunology 64:101-105, 1988. .
Larche et al., "Functional evidence for a monoclonal antibody that
binds to the human IL-4 receptor", Immunology 65:617-622, 1988
(Dec. 1988). .
Jabaari et al., British J. Cancer 59 (6) 910-914, 1989. .
Salari et al., Biochem. J. 262:897-908, 1989. .
Urdal et al., "Studies on Hematopoietic Growth Factor Receptors
Using Human Recombinant IL-3, GM-CSF, G-CSF, M-CSF, IL-1 and IL-4",
Behring Inst. Mitt. 83:27-39, Aug. 1988. .
Harada et al., "Expression cloning of a cDNA encoding the murine
interleukin 4 receptor based on ligand binding", Proc. Natl. Acad.
Sci. USA 87:857-861, 1990. .
Mosley et al., "The Murine Interleukin-4 Receptor: Molecular
Cloning and Characterization of Secreted and Membrane Bound Forms",
Cell 59:335-348, Oct. 1989. .
Finkelman et al., "IL-4 Is Required to Generate and Sustain In Vivo
IgE Responses", J. Immunol. 141:2335-2341, 1988. .
Finkelman et al., "Suppression of in vivo polyclonal IgE responses
by monoclonal antibody to the lymphokine B-cell stimulatory factor
1", Proc. Natl. Acad. Sci. USA 83:9675-9678, 1986. .
Snapper et al., "Differential Regulation of IgG1 and IgE Synthesis
by Interleukin 4", J. Exp. Med. 167:183-196, 1988. .
Finkelman et al., "T Help Requirements for the Generation of an In
Vivo IgE Response: A Late Acting Form of T Cell Help Other Than
IL-4 Is Required for IgE but not for IgG1 Production", J. Immunol.
142:403-408, 1989. .
Galizzi et al., "Purification of a 130-kDA T Cell Glycoprotein That
Binds Human Interleukin 4 with High Affinity", J. Biol. Chem.
265:439-444, 1990. .
Galizzi et al., "Internalization of Human Interleukin 4 and
Transient Down-regulation of Its Receptor in the C23-inducible
Jijoye Cells", J. Biol. Chem. 264:6984-6989, 1989..
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Primary Examiner: Walsh; Stephen G.
Assistant Examiner: Ulm; John D.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a divisional of application Ser. No. 07/480,694, filed Feb.
14, 1990, currently pending, which is a continuation-in-part of
application Ser. No. 07/370,924, filed Jun. 23, 1989, now
abandoned, which is a continuation-in-part of Ser. No. 07/326,156,
filed Mar. 20, 1989, now abandoned, which is a continuation-in-part
of Ser. No. 07/319,438, filed Mar. 2, 1989, now abandoned, which is
a continuation-in-part of Ser. No. 07/265,047, filed Oct. 31, 1988,
now abandoned.
Claims
We claim:
1. A substantially homogeneous IL-4R protein capable of binding
IL-4, wherein said protein is a human IL-4R comprising the amino
acid sequence of amino acids 1-800 of FIGS. 4A-4C.
2. A substantially homogeneous IL-4R protein capable of binding
IL-4, wherein said protein is a soluble human IL-4R protein
comprising an amino acid sequence selected from the group
consisting of amino acids 1-197, 1-198, and 1-207 of FIG. 4A.
3. A substantially homogeneous soluble human IL-4R protein having
the amino acid sequence presented as amino acids 1-207 of FIG.
4A.
4. A pharmaceutical composition comprising soluble IL-4R protein
according to claim 3 in admixture with a suitable diluent,
excipient, or carrier.
5. A purified soluble human IL-4R protein comprising modification
selected from the group consisting of:
(a) alteration of N-glycosylation site(s);
(b) alteration of KEX2 protease processing site(s); and
(c) conservative amino acid substitution(s);
wherein said IL-4 R has an amino acid sequence that is otherwise
identical to the amino acid sequence of residues 1-207 of FIG. 4A,
and
wherein said soluble IL-4R protein is capable of binding IL-4.
6. A pharmaceutical composition comprising a soluble IL-4R protein
according to claim 5 in admixture with a suitable diluent,
excipient, or carrier.
7. A purified IL-4 receptor (IL-4R) capable of binding IL-4,
wherein said IL-4R has an amino acid sequence selected from the
group consisting of:
a) amino acids 1-785 of FIGS. 2A-2C;
b) amino acids 1-800 of FIGS. 4A-4C;
c) amino acids 1-208 of FIG. 2A;
d) amino acids 1-207 of FIG. 4A;
e) amino acids 1-800 of FIGS. 4A-4C, with the proviso that the
residue at position 50 is valine rather than isoleucine;
f) amino acids 1-207 of FIGS. 4A-4C, with the proviso that the
residue at position 50 is valine rather than isoleucine; and
g) an amino acid sequence that includes conservative amino acid
substitutions in a sequence of (a), (b), (c), (d), (e), or (f),
wherein the sequence containing said substitutions is capable of
binding IL-4.
8. A purified soluble human IL-4R protein having the sequence of
amino acids 1-x of FIG. 4A, wherein x represents an integer from
193 through 207.
9. Isolated IL-4receptor (IL-4R) protein capable of binding IL-4,
wherein said I-4R protein comprises an amino acid sequence selected
from the group consisting of:
a) amino acids 1-785 of FIGS. 2A-2C;
b) amino acids 1-800 of FIGS. 4A-4C;
c) amono acids 1-208 of FIG. 2A;
d) amino acids 1-197 of FIG. 4A;
e) amino acids 1-198 of FIG. 4A;
f) amino acids 1-207 of FIG. 4A;
g) amino acids 1-49 and 51-800 of FIGS. 4A-4C, wherein the residue
at position 50 is valine; and
h) amino acids 1-49 and 51-207 of FIG. 4A, wherein the residue at
position 50 is valine.
10. Isolated IL-4R protein according to claim 9, wherein said
protein has a sequence as set forth at amino acid positions 1-800
of FIGS. 4A-4C.
11. Isolated soluble IL-4R protein according to claim 9, wherein
said protein has a sequence selected from the group consisting of
sequences set forth at amino acid positions 1-197 of FIG. 4A, 1-198
of FIG. 4A and 1-207 of FIG. 4A.
12. Isolated IL-4R protein according to claim 9, wherein said
protein has a sequence as set forth at amino acid positions 1-785
of FIG. 2A-2C.
13. Isolated soluble IL-4R protein according to claim 9, wherein
said protein has a sequence as set forth at amino acid positions
1-208 of FIG. 2A.
14. Isolated IL-4R protein according to claim 9, wherein said
protein has a sequence as set forth at amino acid positions 1-49
and 51-800 of FIGS. 4A-4C and position 50 is valine.
15. Isolated soluble IL-4R protein according to claim 9, wherein
said protein has a sequence as set forth at amino acid positions
1-49 and 51-207 of FIG. 4A and position 50 is valine.
16. Isolated interleukin-4 receptor (IL-4R) protein obtainable from
a recombinant expression system comprising a recombinant expression
vector, wherein said recombinant expression vector comprises a DNA
having a sequence selected from the group of sequences consisting
of (i) nucleotides 1-2355 of FIGS. 2A-2C and (ii) nucleotides
1-2400 of FIGS. 4A-4C.
17. Isolated interleukin-4 receptor (IL-4R) protein capable of
binding interleukin-4, wherein said IL-4R is obtainable from a
recombinant expression system comprising a recombinant expression
vector, wherein said recombinant expression vector comprises
nucleotides -75 to 621 of FIG. 4A.
18. Isolated IL-4R protein, wherein said protein is a fragment of
the IL-4R protein according to claim 17, wherein said fragment is
capable of binding IL-4.
19. A pharmaceutical composition comprising an IL-4R protein
according to claim 18, and a suitable diluent, excipient, or
carrier.
20. Isolated interleukin-4 receptor (IL-4R) protein capable of
binding interleukin-4, wherein said IL-4R has a sequence containing
conservative amino acid substitutions in an amino acid sequence
selected from the group consisting of:
(a) amino acid positions 1-785 of FIG. 2A-2C;
(b) amino acid positions 1-800 of FIGS. 4A-4C;
(c) amino acid positions 1-208 of FIG. 2A; and
(d) amino acid positions 1-207 of FIG. 4A.
21. Isolated interleukin-4 receptor (IL-4R) protein, wherein said
IL-4R has an amino acid sequence selected from the group consisting
of:
a) a fragment the amino acid sequence at positions -25 to 785 of
FIG. 2A-2C; and
b) a fragment of the amino acid sequence at positions -25 to 800 of
FIG. 4A-4C; wherein said fragment is capable of binding IL-4.
Description
BACKGROUND OF THE INVENTION
The present invention relates generally to cytokine receptors and,
more specifically, to Interleukin-4 receplors.
Interleukin-4 (IL-4, also known as B cell stimulating factor, or
BSF-1) was originally characterized by its ability to stimulate the
proliferation of B cells in response to low concentrations of
antibodies directed to surface immunoglobulin. More recently, IL-4
has been shown to possess a far broader spectrum of biological
activities, including growth co-stimulation of T cells, mast cells,
granulocytes, megakaryocytes, and erythrocytes. In addition, IL-4
stimulates the proliferation of several IL-2- and IL-3-dependent
cell lines, induces the expression of class II major
histocompatibility complex molecules on resting B cells, and
enhances the secretion of IgE and IgG1 isotypes by stimulated B
cells. Both murine and human IL-4 have been definitively
characterized by recombinant DNA technology and by purification to
homogeneity of the natural murine protein (Yokota et al., Proc.
Natl. Acad. Sci. USA 83:5894, 1986; Noma et al., Nature 319:640,
1986; and Grabstein et al., J. Exp. Med. 163:1405, 1986).
The biological activities of IL-4 are mediated by specific cell
surface receptors for IL-4 which are expressed on primary cells and
in vitro cell lines of mammalian origin. IL-4 binds to the
receptor, which then transduces a biological signal to various
immune effector cells. Purified IL-4 receptor (IL-4R) compositions
will therefore be useful in diagnostic assays for IL-4 or IL-4
receptor, and in raising antibodies to IL-4 receptor for use in
diagnosis or therapy. In addition, purified IL-4 receptor
compositions may be used directly in therapy to bind or scavenge
IL-4, providing a means for regulating the biological activities of
this cytokine.
Although IL-4 has been extensively characterized, little progress
has been made in characterizing its receptor. Numerous studies
documenting the existence of an IL-4 receptor on a wide range of
cell types have been published; however, structural
characterization has been limited to estimates of the molecular
weight of the protein as determined by SDS-PAGE analysis of
covalent complexes formed by chemical cross-linking between the
receptor and radiolabeled IL-4 molecules. Ohara et al. (Nature
325:537, 1987) and Park et al. (Proc. Natl. Acad. Sci. USA 84:1669,
1987) first established the presence of an IL-4 receptor using
radioiodinated recombinant murine IL-4 to bind a high affinity
receptor expressed in low numbers on B and T lymphocytes and a wide
range of cells of the hematopoietic lineage. By affinity
cross-linking .sup.125 I-IL-4 to IL-4R, Ohara et al. and Park et
al. identified receptor proteins having apparent molecular weights
of 60,000 and 75,000 daltons, respectively. It is possible that the
small receptor size observed on the murine cells represents a
proteolytically cleaved fragment of the native receptor. Subsequent
experiments by Park et al. (J. Exp. Med. 166:476, 1987) using
yeast-derived recombinant human IL-4 radiolabeled with .sup.125 I
showed that human IL-4 receptor is present not only on cell lines
of B, T, and hematopoietic cell lineages, but is also found on
human fibroblasts and cells of epithelial and endothelial origin.
IL-4 receptors have since been shown to be present on other cell
lines, including CBA/N splenic B cells (Nakajima et al., J.
Immunol. 139:774, 1987), Burkitt lymphoma Jijoye cells (Cabrillat
et al., Biochem. & Biophys. Res. Commun. 149:995, 1987), a wide
variety of hemopoietic and nonhemopoietic cells (Lowenthal et al.,
J. Immunol. 140:456, 1988), and murine Lyt-2.sup.- /L3T4.sup.-
thymocytes. More recently, Park et al. (UCLA Symposia, J. Cell
Biol., Suppl. 12A, 1988) reported that, in the presence of
sufficient protease inhibitors, .sup.125 I-IL-4-binding plasma
membrane receptors of 138-145 kDa could be identified on several
murine cell lines. Considerable controversy thus remains regarding
the actual size and structure of IL-4 receptors.
Further study of the structure and biological characteristics of
IL-4 receptors and the role played by IL-4 receptors in the
responses of various cell populations to IL-4 or other cytokine
stimulation, or of the methods of using IL-4 receptors effectively
in therapy, diagnosis, or assay, has not been possible because of
the difficulty in obtaining sufficient quantities of purified IL-4
receptor. No cell lines have previously been known to express high
levels of IL-4 receptors constitutively and continuously, and in
cell lines known to express detectable levels of IL-4 receptor, the
level of expression is generally limited to less than about 2000
receptors per cell. Thus, efforts to purify the IL-4 receptor
molecule for use in biochemical analysis or to clone and express
mammalian genes encoding IL-4 receptor have been impeded by lack of
purified receptor and a suitable source of receptor mRNA.
SUMMARY OF THE INVENTION
The present invention provides DNA sequences encoding mammalian
Interleukin-4 receptors (IL-4R) or subunits thereof. Preferably,
such DNA sequences are selected from the group consisting of: (a)
cDNA clones having a nucleotide sequence derived from the coding
region of a native IL-4R gene; (b) DNA sequences capable of
hybridization to the cDNA clones of (a) under moderately stringent
conditions and which encode biologically active IL-4R molecules;
and (c) DNA sequences which are degenerate, as a result of the
genetic code, to the DNA sequences defined in (a) and (b) and which
encode biologically active IL-4R molecules. The present invention
also provides recombinant expression vectors comprising the DNA
sequences defined above, recombinant IL-4R molecules produced using
the recombinant expression vectors, and processes for producing the
recombinant IL-4R molecules using the expression vectors.
The present invention also provides substantially homogeneous
protein compositions comprising mammalian IL-4R. The full length
murine molecule is a glycoprotein having a molecular weight of
about 130,000 to about 140,000 M.sub.r by SDS-PAGE. The apparent
binding affinity (K.sub.a) for COS cells transfected with murine
IL-4 receptor clones 16 and 18 from the CTLL 19.4 library is 1 to
8.times.10.sup.9 M.sup.-1. The K.sub.a for COS cells transfected
with murine IL-4 receptor clones 7B9-2 and 7B 9-4 from the murine
7B9 library is 2'10.sup.9 to 1.times.10.sup.10 M.sup.-1. The mature
murine IL-4 receptor molecule has an N-terminal amino add sequence
as follows: IKVLGEPTCFSDYIRTSTCEW.
The human IL-4R molecule is believed to have a molecular weight of
between about 110,000 and 150,000 M.sub.r and has an N-terminal
amino add sequence, predicted from the cDNA sequence and by analogy
to the biochemically determined N-terminal sequence of the mature
murine protein, as follows: MKVLQEPTCVSDYMSISTCEW.
The present invention also provides compositions for use in
therapy, diagnosis, assay of IL-4 receptor, or in raising
antibodies to IL-4 receptors, comprising effective quantities of
soluble receptor proteins prepared according to the foregoing
processes. Such soluble recombinant receptor molecules include
truncated proteins wherein regions of the receptor molecule not
required for IL-4 binding have been deleted.
The present invention also provides a method for suppressing IL-4
mediated immune or inflammatory responses. This method comprises
administering an effective quantity of soluble IL-4 receptor
(sIL-4R), in association with a pharmaceutical carrier, to a
mammal, including man. sIL-4R suppresses IL-4 dependent immune or
inflammatory responses, including, for example, B cell mediated
activities, such as B cell proliferation, immunoglobulin secretion,
and expression of Fc.epsilon.R which are induced by IL-4. sIL-4R
also suppresses cytotoxic T cell induction. Clinical applications
of sIL-4R include, for example, use in allergy therapy to
selectively suppress IgE synthesis and use in transplantation
therapy to prevent allograft rejection. sIL-4R is also useful to
suppress delayed-type hypersensitivity or contact hypersensitivity
reactions. sIL-4R is highly specific in its immunosuppressive
activity because it suppresses only IL-4 mediated immune
responses.
These and other aspects of the present invention will become
evident upon reference to the following detailed ddescription and
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows restriction maps of cDNA clones containing the coding
regions (denoted by a bar) of the murine and human IL-4R cDNAs. The
restriction sites EcoRI, PvuII, Hinc II and SstI are represented by
the letters R, P, H and S, respectively.
FIGS. 2A-C depict the cDNA sequence and the derived amino acid
sequence of the coding region of a murine IL-4 receptor, as derived
from clone 7B9-2 of the 7B9 library. The N-terminal isoleucine of
the mature protein is designated amino acid number 1. The coding
region of the full-length membrane-bound protein from clone 7B9-2
is defined by amino acids 1-785. The ATC codon specifying the
isoleucine residue constituting the mature N-terminus is underlined
at position 1 of the protein sequence; the putative transmembrane
region at amino acids 209-232 is also underlined. The sequences of
the ceding regions of clones 7B9-4 and clones CTLL-18 and CTLL-16
of the CTLL 19.4 library are identical to that of 7B9-2 except as
follows. The coding region of CTLL-16 encodes a membrane-bound
IL-4-binding receptor defined by amino acids -25 through 233
(including the putative 25 amino acid signal poptide sequence), but
is followed by a TAG terminator codon (not shown) which ends the
opon reading frame. The nucleic acid sequence indicates the
presence of a splice donor site at this position (indicated by an
arrow in FIG. 1) and a splice acceptor site near the 3' end
(indicated by a second arrow), suggesting that CTLL-16 was derived
from an unspliced mRNA intermediate. Clones 7B9-4 and CTLL-18
encode amino acids 23 through 199 and -25 through 199,
respectively. After amino acid 199, a 114-base pair insert
(identical in both clones and shown by an open box in FIG. 1)
introduces six new amino acids, followed by a termination codon.
This form of the receptor is soluble.
FIG. 3 is a schematic illustration of the mammalian high expression
plasmid pCAV/NOT, which is described in greater detail in Example
8.
FIGS. 4A-C depict the coding sequence of a human IL-4 receptor cDNA
from clone T22-8, which was obtained from a cDNA library derived
from the T cell line T22. The predicted N-terminal methionine of
the mature protein and the transmembrane region are underlined.
FIGS. 5A-B are a comparison of the predicted amino acid sequences
of human (top line). and murine (bottom line) IL-4 receptor cDNA
clones.
FIG. 6A-6B shows the inhibition of B cell proliferation with IL-4
(FIG. 6A) or IL-1 (FIG. 6 B) at various doses either alone
(.smallcircle.) or in the presence of sIL-4R (.quadrature.), sIL-1R
(.box-solid.) or anti-IL-4 antibody (.circle-solid.) as described
in Example 15.
FIG. 7A-7F shows the inhibition of B cell proliferation with fixed
concentrations of 10 (.circle-solid.), 1 (.quadrature.), 0.1
(.smallcircle.) or 0 (.DELTA.) ng/ml of IL-4 (FIGS. 7A-7C) and Il-1
(FIGS. 7D-7F) at various doses of sIL-4R (FIGS. 7A and 7D),
anti-IL-4 antibody (FIGS. 7B and 7E) and sIL-1R (FIGS. 7C and 7F)
as described in Example 15.
FIG. 8A-8C shows the inhibition of immunoglobulin class switching
with various doses of IL-4 and with sIL-4R (.quadrature.), sIL-1R
(.box-solid.), or anti-IL-4 antibody (.circle-solid.) or medium
control (.DELTA.) as described in Example 16.
FIG. 9A-9C shows the inhibition of IL-4-induced immunoglobulin
class switching with fixed concentration of IL-4 and various doses
of sIL-4R (.quadrature.), sIL-1R (.box-solid.), anti-IL-4 antibody
(.circle-solid.) or medium control (.DELTA.) as described in
Example 16.
FIG. 10A-10D shows the inhibition of MHC class II antigen
expression with (dashed line) or without (solid line) IL-4 in the
presence of medium control (FIG. 10A), sIL-4R (FIG. 10B), anti-IL-4
antibody (FIG. 10C) or sIL-1R (FIG. 10D) as described in Example
17.
FIG. 11A-11D shows the inhibition of Fc.epsilon.R (CD23) expression
with (dashed line) or without (solid line) IL-4 in the presence of
medium control (FIG. 11A), sIL-4R (FIG. 11B), anti-IL-4 antibody
(FIG. 11C) or sIL-1R (FIG. 11D) as described in Example 17.
FIG. 12 shows the inhibition of antigen specific polyclonal IgE
levels by sIL-4R as described in Example 18.
FIG. 13 shows the inhibition of antigen specific anti-TNP-KLH IgE
levels by sIL-4R as described in Example 18.
FIG. 14 shows that sIL-4R doses of 1, 5 or 25 ug on days -1, 0 and
+1, as described in Example 19, do not significantly inhibit
antigert specific anti-IgD IgE levels.
FIG. 15 shows the inhibition of contact hypersensitivity responses
to DNFB with sIL-4R as described in Example 20.
FIG. 16 shows the inhibition of delayed-type hypersensitivity
responses to SRBC with sIL-4R as described in Example 21.
FIG. 17 shows that sIL-4R, administered in Example 22, inhibits
alloantigen induced proliferation (measured by weight gain) of
lymph node cells. Mice are injected in the footpad with syngeneic
cells (from the same species) and in the contralateral footpad with
allogeneic cells (from another species). Large .DELTA. values
(e.g., for MSA or no reaction) indicate that the treatment did not
inhibit alloantigen induced lymphocyte proliferation and
inflammation. Small .DELTA. values indicate that the treatment
inhibited alloantigen induced lymphocyte proliferation and
inflammation.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
"Interleukin-4" and "IL-4" (also referred to as B cell stimulating
factor, or BSF-1) is a T cell-derived cytokine involved in the
regulation of immune and inflammatory responses. The biological
activities of IL-4 are mediated through binding to specific cell
surface receptors, referred to as "Interleukin-4 receptors", "IL-4
receptors" or simply "IL-4R". "IL-4 mediated" immune or
inflammatory responses include all biological responses which are
caused by the binding of IL-4 to a native IL-4 receptor (bound to a
cell surface) or which may be inhibited or suppressed by preventing
IL-4 from binding to a native IL-4 receptor. IL-4 mediated
biological responses include, for example. IL-4 induced
proliferation of antigen-primed B lymphocytes, expression of class
II major histocompatibility complex molecules on resting B cells,
secretion and expression of antibodies of the IgE and IgG1 isotype,
and regulation of the expression of the low affinity Fc receptor
for IgE (CD23) on lymphocytes and monocytes. Outside the B
lymphocyte compartment, IL-4 mediated biological responses include
the proliferation of a variety of primary cells and in vitro cell
lines, including factor-dependent T cell and mast cell lines,
murine and human T lymphocytes, thymocytes, and connective
tissue-type mast cells. IL-4 also induces both murine and human
cytotoxic T cells. Under certain conditions, IL-4 inhibits the
response of lymphold cells to IL-2. IL-4 acts on both murine and
human hematopoietic progenitor cells to either stimulate or
suppress in vitro formation of colonies in combination with known
colony stimulating factors. IL-4 also induces class I and class II
MHC molecules on more mature cells of the monocytic lineage,
enhances antigen presenting ability and promotes formation of giant
multinucleated cells. Specific clinical conditions which may be
mediated by IL-4 include, for example, graft rejection, graft
versus host disease, allergy, asthma and delayed-type
hypersensitivity responses.
As used herein, the terms "IL-4 receptor" or "IL-4R" refer to
proteins which bind intedeukin-4 (IL-4) molecules and, in their
native configuration as intact human plasma membrane proteins, play
a role in transducing the biological signal provided by IL-4 to a
cell. Intact receptor proteins generally include an extracellular
region which binds to a ligand, a hydrophobic transmembrane region
which causes the protein to be immobilized within the plasma
membrane lipid bilayer, and a cytoplasmic or intracellular region
which interacts with cytoplasmic proteins and/or chemicals to
deliver a biological signal to effector cells via a cascade of
chemical reactions within the cytoplasm of the cell. The
hydrophobic transmembrane region and a highly charged sequence of
amino acids in the cytoplasmic region immediately following the
transmembrane region cooperatively function to halt transport of
the IL-4 receptor across the plasma membrane.
"IL-4 receptors" are proteins having amino acid sequences which are
substantially similar to the native mammalian Interleukin-4
receptor amino acid sequences disclosed in FIGS. 2 and 4 or
fragments thereof, and which are biologically active as defined
below, in that they are capable of binding Interleukin-4 (IL-4)
molecules or transducing a biological signal initiated by an IL-4
molecule binding to a cell, or cross-reacting with anti-IL-4R
antibodies raised against IL-4R from natural (i.e., nonrecombinant)
sources. The native human IL-4 receptor molecule has an apparent
molecular weight by SDS-PAGE of about 140 kilodaltons (kDa). The
native murine IL-4 receptor molecule has an apparent molecular
weight by SDS-PAGE of about 140 kilodaltons (kDa). The terms "IL-4
receptor" or "IL-4R" include, but are not limited to, soluble IL-4
receptors, as defined below. Specific IL-4 receptor polypeptides
are designated herein by parenthetically indicating the amino acid
sequence numbers, followed by any additional amino acid sequences.
For Example, human IL-4R (1-207) refers to a human IL-4R protein
having the sequence of amino acids 1-207 as shown in FIG. 4A. Human
IL-4R (1-184) Pro Ser Asn Glu Asn refers to a human IL-4R protein
having the sequence of amino acids 1-184 as shown in FIG. 4A,
followed by the amino acid sequence Pro Ser Asn Glu Asn. As used
throughout the specification, the term "mature" means a protein
expressed in a form lacking a leader sequence as may be present in
full-length transcripts of a native gene. Various bioequivalent
protein and amino acid analogs are described in the detailed
description of the invention.
"Substantially similar" IL-4 receptors include those whose amino
acid or nucleic acid sequences vary from the native sequences by
one or more substitutions, deletions, or additions, the net effect
of which is to retain biological activity of the IL-4R protein. For
example, nucleic acid subunits and analogs are "substantially
similar" to the specific DNA sequences disclosed herein if: (a) the
DNA sequence is derived from the coding region of a native
mammalian IL-4R gene; (b) the DNA sequence is capable of
hybridization to DNA sequences of (a) under moderately stringent
conditions and which encode biologically active IL-4R molecules; or
DNA sequences which are degenerate as a result of the genetic code
to the DNA sequences defined in (a) or (b) and which encode
biologically active IL-4R molecules. Substantially similar analog
proteins will generally be greater than about 30 percent similar to
the corresponding sequence of the native IL-4R. Sequences having
lesser degrees of similarity but comparable biological activity are
considered to be equivalents. More preferably, the analog proteins
will be greater than about 80 percent similar to the corresponding
sequence of the native IL-4R, in which case they are defined as
being "substantially identical." In defining nucleic acid
sequences, all subject nucleic add sequences capable of encoding
substantially similar amino acid sequences are considered
substantially similar to a reference nucleic acid sequence. Percent
similarity may be determined, for example, by comparing sequence
information using the GAP computer program, version 6.0, available
from the University of Wisconsin Genetics Computer Group (UWGCG).
The GAP program utilizes the alignment method of Needleman and
Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and
Waterman (Adv. Appl Math. 2:482, 1981). Briefly, the GAP program
defines similarity as the number of aligned symbols (i.e.,
nucleotides or amino acids) which are similar, divided by the total
number of symbols in the shorter of the two sequences. The
preferred default parameters for the GAP program include: (1) a
unary comparison matrix (containing a value of 1 for identities and
0 for non-identities) for nucleotides, and the weighted comparison
matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as
described by Schwartz and Dayhoff, ed., Atlas of Protein Sequence
and Structure, National Biomedical Research Foundation, pp.
353-358, 1979; (2) a penalty of 3.0 for each gap and an additional
0.10 penalty for each symbol in each gap; and (3) no penalty for
end gaps.
"Soluble IL-4 receptor" or "sIL4-R" as used in the context of the
present invention refers to a protein, or a substantially
equivalent analog, having an amino acid sequence corresponding to
the extracellular region of native IL-4 receptors, for example,
polypeptides having the amino acid sequences substantially
equivalent to the sequences of amino acids 1-208 of FIG. 2, amino
acids 1-207 of FIG. 4 or to the amino acid sequences discussed in
Examples 8C and 9. Equivalent sIL-4Rs include polypeptides which
vary from the sequences shown in FIGS. 2 or 4 by one or more
substitutions, deletions, or additions, and which retain the
ability to bind IL-4 and inhibit the ability of IL-4 to transduce a
signal via cell surface bound IL-4 receptor proteins. Because
sIL-4R proteins are devoid of a transmembrane region, they are
secreted from the host cell in which they are produced. When
administered in therapeutic formulations, sIL-4R proteins circulate
in the body and bind to circulating IL-4 molecules, preventing
interaction of IL-4 with natural IL-4 receptors and inhibiting
transduction of IL-4 mediated biological signals, such as immune or
inflammatory responses. The ability of a polypoptide to inhibit
IL-4 signal transduction can be determined by transfecting cells
with recombinant IL-4 receptor DNAs to obtain recombinant receptor
expression. The cells are then contacted with IL-4 and the
resulting metabolic effects examined. If an effect results which is
attributable to the action of the ligand, then the recombinant
receptor has signal transducing activity. Exemplary procedures for
determining whether a polypeptide has signal transducing activity
are disclosed by Idzerda et al., J. Exp. Med., March 1990 in press,
Curtis et al., Proc. Natl. Acad. Sci. USA 86:3045 (1989), Prywes et
al., EMBO J. 5:2179 (1986) and Chou et al., J. Biol. Chem. 262:1842
(1987). Alternatively primary cells of cell lines which express an
endogenous IL-4 receptor and have a detectable biological response
to IL-4 could also be utilized. Such is the case with the CTLL-2
cell line which responds by short term proliferation in response to
either IL-2 or IL-4; the IL-4 induced proliferation can be blocked
specifically by the addition of exogenous soluble IL-4R (Mosley et
al., Cell 59:335 (1989). In addition, any one of the in vivo or in
vitro assays described in Examples 14-23 can be utilized to
determine whether a soluble IL-4R inhibits transduction of a
specific IL-4 mediated biological signal. The cloning, sequencing
and expression of full-length and soluble forms of the receptor for
murine IL-4 have recently been described by the applicants, Mosley
et al., Cell 59:335, 1989, which publication is incorporated herein
by reference.
"Recombinant," as used herein, means that a protein is derived from
recombinant (e.g., microbial or mammalian) expression systems.
"Microbial" refers to recombinant proteins made in bacterial or
fungal (e.g., yeast) expression systems. As a product, "recombinant
microbial" defines a protein produced in a microbial expression
system which is essentially free of native endogenous substances.
Protein expressed in most bacterial cultures, e.g., E. coli, will
be free of glycan. Protein expressed in yeast may have a
glycosylation pattem different from that expressed in mammalian
cells.
"Biologically active," as used throughout the specification as a
characteristic of IL-4 receptors, means that a particular molecule
shares sufficient amino acid sequence similarity with the
embodiments of the present invention disclosed herein to be capable
of binding detectable quantities of IL-4, transducing an IL-4
signal to a cell, for example, as a component of a hybrid receptor
construct, or cross-reacting with anti-IL-4R antibodies raised
against IL-4R from natural (i.e., nonrecombinant) sources.
Preferably, biologically active IL-4 receptors within the scope of
the present invention are capable of binding greater than 0.1
nmoles IL-4 per nmole receptor, and most preferably, greater than
0.5 nmole IL-4 per nmole receptor in standard binding assays (see
below).
"DNA sequence" refers to a DNA molecule, in the form of a separate
fragment or as a component of a larger DNA construct, which has
been derived from DNA isolated at least once in substantially pure
form, i.e., free of contaminating endogenous materials and in a
quantity or concentration enabling identification, manipulation,
and recovery of the sequence and its component nucleotide sequences
by standard biochemical methods, for example, using a cloning
vector. Such sequences are preferably provided in the form of an
open reading frame uninterrupted by internal nontranslated
sequences, or introns, which are typically present in eukaryotic
genes. Genomic DNA containing the relevant sequences could also be
used. Sequences of non-translated DNA may be present 5' or 3' from
the open reading frame, where the same do not interfere with
manipulation or expression of the coding regions.
"Nucleotide sequence" refers to a heteropolymer of
deoxydbonucleotides. DNA sequences encoding the proteins provided
by this invention can be assembled from cDNA fragments and short
oligonucleotide linkers, or from a series of oligonucleotides, to
provide a synthetic gene which is capable of being expressed in a
recombinant transcriptional unit.
"Recombinant expression vector" refers to a replicable DNA
construct used either to amplify or to express DNA which encodes
IL-4R and which includes a transcriptional unit comprising an
assembly of (1) a genetic element or elements having a regulatory
role in gene expression, for example, promoters or enhancers, (2) a
structural or coding sequence which is transcribed into mRNA and
translated into protein, and (3) appropriate transcription and
translation initiation and termination sequences. Structural
elements intended for use in yeast expression systems preferably
include a leader sequence enabling extracellular secretion of
translated protein by a host cell. Alternatively, where recombinant
protein is expressed without a leader or transport sequence, it may
include an N-terminal methionine residue. This residue may
optionally be subsequently cleaved from the expressed recombinant
protein to provide a final product.
"Recombinant microbial expression system" means a substantially
homogeneous monoculture of suitable host microorganisms, for
example, bacteria such as E. coli or yeast such as S. cerevisiae,
which have stably integrated a recombinant transcriptional unit
into chromosomal DNA or carry the recombinant transcriptional unit
as a component of a resident plasmid. Generally, cells constituting
the system are the progeny of a single ancestral transformant.
Recombinant expression systems as defined herein will express
heterologous protein upon induction of the regulatory elements
linked to the DNA sequence or synthetic gene to be expressed.
Proteins and Analogs
The present invention provides substantially homogeneous
recombinant mammalian IL-4R polypeptides substantially free of
contaminating endogenous materials and, optionally, without
associated native-pattern glycosylation. The native murine and
human IL-4 receptor molecules are recovered from cell lysates as
glycoproteins having an apparent molecular weight by SDS-PAGE of
about 130-145 kilodaltons (kDa). Mammalian IL-4R of the present
invention includes, by way of example, primate, human, murine,
canine, feline, bovine, ovine, equine and porcine IL-4R.
Derivatives of IL-4R within the scope of the invention also include
various structural forms of the primary protein which retain
biological activity. Due to the presence of ionizable amino and
carboxyl groups, for example, an IL-4R protein may be in the form
of acidic or basic salts, or in neutral form. Individual amino acid
residues may also be modified by oxidation or reduction.
The primary amino acid structure may be modified by forming
covalent or aggregative conjugates with other chemical moieties,
such as glycosyl groups, lipids, phosphate, acetyl groups and the
like, or by creating amino acid sequence mutants. Covalent
derivatives are prepared by linking particular functional groups to
IL-4R amino acid side chains or at the N- or C-termini. Other
derivatives of IL-4R within the scope of this invention include
covalent or aggregative conjugates of IL-4R or its fragments with
other proteins or polypeptides, such as by synthesis in recombinant
culture as N-terminal or C-terminal fusions. For example, the
conjugated peptide may be a signal (or leader) polypeptide sequence
at the N-terminal region of the protein which co-translationally or
post-translationally directs transfer of the protein from its site
of synthesis to its site of function inside or outside of the cell
membrane or wall (e.g., the yeast G-factor leader). IL-4R protein
fusions can comprise peptides added to facilitate purification or
identification of IL-4R (e.g., poly-His). Specific examples of a
poly-HIS fusion construct that is biologically active are soluble
human IL-4R (1-207) His His and soluble human IL-4R (1-207) His His
His His His His. The amino acid sequence of IL-4receptor can also
be linked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK)
(Hopp et al., Bio/Technology 6:1204, 1988.) The latter sequence is
highly antigenic and provides an epitope reversibly bound by a
specific monoclonal antibody, enabling rapid assay and facile
purification of expressed recombinant protein. This sequence is
also specifically cleaved by bovine mucosal enterokinase at the
residue immediately following the Asp-Lys pairing. Fusion proteins
capped with this peptide may also be resistant to intracellular
degradation in E. coli. A specific example of such a peptide is
soluble human IL-4R (1-207) Asp Tyr Lys Asp Asp Asp Asp Lys.
IL-4R derivatives may also be used as immunogens, reagents in
receptor-based immunoassays, or as binding agents for affinity
purification procedures of IL-4 or other binding ligands. IL-4R
derivatives may also be obtained by cross-linking agents, such as
M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at
cysteine and lysine residues. IL-4R proteins may also be covalently
bound through reactive side groups to various insoluble substrates,
such as cyanogen bromide-activated, bisoxirane-activated,
carbonyldiimidazole-activated or tosyl-activated agarose
structures, or by adsorbing to polyolefin surfaces (with or without
glutaraldehyde cross-linking). Once bound to a substrate, IL-4R may
be used to selectively bind (for purposes of assay or purification)
anti-IL-4R antibodies or IL-4.
The present invention also includes IL-4R with or without
associated native-pattern glycosylation. IL-4R expressed in yeast
or mammalian expression systems, e.g., COS-7 cells, may be similar
or significantly different in molecular weight and glycosylation
pattern than the native molecules, depending upon the expression
system. Expression of IL-4R DNAs in bacteda such as E. coli
provides non-glycosylated molecules. Functional mutant analogs of
mammalian IL-4R having inactivated N-glycosylation sites can be
produced by oligonucleotide synthesis and ligation or by
site-specific mutagenesis techniques. These analog proteins can be
produced in a homogeneous, reduced-carbohydrate form in good yield
using yeast expression systems. N-glycosylation sites in eukaryotic
proteins are characterized by the amino acid triplet Asn-A.sub.1
-Z, where A.sub.1 is any amino acid except Pro, and Z is Ser or
Thr. In this sequence, asparagine provides a side chain amino group
for covalent attachment of carbohydrate. Such a site can be
eliminated by substituting another amino acid for Asn or for
residue Z, deleting Asn or Z, or inserting a non-Z amino acid
between A.sub.1 and Z, or an amino acid other than Asn between Asn
and A.sub.1.
IL-4R derivatives may also be obtained by mutations of IL-4R or its
subunits. An IL-4R mutant, as referred to herein, is a pelypeptide
homologous to IL-4R but which has an amino acid sequence different
from native IL-4R because of a deletion, insertion or substitution.
Like most mammalian genes, mammalian IL-4 receptors are presumably
encoded by multi-exon genes. Alternative mRNA constructs which can
be attributed to different mRNA splicing events following
transcription, and which share large regions of identity or
similarity with the cDNAs claimed herein, are considered to be
within the scope of the present invention.
Bioequivalent analogs of IL-4R proteins may be constructed by, for
example, making various substitutions of residues or sequences or
deleting terminal or internal residues or sequences not needed for
biological activity. For example, cysteine residues can be deleted
or replaced with other amino acids to prevent formation of
incorrect intramolecular disulfide bridges upon renaturation. Other
approaches to mutagenesis involve modification of adjacent dibasic
amino acid residues to enhance expression in yeast systems in which
KEX2 protease activity is present. Generally, substitutions should
be made conservatively; i.e., the most preferred substitute amino
acids are those having physicochemical characteristics resembling
those of the residue to be replaced. Similarly, when a deletion or
insertion strategy is adopted, the potential effect of the deletion
or insertion on biological activity should be considered.
Subunits of IL-4R may be constructed by deleting terminal or
internal residues or sequences. Particularly preferred subunits
include those in which the transmembrane region and intracellular
domain of IL-4R are deleted or substituted with hydrophilic
residues to facilitate secretion of the receptor into the cell
culture medium. The resulting protein is a soluble IL-4R molecule
which may retain its ability to bind IL-4. Particular examples of
soluble IL-4R include polypeptides having substantial identity to
soluble murine IL-4R (1-208), soluble human IL-4R (1-207) and
soluble human IL-4R (1-198), all of which retain the biological
activity of soluble human IL-4R (1-207). Chimeric polypeptides
comprising fragments of human and murine IL-4R may also be
constructed, for example, IL-4R (1-197) Pro Ser Asn Glu Asn Leu,
which is comprised of the sequence of amino acids 1-197 of human
IL-4R followed by the N-terminal six amino acids of soluble murine
IL-4R clone 18. This polypeptide has been found to retain the
biological activity of soluble IL-4R (1-207).
Mutations in nucleotide sequences constructed for expression of
analog IL-4Rs must, of course, preserve the reading frame phase of
the coding sequences and preferably will not create complementary
regions that could hybridize to produce secondary mRNA structures,
such as loops or hairpins, which would adversely affect translation
of the receptor mRNA. Although a mutation site may be
predetermined, it is not necessary that the nature of the mutation
per se be predetermined. For example, in order to select for
optimum characteristics of mutants at a given site, random
mutagenesis may be conducted at the target codon and the expressed
IL-4R mutants screened for the desired activity.
Not all mutations in the nucleotide sequence which encodes IL-4R
will be expressed in the final product, for example, nucleotide
substitutions may be made to enhance expression, primarily to avoid
secondary structure bops in the transcribed mRNA (see EPA 75,444A,
incorporated herein by reference), or to provide codons that are
more readily translated by the selected host, e.g., the well-known
E. coli preference codons for E. coli expression.
Mutations can be introduced at particular loci by synthesizing
oligonucleotides containing a mutant sequence, flanked by
restriction sites enabling ligation to fragments of the native
sequence. Following ligation, the resulting reconstructed sequence
encodes an analog having the desired amino add insertion,
substitution, or deletion.
Alternatively, oligonucleotide-directed site-specific mutagenesis
procedures can be employed to provide an altered gene having
particular codons altered according to the substitution, deletion,
or insertion required. Exemplary methods of making the alterations
set forth above are disclosed by Walder et al. (Gene 42:133, 1986);
Bauer et al. (Gene 37:73, 1985); Craik (Bio Techniques, January
1985, 12-19); Smith et al. (Genetic Engineering: Principles and
Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and
4,737,462, which are incorporated by reference herein.
Expression of Recombinant IL4R
The present invention provides recombinant expression vectors which
include synthetic or cDNA-derived DNA fragments encoding mammalian
IL-4R or bidequivalent analogs operably linked to suitable
transcriptional or translational regulatory elements derived from
mammalian, microbial, viral or insect genes. Such regulatory
elements include a transcriptional promoter, an optional operator
sequence to control transcription, a sequence encoding suitable
mRNA ribosomal binding sites, and sequences which control the
termination of transcription and translation, as described in
detail below. The ability to replicate in a host, usually conferred
by an origin of replication, and a selection gene to facilitate
recognition of transformants may additionally be incorporated. DNA
regions are operably linked when they are functionally related to
each other. For example, DNA for a signal peptide (secretory
leader) is operably linked to DNA for a polypeptide if it is
expressed as a precursor which participates in the secretion of the
polypeptide; a promoter is operably linked to a coding sequence if
it controls the transcription of the sequence; or a ribosome
binding site is operably linked to a coding sequence if it is
positioned so as to permit translation. Generally, operably linked
means contiguous and, in the case of secretory leaders, contiguous
and in reading frame.
DNA sequences encoding mammalian IL-4 receptors which are to be
expressed in a microorganism will preferably contain no introns
that could prematurely terminate transcription of DNA into mRNA;
however, premature termination of transcription may be desirable,
for example, where it would result in mutants having advantageous
C-terminal truncations, for example, deletion of a transmembrane
region to yield a soluble receptor not bound to the cell membrane.
Due to code degeneracy, there can be considerable variation in
nucleotide sequences encoding the same amino acid sequence;
exemplary DNA embodiments are those corresponding to the nucleotide
sequences shown in the Figures. Other embodiments include sequences
capable of hybridizing to the sequences of the Figures under
moderately stringent conditions (50.degree. C., 2.times. SSC) and
other sequences hybridizing or degenerate to those described above,
which encode biologically active IL-4 receptor polypeptides.
DNA which codes for soluble IL-4R proteins may be isolated using
the cloning techniques described in the examples or may be made by
constructing cDNAs which encode only the extracellular domain of
IL-4 receptor (devoid of a transmembrane region) using well-known
methods of mutagenesis. Soluble forms of human IL-4 receptor are
not yet known to exist and must therefore be constructed from
isolated recombinant IL-4 receptor cDNAs. cDNAs which encode sIL-4R
may be constructed, for example, by truncating a cDNA encoding the
full length IL-4 receptor 5' of the transmembrane region, ligating
synthetic oligonucleotides to regenerate truncated portions of the
extracellular domain, if necessary, and providing a stop codon to
terminate transcription. DNA sequences encoding the soluble IL-4
receptor proteins can be assembled from cDNA fragments and short
oligonucleotide linkers, or from a series of oligonucleotides, to
provide a synthetic gene which is capable of being expressed in a
recombinanl transcriptional unit. Such DNA sequences are preferably
provided in the form of an open reading frame uninterrupted by
internal nontranslated sequences, or introns, which are typically
present in eukaryotic genes. Genomic DNA containing the relevant
sequences could also be used. Sequences of non-translated DNA may
be present 5' or 3' from the open reading frame, where the same do
not interfere with manipulation or expression of the coding
regions.
Transformed host cells are cells which have been transformed or
transfected with IL-4R vectors constructed using recombinant DNA
techniques. Transformed host cells ordinarily express IL-4R, but
host cells transformed for purposes of cloning or amplifying IL-4R
DNA do not need to express IL-4R. Expressed IL-4R will be deposited
in the cell membrane or secreted into the culture supernatant,
depending on the IL-4R DNA selected. Suitable host cells for
expression of mammalian IL-4R include prokaryotes, yeast or higher
eukaryotic cells under the control of appropriate promoters.
Prokaryotes include gram negative or gram positive organisms, for
example E. coil or bacilli. Higher eukaryotic cells include
established cell lines of mammalian odgin as described below.
Cell-free translation systems could also be employed to produce
mammalian IL-4R using RNAs derived from the DNA constructs of the
present invention. Appropriate cloning and expression vectors for
use with bacterial, fungal, yeast, and mammalian cellular hosts are
described by Pouwels et al. (Cloning Vectors: A Laboratory Manual,
Elsevier, New York, 1985), the relevant disclosure of which is
hereby incorporated by reference.
Prokaryotic expression hosts may be used for expression of IL-4Rs
that do not require extensive proteolytic and disulfide processing.
Prokaryotic expression vectors generally comprise one or more
phenotypic selectable markers, for example a gene encoding proteins
conferring antibiotic resistance or supplying an autotrophic
requirement, and an origin of replication recognized by the host to
ensure amplification within the host. Suitable prokaryotic hosts
for transformation include E. coli, Bacillus subfills, Salmonella
typhimurium, and various species within the genera Pseudornonas,
Streptomyces, and Staphylococcus, although others may also be
employed as a matter of choice.
Useful expression vectors for bacterial use can compdse a
selectable marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed. E. coil is
typically transformed using derivatives of pBR322, a plasmid
derived from an E. coil species (Bolivar et al., Gene 2:95, 1977).
pBR322 contains genes for ampicillin and tetracycline resistance
and thus provides simple means for identifying transformed
cells.
Promoters commonly used in recombinant microbial expression vectors
include the .beta.-lactamase (penicillinase) and lactose promoter
system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,
Nature 281:544, 1979), the tryptophan (trp) promoter system
(Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EPA 36,776) and
tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful
bacterial expression system employs the phage .lambda. P.sub.L
promoter and cl857ts thermolabile repressor. Plasmid vectors
available from the American Type Culture Collection which
incorporate derivatives of the .lambda. P.sub.L promoter include
plasmid pHUB2, resident in E. coil strain JMB9 (ATCC 37092) and
pPLc28, resident in E. coil RR1 (ATCC 53082).
Recombinant IL-4R proteins may also be expressed in yeast hosts,
preferably from the Saccharomyces genus, such as S. cerevisiae.
Yeast of other genera, such as Pichia or Kluyveromyces may also be
employed. Yeast vectors will generally contain an odgin of
replication from the 2.mu. yeast plasmid or an autonomously
replicating sequence (ARS), promoter, DNA encoding IL-4R, sequences
for polyadenylation and transcription termination and a selection
gene. Preferably, yeast vectors will include an origin of
replication and selectable marker permitting transformation of both
yeast and E. coli, e.g., the ampicillin resistance gene of E. coil
and S. cerevisiae trp1 gene, which provides a selection marker for
a mutant strain of yeast lacking the ability to grow in tryptophan,
and a promoter derived from a highly expressed yeast gene to induce
transcription of a structural sequence downstream. The presence of
the trp1 lesion in the yeast host cell genome then provides an
effective environment for detecting transformation by growth in the
absence of tryptophan.
Suitable promoter sequences in yeast vectors include the promoters
for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.
Bio. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et
al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem.
17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase. Suitable
vectors and promoters for use in yeast expression are further
described in Hitzeman, EPA 73,657.
Preferred yeast vectors can be assembled using DNA sequences from
pBR322 for selection and replication in E. coil (Amp.sup.r gene and
origin of replication) and yeast DNA sequences including a
glucose-repressible ADH2 promoter and .alpha.-factor secretion
leader. The ADH2 promoter has been described by Russell et al. (J.
Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724,
1982). The yeast .alpha.-factor leader, which directs secretion of
heterologous proteins, can be inserted between the promoter and the
structural gene to be expressed. See, e.g., Kurjan et al., Cell
30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA
81:5330, 1984. The leader sequence may be modified to contain, near
its 3' end, one or more useful restriction sites to facilitate
fusion of the leader sequence to foreign genes.
Suitable yeast transformation protocols are known to those of skill
in the art; an exemplary technique is described by Hinnen et al.,
Proc. Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp.sup.+
transformants in a selective medium consisting of 0.67% yeast
nitrogen base, 0.5% casamino acids, 2% glucose, 10 .mu.g/ml adenine
and 20 .mu.g/ml uracil.
Host strains transformed by vectors comprising the ADH2 promoter
may be grown for expression in a rich medium consisting of 1% yeast
extract, 2% peptone, and 1% glucose supplemented with 80 .mu.g/ml
adenine and 80 .mu.g/ml uracil. Derepression of the ADH2 promoter
occurs upon exhaustion of medium glucose. Crude yeast supernatants
are harvested by filtration and held at 4.degree. C. prior to
further purification.
Various mammalian or insect cell culture systems can be employed to
express recombinant protein. Baculovirus systems for production of
heterologous proteins in insect cells are reviewed by Luckow and
Summers, Bio/Technology 6:47 (1988). Examples of suitable mammalian
host cell lines include the COS-7 lines of monkey kidney cells,
described by Gluzman (Cell 23:175, 1981), and other cell lines
capable of expressing an appropriate vector including, for example,
L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell
lines. Mammalian expression vectors may comprise nontranscribed
elements such as an odgin of replication, a suitable promoter and
enhancer linked to the gene to be expressed, and other 5' or 3'
flanking nontranscribed sequences, and 5' or 3' nontranslated
sequences, such as necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites, and
transcriptional termination sequences.
The transcriptional and translational control sequences in
expression vectors to be used in transforming vertebrate cells may
be provided by viral sources. For example, commonly used promoters
and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus
40 (SV40), and human cytomegalovirus. DNA sequences derived from
the SV40 viral genome, for example, SV40 origin, early and late
promoter, enhancer, splice, and polyadenylation sites may be used
to provide the other genetic elements required for expression of a
heterologous DNA sequence. The early and late promoters are
particularly useful because both are obtained easily from the virus
as a fragment which also contains the SV40 viral odgin of
replication (Fiers et al., Nature 273:113, 1978). Smaller or larger
SV40 fragments may also be used, provided the approximately 250 bp
sequence extending from the Hind III site toward the Bgl I site
located in the viral origin of replication is included. Further,
mammalian genomic IL-4R promoter, control and/or signal sequences
may be utilized, provided such control sequences are compatible
with the host cell chosen. Additional details regarding the use of
mammalian high expression vectors to produce a recombinant
mammalian IL-4 receptor are provided in Example 8 below. Exemplary
vectors can be constructed as disclosed by Okayama and Berg (Mol.
Cell. Biol. 3:280, 1983).
A useful system for stable high level expression of mammalian
receptor cDNAs in C127 murine mammary epithelial cells can be
constructed substantially as described by Cosman et al. (Mol.
Immunol. 23:935, 1986).
A particularly preferred eukaryotic vector for expression of IL-4R
DNA is disclosed below in Example 2. This vector, referred to as
pCAV/NOT, was derived from the mammalian high expression vector
pDC201 and contains regulatory sequences from SV40, adenovirus-2,
and human cytomegalovirus. pCAV/NOT containing a human IL-7
receptor insert has been deposited with the American Type Culture
Collection (ATCC) under deposit accession number 68014.
Purification of IL-4 Receptors
Purified mammalian IL-4 receptors or analogs are prepared by
culturing suitable host/vector systems to express the recombinant
translation products of the DNAs of the present invention, and
purifying IL-4 receptor from the culture media or cell
extracts.
For example, supernatants from systems which secrete recombinant
protein into culture media can be first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. Following the
concentration step, the concentrate can be applied to a suitable
purification matrix. For example, a suitable affinity matrix can
comprise an IL-4 or lectin or antibody molecule bound to a suitable
support. Alternatively, an anion exchange resin can be employed,
for example, a matrix or substrate having pendant diethylaminoethyl
(DEAE) groups. The matrices can be acrylamide, agarose, dextran,
cellulose or other types commonly employed in protein purification.
Alternatively, a cation exchange step can be employed. Suitable
cation exchangers include various insoluble matrices comprising
sulfopropyl or carboxymethyl groups. Sulfopropyl groups are
preferred.
Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify an IL-4R composition. Some or all
of the foregoing purification steps, in various combinations, can
also be employed to provide a homogeneous recombinant protein.
Recombinant protein produced in bacterial culture is usually
isolated by initial extraction from cell pellets, followed by one
or more concentration, salting-out, aqueous ion exchange or size
exclusion chromatography steps. Finally, high performance liquid
chromatography (HPLC) can be employed for final purification steps.
Microbial cells employed in expression of recombinant mammalian
IL-4R can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
Fermentation of yeast which express mammalian IL-4R as a secreted
protein greatly simplifies purification. Secreted recombinant
protein resulting from a large-scale fermentation can be purified
by methods analogous to those disclosed by Urdal et al. (J.
Chromatog. 296:171, 1984). This reference describes two sequential,
reversed-phase HPLC steps for purification of recombinant human
IL-2 on a preparative HPLC column.
Human IL-4R synthesized in recombinant culture is characterized by
the presence of non-human cell components, including proteins, in
amounts and of a character which depend upon the purification steps
taken to recover human IL-4R from the culture. These components
ordinarily will be of yeast, prokaryotic or non-human higher
eukaryotic odgin and preferably are present in innocuous
contaminant quantities, on the order of less than about 1 percent
by weight. Further, recombinant cell culture enables the production
of IL-4R free of proteins which may be normally assodated with
IL-4R as it is found in nature in its spedes of origin, e.g. in
cells, cell exudates or body fluids.
Administration of Soluble IL-4 Receptor Compositions
The present invention provides methods of using therapeutic
compositions comprising an effective amount of soluble IL-4
receptor proteins and a suitable diluent and carrier, and methods
for suppressing IL-4-dependent immune responses in humans
comprising administering an effective amount of soluble IL-4
receptor protein.
For therapeutic use, purified soluble IL-4 receptor protein is
administered to a patient, preferably a human, for treatment in a
manner appropriate to the indication. Thus, for example, soluble
IL-4 receptor protein compositions administered to suppress immune
function can be given by bolus injection, continuous infusion,
sustained release from implants, or other suitable technique.
Typically, a soluble IL-4 receptor therapeutic agent will be
administered in the form of a composition comprising purified
protein in conjunction with physiologically acceptable carriers,
excipients or diluents. Such carriers will be nontoxic to
recipients at the dosages and concentrations employed. Ordinarily,
the preparation of such compositions entails combining the IL-4R
with buffers, antioxidants such as ascorbic acid, low molecular
weight (less than about 10 residues) polypeptides, proteins, amino
acids, carbohydrates including glucose, sucrose or dextrins,
chelating agents such as EDTA, glutathione and other stabilizers
and excipients. Neutral buffered saline or saline mixed with
conspecific serum albumin are exemplary appropriate diluents.
Preferably, product is formulated as a lyophilizate using
appropriate excipient solutions (e.g., sucrose) as diluents.
Appropriate dosages can be determined in trials; generally, soluble
IL-4 receptor dosages of from about 1 ng/kg/day to about 10
mg/kg/day, more preferably from about 500 ng/kg/day to about 5
mg/kg/day, and most preferably from about 5 ug/kg/day to about 2
mg/kg/day, are appropriate for inducing a biological effect. The
amount and frequency of administration will depend, of course, on
such factors as the nature and severity of the indication being
treated, the desired response, the condition of the patient, and so
forth.
Soluble IL-4R proteins are administered for the purpose of
inhibiting IL-4 dependent responses, such as suppressing immune
responses in a human. A variety of diseases or conditions are
caused by IL-4 dependent immune responses as determined by the
ability of sIL-4R to inhibit the response. Soluble IL-4R
compositions may be used, for example, to regulate the function of
B cells. Soluble IL-4R inhibits IL-4 dependent B cell proliferation
and isotype specific (IgGI and IgE) secretions. sIL-4R may
therefore be used to suppress IgE antibody formation in the
treatment of IgE-induced immediate hypersensitivity reactions, such
as allergic rhinitis (common hay fever), bronchial asthma, atopic
dermatitis and gastrointestinal food allergy.
sIL-4R compositions may also be used to regulate the function of T
cells. Although T cell dependent functions were formerly thought to
be mediated principally by IL-2, recent studies have shown that
under some circumstances T cell growth and proliferation can be
mediated by growth factors such as IL-4. Examples 20 through 23
below, for example, indicate that sIL-4R suppresses or inhibits
T-cell dependent responses to alloantigen. A variety of diseases or
conditions are caused by an immune response to alloantigen,
including allograft rejection and graft-versus-host reaction. In
alloantigen-induced immune responses, sIL-4R suppresses
lymphoproliferation and inflammation which result upon activation
of T cells. sIL-4R has therefore been shown to be potentiatty
effective in the clinical treatment of, for example, rejection of
aliografts (such as skin, kidney, head, lung liver and pancreas
transplants), and graft-versus-host reactions in patients who have
received bone marrow transplants.
sIL-4R may also be used in clinical treatment of autoimmune
dysfunctions, such as rheumatoid arthritis, diabetes, which are
dependent upon the activation of T cells against antigens not
recognized as being indigenous to the host.
Because of the primary role IL-2 plays in the proliferation and
differentiation of T cells, combination therapy using both IL-4 and
IL-2 may be used in the treatment of T cell dependent dysfunctions.
Use in connection with other soluble cytokine receptors, e.g., IL-1
receptor, is also contemplated.
The following examples are offered by way of illustration, and not
by way of limitation.
EXAMPLES
Example 1
Binding Assays for IL-4 Receptor
A. Radiolabeling of IL-4
Recombinant murine and human IL-4 were expressed in yeast and
purified to homogeneity as described by Park, et al., Proc. Natl.
Acad. Sci. USA 84:5267 (1987) and Park et al., J. Exp. Med. 166:476
(1987), respectively. The purified protein was radiolabeled using a
commercially available enzymobead radioiodination reagent (BioRad).
In this procedure 2.5 .mu.g rIL-4 in 50 .mu.l 0.2M sodium
phosphate, pH 7.2 are combined with 50 .mu.l enzymobead reagent, 2
MCi of sodium iodide in 20 .mu.l of 0.05M sodium phosphate pH 7.0
and 10 .mu.l of 2.5% b-D-glucose. After 10 min at 25.degree. C.,
sodium azide (10 .mu.l of 50 mM) and sodium metabisulfite (10 .mu.l
of 5 mg/ml) were added and incubation continued for 5 min. at
25.degree. C. The reaction mixture was fractionated by gel
filtration on a 2 ml bed volume of Sephadex.RTM. G-25 (Sigma)
equilibrated in Roswell Park Memodal Institute (RPMI) 1640 medium
containing 2.5% (w/v) bovine serum albumin (BSA), 0.2% (w/v) sodium
azide and 20 mM Hepes pH 7.4 (binding medium). The final pool of
.sup.125 I-IL-4 was diluted to a working stock solution of
2.times.10.sup.-8 M in binding medium and stored for up to one
month at 4.degree. C. without detectable loss of receptor binding
activity. The specific activity is routinely in the range of
1-2.times.10.sup.16 cpm/mmole IL-4.
B. Binding to Adherent Cells
Binding assays done with cells grown in suspension culture (i.e.,
CTLL and CTLL-19.4) were performed by a phthalate oil separation
method (Dower et al., J. Immunol. 132:751, 1984) essentially as
described by Park et al., J. Biol. Chem. 261:4177, 1986 and Park et
al., supra. Binding assays were also done on COS cells transfected
with a mammalian expression vector containing cDNA encoding an IL-4
receptor molecule. For Scatchard analysis of binding to adherent
cells, COS ceils were transfected with plasmid DNA by the method of
Luthman et al., Nucl. Acids. Res. 11:1295, 1983, and McCutchan et
al., J. Natl. Cancer Inst. 41:351, 1968. Eight hours following
transfection, cells were trypsinized, and reseeded in six well
plates (Costar, Cambridge, Mass.) at a density of 1.times.10.sup.4
COS-IL-4 receptor transfectants/well mixed with 5.times.10.sup.5
COS control transfected cells as carriers. Two days later
monolayers were assayed for .sup.125 I-IL-4 binding at 4.degree. C.
essentially by the method described by Park et al., J. Exp. Med.
166:476, 1987. Nonspecific binding of .sup.125 I-IL-4 was measured
in the presence of a 200-fold or greater molar excess of unlabeled
IL-4. Sodium azide (0.2%) was included in all binding assays to
inhibit internalization of .sup.125 I-IL-4 by cells at 37.degree.
C.
For analysis of inhibition of binding by soluble IL-4R, supematants
from COS cells transfected with recombinant IL-4R constructs were
harvested three days after transfection. Sedal two-fold dilutions
of conditioned media were pre-incubated with 3.times.10.sup.-10 M
.sup.125 I-Il-4 (having a specific activity of about
1.times.10.sup.16 cpm/mmol) for one hour at 37.degree. C. prior to
the addition of 2.times.10.sup.6 CTLL cells. Incubation was
continued for 30 minutes at 37.degree. C. prior to separation of
free and cell-bound murine .sup.125 I-IL-4.
C. Solid Phase Binding Assays
The ability of IL-4 receptor to be stably adsorbed to
nitrocellulose from detergent extracts of CTLL 19.4 cells yet
retain IL-4 binding activity provided a means of monitoring
purification. One ml aliquots of cell extracts (see Example 3),
IL-4 affinity column fractions (see Example 4) or other samples are
placed on dry BA85/21 nitrocellulose membranes (Schleicher and
Schuesurfacell, Keene, N.H.) and allowed to dry. The membranes are
incubated in tissue culture dishes for 30 minutes in Tris (0.05M)
buffered saline (0.15M) pH 7.5 containing 3% w/v BSA to block
nonspecific binding sites. The membrane is then covered with
4.times.10.sup.-11 M .sup.125 I-IL-4 in PBS+3% BSA with or without
a 200 fold molar excess of unlabeled IL-4 and incubated for 2 hr at
4.degree. C. with shaking. At the end of this time, the membranes
are washed 3 times in PBS, dded and placed on Kodak X-Omat.TM. AR
film for 18 hr at -70.degree. C.
Example 2
Selection of CTLL Cells with High IL-4 Receptor Expression by
Fluorescence Activated Cell Sorting (FACS)
The preferred cell line for obtaining high IL-4 receptor selection
is CTLL, a murine IL-2 dependent cytotoxic T cell line (ATCC TIB
214) which typically exhibits 2,000 to 5,000 IL-4 receptors per
cell and responds to IL-4 by short-term proliferation. To obtain
higher levels of IL-4 receptor expression, CTLL cells (parent
cells) were subjected to multiple rounds of fluorescence-activated
cell sorting with labeled IL-4. A highly fluorescent derivative of
IL-4 was derived by conjugating rmIL-4 fluorescein hydrazide to
periodate oxidized sugar moieties of IL-4 which was produced in
yeast as described by Park et al., Proc. Natl. Acad. Sci. USA
84:1669 (1987). The fluorescein-conjugated IL-4 was prepared by
combining aliquots of hyperglycosylated rmIL-4 (300 .mu.g in 300
.mu.l of 0.1M citrate-phosphate buffer, pH 5.5) with 30 .mu.l of 10
sodium m-periodiate (Sigma), freshly prepared in 0.1M
citrate-phosphate, pH 5.5 and the mixture incubated at 4.degree. C.
for 30 minutes in the dark. The reaction was quenched with 30 .mu.l
of 0.1M glycerol and dialyzed for 18 hours at 4.degree. C. against
0.1M citrate-phosphate pH 5.5. Following dialysis, a 1/10 volume of
100 mM 5-(((2-(carbohydrazino)methyl)thio)acetyl)-aminofluorescein
(Molecular Probes, Eugene Oreg.) dissolved in DMSO was added to the
sample and incubated at 25.degree. C. for 30 minutes. The
IL-4-fluorescein was then exhaustively dialyzed at 4.degree. C.
against PBS, pH 7.4 and protein concentration determined by amino
acid analysis. The final product was stored at 4.degree. C.
following the addition of 1% (w/v) BSA and sterile filtration.
CTLL cells (5.times.10.sup.6) were then incubated for 30 min at
37.degree. C. in 150 .mu.l PBS+1% BSA containing 1.times.10.sup.-9
M IL-4-fluorescein under sterile conditions. The mixture was then
chilled to 4.degree. C., washed once in a large volume of PBS+1%
BSA and sorted using an EPICS.RTM. C flow cytometer (Coulter
Instruments). The cells providing the highest level fluorescence
signal (top 1.0%) were collected in bulk and the population
expanded in liquid cell culture and subjected to additional rounds
of sorting as described below. Alternatively, for single cell
cloning, cells exhibiting a fluorescence signal in the top 1.0%
were sorted into 96 well tissue culture microtiter plates at 1 cell
per well.
Progress was monitored by doing binding assays with .sup.125 I-IL-4
following each round of FACS selection. Unsorted CTLL cells (CTLL
parent) typically exhibited 1000-2000 IL-4 receptors per cell. CTLL
cells were subjected to 19 rounds of FACS selection. The final CTLL
cells selected (CTLL-19) exhibited 5.times.10.sup.5 to
1.times.10.sup.6 IL-4 receptors per cell. At this point the CTLL-19
population was subjected to EPICS.RTM. C-assisted single cell
cloning and individual clonal populations were expanded and tested
for .sup.125 I-IL-4 binding. A single clone, designated CTLL-19.4,
exhibited 1.times.10.sup.6 IL-4 receptors per cell and was selected
for purification and cloning studies. While the calculated apparent
K.sub.a values are similar for the two lines, CTLL-19.4 expresses
approximately 400-fold more receptors on its surface than does the
CTLL parent.
Example 3
Detergent Extraction of CTLL Cells
CTLL 19.4 cells were maintained in RPMI 1640 containing 10% fetal
bovine serum, 50 U/ml penicillin, 50 ug/ml streptomycin and 10
ng/ml of recombinant human IL-2. Cells were grown to
5.times.10.sup.5 cells/ml in roller bottles, harvested by
centrifugation, washed twice in serum free DMEM and sedimented at
2000.times.g for 10 minutes to form a packed pellet (about
2.times.10.sup.8 cells/ml). To the pellet was added an equal volume
of PBS containing 1% Triton.RTM. X-100 and a cocktail of protease
inhibitors (2 mM phenylmethysulfonylfluoride, 10 .mu.M pepstatin,
10 .mu.M leupeptin, 2 mM o-phenanthroline and 2 mM EGTA). The cells
were mixed with the extraction buffer by vigorous vortexing and the
mixture incubated on ice for 20 minutes after which the mixture was
centrifuged at 12,000.times.g for 20 minutes at 8.degree. C. to
remove nuclei and other debds. The supernatant was either used
immediately or stored at -70.degree. C. until use.
Example 4
IL-4 Receptor Purification by IL-4 Affinity Chromatography
In order to obtain sufficient quantities of murine IL-4R to
determine its N-terminal sequence or to further characledze human
IL-4R, prosurfacelein obtained from the detergent extraction of
cells was further purified by affinity chromatography. Recombinant
murine or human IL-4 was coupled to Affigel.RTM.-10 (BioRad)
according to the manufacturer's suggestions. For example, to a
solution of IL-4 (3.4 mg/ml in 0.4 ml of 0.1M Hepes pH 7.4) was
added 1.0 ml of washed Affigel.RTM.-10. The solution was rocked
overnight at 4.degree. C. and an aliquot of the supernatant tested
for protein by a BioRad protein assay per the manufacturers
instructions using BSA as a standard. Greater than 95% of the
protein had coupled to the gel, suggesting that the column had a
final load of 1.3 mg IL-4 per ml gel. Glycine ethyl ester was added
to a final concentration of 0.05M to block any unreacted sites on
the gel. The gel was washed extensively with PBS-1% Triton.RTM.
followed by 0.1 Glycine-HCl, pH 3.0. A 0.8.times.4.0 cm column was
prepared with IL-4-coupled Affigel.RTM. prepared as described (4.0
ml bed volume) and washed with PBS containing 1% Triton.RTM. X-100
for purification of murine IL-4R. Alternatively, 50 .mu.l aliquots
of 20% suspension of IL-4-coupled Affigel.RTM. were incubated with
.sup.35 S-cysteine/methionine-labeled cell extracts for small-scale
affinity purifications and gel electrophoresis.
Aliquots (25 ml) of detergent extracted IL-4 receptor bearing CTLL
19.4 cells were slowly applied to the murine IL-4 affinity column
at 4.degree. C. (flow rate of 3.0 ml/hr). The column was then
washed sequentially with PBS containing 1% Triton.RTM. X-100, RIPA
buffer (0.05M Tris, 0.15M NaCl, 1% NP-40, 1% deoxycholate and 0.1%
SDS), PBS containing 0.1% Triton.RTM. X-100 and 10 mM ATP, and PBS
with 1% Triton.RTM. X-100 to remove all contaminating material
except the mIL-4R. The column was then eluted with pH 3.0 glycine
HCl buffer containing 0.1% Triton.RTM. X-100 to remove the IL-4R
and washed subsequently with PBS containing 0.1% Triton.RTM. X-100.
One milliliter fractions were collected for the elution and 2 ml
fractions collected during the wash. Immediately following elution,
samples were neutralized with 80 .mu.l of 1M Hepes, pH 7.4. The
presence of receptor in the fractions was detected by the solid
phase binding assay as described above, using .sup.125 l-labeled
IL-4. Aliquots were removed from each fraction for analysis by
SDS-PAGE and the remainder frozen at -70.degree. C. until use. For
SDS-PAGE, 40 .mu.l of each column fraction was added to 40 .mu.l of
2.times.SDS sample buffer (0.125M Tris HCl pH 6.8, 4% SDS, 20%
glycerol, 10% 2-mercaptoethanol). The samples were placed in a
boiling water bath for 3 minutes and 80 .mu.l aliquots applied to
sample wells of a 10% polyacrylamide gel which was set up and run
according to the method of Laemmli (Nature 227:680, 1970).
Following electrophoresis, gels were silver stained as previously
described by Urdal et al. (Proc. Natl. Acad. Sci. USA 81:6481,
1984).
Purification by the foregoing process permitted identification by
silver staining of polyacrylamide gels of two mIL-4R protein bands
averaging 45-55 kDa and 30-40 kDa that were present in fractions
exhibiting IL-4 binding activity. Experiments in which the cell
surface proteins of CTLL-19.4 cells were radiolabeled and .sup.125
l-labeled receptor was purified by affinity chromatography
suggested that these two proteins were expressed on the cell
surface. The ratio of the lower to higher molecular weight bands
increased upon storage of fractions at 4.degree. C., suggesting a
precursor product relationship, possibly due to slow proteolytic
degradation. The mIL-4 receptor protein purified by the foregoing
process remains capable of binding IL-4, both in solution and when
adsorbed to nitrocellulose.
Example 5
Seauencing of IL-4 Receptor Protein
CTLL 19.4 mIL-4 receptor containing fractions from the mIL-4
affinity column purification were prepared for amino terminal
protein sequence analysis by fractionating on an SDS-PAGE gel and
then transferred to a PVDF membrane. Prior to running the protein
fractions on polyacrylamide gels, it was first necessary to remove
residual detergent from the affinity purification process.
Fractions containing proteins bound to the mIL-4 affinity column
from three preparations were thawed and concentrated individually
in a speed vac under vacuum to a final volume of 1 ml. The
concentrated fractions were then adjusted to pH 2 by the addition
of 50% (v/v) TFA and injected onto a Brownlees RP-300
reversed-phase HPLC column (2.1.times.30 mm) equilibrated with 0.1%
(v/v) TFA in H.sub.2 O at a flow rate of 200 .mu.l/min running on a
Hewlett Packard Model 1090M HPLC. The column was washed with 0.1%
TFA in H.sub.2 O for 20 minutes post injection. The HPLC column
containing the bound protein was then developed with a gradient as
follows:
______________________________________ Time % Acetonitrile in 0.1%
TFA ______________________________________ 0 0 5 30 15 30 25 70 30
70 35 100 40 0 ______________________________________
1 ml fractions were collected every five minutes and analyzed for
the presence of protein by SDS PAGE followed by silver
staining.
Each fraction from the HPLC run was evaporated to dryness in a
speed vac and then resuspended in Laemmli reducing sample buffer,
prepared as described by Laemmli, U. K. Nature 227:680, 1970.
Samples were applied to a 5-20% gradient Laemmli SDS gel and run at
45 mA until the dye front reached the bottom of the gel. The gel
was then transferred to PVDF paper and stained as described by
Matsudaira, J. BioL Chem. 262:10035, 1987. Staining bands were
clearly identified in fractions from each of the three preparations
at approximately 30,000 to 40,000 M.sub.r.
The bands from the previous PVDF blotting were excised and
subjected to automated Edman degradation on an Applied Biosystems
Model 477A Protein Sequencer essentially as described by March et
al. (Nature 315:641, 1985), except that PTH amino acids were
automatically injected and analyzed on line with an Applied
Biosystems Model 120A HPLC using a gradient and detection system
supplied by the manufacturer. The following amino terminal sequence
was determined from the results of sequencing: NH.sub.2
-lle-Lys-Val-Leu-Gly-Glu-Pro-Thr-(Cys/Asn)-Phe-Ser-Asp-Tyr-Ile.
Position 9 was assigned as a cysteine or glycosylated asparagine
owing to the lack of an observable PTH-amino acid signal in the
cycle. The bands from the second preparation used for amino
terminal sequencing were treated with CNBr using the in situ
technique described by March et al. (Nature 315: 641, 1985) to
cleave the protein after intemal methionine residues. Sequencing of
the resulting cleavage products yielded the following data,
indicating that the CNBr cleaved the protein after two internal
methionine residues:
______________________________________ Cycle Residues Observed
______________________________________ 1 Val, Ser 2 Gly, Leu 3 Ile,
Val 4 Tyr, Ser 5 Arg, Tyr 6 Glu, Thr 7 Asp, Ala 8 Asn, Leu 9 Pro,
Val 10 Ala 11 Glu, Val 12 Phe, Gly 13 Ile, Asn 14 Val, Gln 15 Tyr,
Ile 16 Lys, Asn 17 Val, Thr 18 Thr, Gly
______________________________________
When compared with the protein sequences derived from clones 16 and
18 (see FIG. 2), the sequences matched as follows: ##STR1##
Identical matches were found for all positions of sequence 1 except
Asn(2) and sequence 2, except Arg at positions 8, 10, and 12, Ser
at position 13, and Leu at position 16. The above sequences
correspond to amino acid residues 137-154 and 169-187 of FIG.
2A.
In addition, the amino terminal sequence matched a sequence derived
from the clone with position 9 being defined as a Cys.
The above data support the conclusion that clones 16 and 18 are
derived from the message for the IL-4 receptor.
Example 6
Synthesis of Hybrid-Subtracted cDNA Probe
In order to screen a library for clones encoding a murine IL-4
receptor, a highly endched IL-4 receptor cDNA probe was obtained
using a subtractive hybridization strategy. Polyadenylated
(polyA.sup.+) mRNA was isolated from two similar cell lines, the
parent cell line CTLL (which expresses approximately 2,000
receptors per cell) and the soded cell line CTLL 19.4 (which
expresses 1.times.10.sup.6 receptors per cell). The mRNA content of
these two cell lines is expected to be identical except for the
relative level of IL-4 receptor mRNA. A radiolabeled
single-stranded cDNA preparation was then made from the mRNA of the
sorted cell line CTLL 19.4 by reverse transcription of
polyadenylated mRNA from CTLL 19.4 cells by a procedure similar to
that described by Maniatis et al., Molecular Cloning, A Laboratory
Manul (Cold Spdng Harbor Laboratory, New York, 1982). Briefly,
polyA.sup.+ mRNA was purified as described by March et al. (Nature
315:641-647, 1985) and copied into cDNA by reverse transcriptase
using oligo dT as a primer. To obtain a high level of .sup.32
P-labeling of the cDNA, 100 .mu.Ci of .sup.32 P-dCTP (s.a.=3000
Ci/mmol) was used in a 50 .mu.l reaction with non-radioactive dCTP
at 10 .mu.l. After reverse transcription at 42.degree. C. for 2
hours, EDTA was added to 20 mM and the RNA was hydrolyzed by adding
NaOH to 0.2M and incubating the cDNA mixture at 68.degree. C. for
20 minutes. The single-stranded cDNA was extracted with a
phenol/chloroform (50/50) mixture previously equilibrated with 10
mM Tris-Cl, 1 mM EDTA. The aqueous phase was removed to a clean
tube and made alkaline again by the addition of NaOH to 0.5M. The
cDNA was then size-fractionated by chromatography on a 6 ml
Sephadex.RTM. G50 column in 30 mM NaOH and 1 mM EDTA to remove
small molecular weight contaminants.
The resulting size-fractionated cDNA generated from the sorted CTLL
19.4 cells was then hybridized with an excess of mRNA from the
unsorted parental CTLL cells by ethanol-precipitating the cDNA from
CTLL 19.4 cells with 30 .mu.g of polyA.sup.+ mRNA isolated from
unsorted CTLL cells, resuspending in 16 .mu.l of 0.25M NaPO.sub.4,
pH 6.8, 0.2% SDS, 2 mM EDTA and incubating for 20 hours at
68.degree. C. The cDNAs from the sorted CTLL 19.4 cells that are
complementary to mRNAs from the unsorted CTLL cells form double
stranded cDNA/mRNA hybrids, which can then be separated from the
single stranded cDNA based on their different binding affinities on
hydroxyapatite. The mixture was diluted with 30 volumes of 0.02M
NaPO.sub.4, pH 6.8, bound to hydroxyapatite at room temperature,
and single-stranded cDNA was then eluted from the resin with 0.12M
NaPO.sub.4, pH 6.8, at 60.degree. C., as described by Sims et al.,
Nature 312:541, 1984. Phosphate buffer was then removed by
centrifugation over 2 ml Sephadex.RTM. G50 spin columns in water.
This hybrid subtraction procedure removes a majority of common
sequences between CTLL 19.4 and unsorted CTLL cells, and leaves a
single-stranded cDNA pool endched for radiolabeled IL-4 receptor
cDNA which can be used to probe a cDNA tbrary (as described
below).
Example 7
Synthesis of cDNA Library and Plaque Screening
A cDNA library was constructed from polyadenylated mRNA isolated
from CTLL 19.4 cells using standard techniques (Gubler, et al.,
Gene 25:263, 1983; Ausubel et al., eds., Current Protocols in
Molecular Biology, Vol. 1, 1987). After reverse transcription using
oligo dT as primer, the single-stranded cDNA was rendered
double-stranded with DNA polymerase I, blunt-ended with T4 DNA
polymerase, methylated with EcoR I methylase to protect EcoR I
cleavage sites within the cDNA, and ligated to EcoR I linkers. The
resulting constructs were digested with EcoR I to remove all but
one copy of the linkers at each end of the cDNA, and ligated to an
equimolar concentration of EcoR I cut and dephosphorylated
.lambda.ZAP.RTM. arms and the resulting ligation mix was packaged
in vitro (Gigapack.RTM.) according to the manufacturers
instructions. Other suitable methods and reagents for generating
cDNA libraries in .lambda. phage vectors are described by Huynh et
al., DNA Cloning Techniques: A Practical Approach, IRL Press,
Oxford (1984); Meissner et al., Proc. Natl. Acad. Sci. USA 84:4171
(1987), and Ausubel et al., supra. .lambda.ZAP.RTM. is a phage
.lambda. cloning vector similar to .lambda.gtl11 (U.S. Pat. No.
4,788,135) containing plasmid sequences from pUC19 (Norrander et
al., Gene 26:101, 1987), a polylinker site located in a lacZ gene
fragment, and an f1 phage origin of replication permitting recovery
of ssDNA when host bacteria are superinfected with f1 helper phage.
DNA is excised in the form of a plasmid comprising the foregoing
elements, designated Bluescript.RTM.. Gigapack.RTM. is a sonicated
E. coli extract used to package .lambda. phage DNA.
.lambda.ZAP.RTM., Bluescript.RTM., and Gigapack.RTM. are registered
trademarks of Stratagene, San Diego, Calif., USA.
The radiolabeled hybrid-subtracted cDNA from Example 6 was then
used as a probe to screen the cDNA library. The amplified library
was plated on BB4 cells at a density of 25,000 plaques on each of
20 150 mm plates and incubated overnight at 37.degree. C. All
manipulations of .lambda.ZAP.RTM. and excision of the
Bluescript.RTM. plasmid were as described by Short et al., (Nucl.
Acids Res. 16:7583, 1988) and Stratagene product literature.
Duplicate plaque lift filters were incubated with hybrid-subtracted
cDNA probes from Example 6 in hybridization buffer containing 50%
formamide, 5.times.SSC, 5.times.Denhardt's reagent and 10% dextran
sulfate at 42.degree. C. for 48 hours as described by Wahl et al.,
Proc. Natl. Acad. Sci. USA 76:3683, 1979. Filters were then washed
at 68.degree. C. in 0.2.times.SSC. Sixteen positive plaques were
purified for further analysis.
Bluescript.RTM. plasmids containing the cDNA inserts were excised
from the phage as described by the manufacturer and transformed
into E. coli Plasmid DNA was isolated from individual colonies,
digested with EcoR I to release the cDNA inserts and
electrophoresed on standard 1% agarose gels. Four duplicate gels
were blotted onto nylon filters to produce identical Southern blots
for analysis with various probes which were (1) radiolabeled cDNA
from unsorted CTLL cells, (2) radiolabeled cDNA from CTLL 19.4
sorted cells, (3) hybrid subtracted cDNA from CTLL 19.4 sorted
cells, and (4) hybrid subtracted cDNA from CTLL 19.4 sorted cells
after a second round of hybridization to poly A.sup.+ mRNA from an
IL-4 receptor negative mouse cell line (LBRM 33 1A5B6). These
probes were increasingly enriched for cDNA copies of mRNA specific
for the sorted cell line CTLL 19.4 . Of the 16 positive plaques
isolated from the library, four clones (11A, 14, 16 and 18) showed
a parallel increase in signal strength with enrichment of the
probe.
Restriction mapping (shown in FIG. 1) and DNA sequencing of the
isolated CTLL clones indicated the existence of at least two
distinct mRNA populalions. Both mRNA types have homosurfaceJogous
open reading frames over most of the coding region yet diverge at
the 3' end, thus encoding homologous proteins with different
COOH-terminal sequences. DNA sequence from inside the open reading
frames of both clones code for protein sequence that is identical
to protein sequence derived from sequencing of the purified IL-4
receptor described in more detail in Example 5. Clone 16 and clone
18 were used as the prototypes for these two distinct message
types. Clone 16 contains an open reading frame that encodes a
258-amino add polypeptide which includes amino acids -25 to 233 of
FIG. 2A. Clone 18 encodes a 230-amino acid soluble receptor
protein, the N-terminal 224 amino acids of which are identical to
the N-terminus of clone 16 but diverge 9 amino acids upstream of
the putative transmembrane region beginning with nucleotide number
598. This insertion acids the 3' nucleotide sequence
CCAAGTAATGAAAATCTG which encodes the C-terminal amino acids,
Pro-Ser-Asn-Glu-Asn-Leu, followed by a termination codon TGA. Both
clones were expressed in a mammalian expression system, as
described in Example 8.
Example 8
Expression of IL-4R in Mammalian Cells
A. Expression in COS-7 Cells.
A eukaryotic expression vector pCAV/NOT, shown in FIG. 3, was
derived from the mammalian high expression vector pDC2010 described
by Sims et al., Science 241:585, 1988). pDC201 is a derivative of
pMLSV, previously described by Cosman et al., Nature 312:768, 1984.
pCAV/NOT is designed to express cDNA sequences inserted at its
multiple cloning site (MCS) when transfected into mammalian cells
and includes the following components: SV40 (hatched box) contains
SV40 sequences from coordinates 5171-270 including the origin of
replication, enhancer sequences and early and late promoters. The
fragment is oriented so that the direction of transcription from
the early promoter is as shown by the arrow. CMV contains the
promoter and enhancer regions from human cytomegalovirus
(nucleotides -671 to +7 from the sequence published by Boshart et
al., Cell 41:521-530, 1985). The tripartite leader (stippled box)
contains the first exon and part of the intron between the first
and second exons of the adenovirus-2 tripartite leader, the second
exon and part of the third exon of the tripartite leader and a
multiple cloning site (MCS) containing sites for Xho I, Kpn I, Sma
I, Not I and Bgi II. pA (hatched box) contains SV40 sequences from
4127-4100 and 2770-2533 that include the polyadenylation and
termination signals for early transcription. Clockwise from pA are
adenovirus-2 sequences 10532-11156 containing the VAI and VAIl
genes (designated by a black bar), followed by pBR322 sequences
(solid line) from 4363-2486 and 1094-375 containing the ampicillin
resistance gene and origin of replication. The resulting expression
vector was designated pCAV/NOT.
Inserts in clone 16 and clone 18 were both released from
Bluescript.RTM. plasmid by digestion with Asp 718 and Not I. The
3.5 kb insert from clone 16 was then ligated directly into the
expression vector pCAV/NOT also cut at the Asp 718 and Not I sites
in the polylinker region. The insert from clone 18 was
bsurfacebnt-ended with T4 polymerass followed by ligation into the
vector pCAV/NOT cut with Sma I and dephosphorylated.
Plasmid DNA from both IL-4 receptor expression plasmids were used
to transfect a sub-confluent layer of monkey COS-7 cells using
DEAE-dextran followed by chloroquine treatment, as described by
Luthman et al. (Nucl. Acids Res. 11:1295, 1983) and McCutchan et
al. (J. Natl. Cancer Inst. 41:351, 1968). The cells were then grown
in culture for three days to permit transient expression of the
inserted sequences. After three days, cell culture supernatants and
the cell monolayers were assayed (as described in Example 1) and
IL-4 binding was confirmed.
B. Expression in CHO Cells.
IL-4R was also expressed in the mammalian CHO cell line by first
ligating an Asp718/Notl restriction fragment of clone 18 into the
pCAV/NOT vector as described in Example 8. The pCAV/NOT vector
containing the insert from clone 18 was then co-transfected using a
standard calcium phosphate method into CHO cells with the
dihydrofolate reductase (DHFR) cDNA selectable marker under the
control of the SV40 early promoter. The DHFR sequence enables
methotrexate selection for mammalian cells harboring the plasmid.
DHFR sequence amplification events in such cells were selected
using elevated methotrexate concentrations. In this way, the
contiguous DNA sequences are also amplified and thus enhanced
expression is achieved. Mass cell cultures of the transfectants
secreted active soluble IL-4R at approximately 100 ng/ml.
C. Express/on in HeLa Cells
IL-4R was expressed in the human HeLa-EBNA cell line 653-6, which
constitutively expresses EBV nuclear antigen-1 driven from the CMV
immediate-early enhancer/promoter. The expression vector used was
pHAV-EO-NEO, described by Dower et al., J. Immunol 142:4314, 1989),
a derivative of pDC201, which contains the EBV odgin of replication
and allows high level expression in the 653-6 cell line.
pHAV-EO-NEO is derived from pDC201 by replacing the adenovirus
major late promoter with synthetic sequences from HIV-1 extending
from -148 to +78 relative to the cap site of the viral mRNA, and
including the HIV-1 tat gene under the control of the SV-40 early
promoter. It also contains a Bgl II-Sma I fragment containing the
neomycin resistance gene of pSV2NEO (Southern & Berg, J. Mol.
App. Genet. 1:332, 1982) inserted into the Bgl II and Hpa I sites
and subcloning downstream of the Sal I cloning site. The resulting
vector permits selection of transfected cells for neomycin
resistance.
A 760 bp IL-4R fragmenf of clone C-18 from the CTLL 19.4 library
was released from the Bluescript.RTM. plasmid of the
.lambda.ZAP.RTM. cloning system (Stratagene, San Diego, Calif.,
USA) by digesting with EcoN I and Sst I restriction enzymes. This
fragment of clone C-18 corresponds to the nucleotide sequence set
forth in FIG. 2, with the addition of a 5' terminal nucleotide
sequence of TGCAGGCACCTTTTGTGTCCCCA, a TGA stop codon which follows
nucleotide 615 of FIG. 2A, and a 3' terminal nucleotide sequence of
CTGAGTGACCTTGGGGGCTGCGGTGGTGAGGAGAGCT. This fragment was then
blunt-ended using T4 polymerass and subcloned into the Sal I site
of pHAV-EO-NEO. The resulting plasmid was then transfected into the
653-6 cell line by a modified polybrene transfection method as
described by Dower et al. (J. Immunol. 142:4314, 1989) or by
electroporation with the exception that the cells were trypsinized
at 2 days post-transfection and split at a ratio of 1:8 into media
containing G418 (Gibco Co.) at a concentration of 1 mg/ml. Culture
media were changed twice weekly until neomycin-resistant colonies
were established. Colonies were then either picked individually
using cloning rings, or pooled together, to generate mass cultures.
These cell lines were maintained under drug selection at a G418
concentration of 250 ug/ml.
In an effort to select cell colonies expressing high levels of
soluble IL-4 receptor, a membrane filter assay was set up as
follows. High expressing cell clones were isolated by seeding 450
HeLa IL-4R transfectant cells in a 20 cm plate and allowing the
cells to grow for 10 days. Cell monolayers were then washed with a
Tris-buffered saline (TBS) solution and overlayed with a
nitrocellulose membrane. The overlay technique is essentially that
of McCracken and Brown, Biotechniques 2:82, 1984, except that the
nitrocesurfaceitulose was overlayed with small glass beads to
ensure that the membrane was kept flat. Cells were incubated for an
additional 24 hours to allow secretion and adsorption of soluble
IL-4 receptor to the nitrocellulose membrane. Finally the membrane
was removed, washed gently in TBS containing 1% bovine serum
albumin (BSA) for 30 minutes at room temperature, then incubated in
TBS with 3% BSA containing .sup.125 l-IL-4 (4.times.10.sup.11 M,
specific activity.about.1.times.10.sup.16 cpm/mmol) for two hours
at 4.degree. C. Membranes were then washed 3 times with PBS, dried
and exposed on Kodak X-omat.TM. film overnight at -70.degree.
C.
The developed film showed spots aligned with cells growing on the
plates in culture. Cell colonies aligned with the darkest spots on
the film (indicating the highest level of IL-4 receptor production
by cells) were harvested, and grown up in culture. When the
individual clones reached confluency, supernatants were tested for
the presence of soluble IL-4 receptor in a binding inhibition assay
as follows. Inhibition assays were performed by first incubating
various concentrations of unlabeled IL-4 or soluble IL-4 receptor
with 50 ul of .sup.125 -IL-4 (1.65.times.10.sup.-10 M) for thirty
minutes at 37.degree. C. in binding medium (RPMI with 2.5% BSA,
0.2% sodium azide, 0.2% M Hepes, pH 7.4). Subsequently
2.times.10.sup.6 CTLL-2 cells were added in 50 ul of binding medium
and the incubation continued for an additional thirty minutes. Free
and cell-bound .sup.125 l-IL-4 were then separated by the pthalate
oil separation method (Dower, S. K. et al., J. Immunol. 132:751,
1984). Percent specific inhibition was calculated using incubation
of .sup.125 l-IL-4 with excess unlabeled IL-4 (4.times.10.sup.-9 M)
as a positive control and 50 ul of binding medium as a negative
control. The binding data were calculated and graphed using RS/1
(BBN Software Products, Cambridge, Mass.) as previously described
(Dower, S. K. et al., J. Exp. Med 162:501, 1985).
Initial cultures of cells produced .about.100-600 ng/ml of soluble
IL-4R protein, and several cell clones isolated with the
membrane-trapping technique produced as much as 2-3 ug/ml of IL-4R
protein. These cell lines are currently maintained under drug
selection in G418 at a concentration of 250 ug/ml. The
establishment of a stable cell clone, HeLa E3C3, producing soluble
IL-4R enabled us to begin scaling up production and purification of
the soluble recombinant IL-4 receptor. For soluble IL-4 receptor
production, the HeLa E3C3 cells were seeded in expanded surface
area roller bottles (1:20 split ratio), and were grown for four
days with 250 ml of modified Dulbecco's Eagles medium, 5% fetal
bovine serum and 1% penicillin, streptomycin and glutamine. Roller
bottles were then switched to serum free media (300 ml/roller
bottle) for three days. Soluble IL-4 receptor protein was purified
from HeLa E3C3 culture supernatants by affinity chromatography on
IL-4 linked to Affigel-10. Recombinant murine IL-4 was coupled to
Affigel.RTM.-10 (BioRad) according to the manufacturer's
suggestions. Briefly, 1.0 ml of washed Affigel.RTM.-10 was added to
a solution of IL-4 (3.4 mg/ml in 0.4 ml of 0.1M Hepes pH 7.4). The
solution was rocked overnight at 4.degree. C. and an aliquot of the
supernatant tested for protein by a BioRad protein assay per the
manufacturers instructions using BSA as a standard. Greater than
95% of the protein had coupled to the gel, suggesting that the
column had a final load of 1.3 mg IL-4 per ml gel. Glycine ethyl
ester was added to a final concentration of 0.05M to block any
unreacted sites on the gel. The gel was washed extensively with
PBS-1% Triton.RTM. followed by 0.1 Glycine-HCl, pH 3.0. A
0.8.times.4.0 cm column was prepared with IL-4-coupled Affigel.RTM.
prepared as described (4.0 ml bed volume) and washed with PBS
containing 1% Triton.RTM. X-100 for purification of murine IL-4R.
Alternatively, 50 .mu.l aliquots of 20% suspension of IL-4-coupled
Affigel.RTM. were incubated with .sup.35
S-cysteine/methionine-labeled cell extracts for small-scale
affinity purifications and gel electrophoresis.
Aliquots (25 ml) of HeLa E3C3 culture supematants (containing
soluble IL-4 receptor) were slowly applied to the murine IL-4
affinity column at 4.degree. C. (flow rate of 3.0 ml/hr). The
column was then washed sequentially with PBS to remove all
contaminating matedal except the bound mIL-4R. The column was then
eluted with 0.01M acetic acid, 0.15M sodium chloride, pH 3.0 to
remove the IL-4R and washed subsequently with PBS. One ml fractions
were collected for the elution and 2 ml fractions collected during
the wash. Immediately following elution, samples were neutralized
with 80 ul of 1M Hepes, pH 7.4. The presence of receptor in the
fractions was detected by the inhibition binding assay described
above.
From 100 ml of HeLa E3C3 culture supernatant approximately 600 ug
of soluble IL-4 receptor protein was purified on a 4.0 ml affinity
column. Purified receptor consisted of three major bands ranging
from 30-39,000 daltons on SDS-PAGE. Heterogeneity in size of this
preparation is due to variability in protein glycosylation, as
treatment of the protein with N-glycanase to remove N-linked
carbohydrates reduces the size of the protein to .about.25,000
daltons on SDS-PAG E. In addition, amino acid sequencing confirmed
that the bands have the same N-terminal sequence. Pudty and protein
concentrations were also confirmed by amino add analysis.
Example 9
Expression of IL-4R in Yeast Cells
For expression of mIL-4R, a yeast expression vector derived from
plXY120 was constructed as follows. plXY120 is identical to
pY.alpha.HuGM (ATCC 53157), except that it contains no cDNA insert
and includes a polylinker/multiple cloning site with an Nco I site.
This vector includes DNA sequences from the following sources: (1)
a large Sph I (nucleotide 562) to EcoR I (nucleotide 4361) fragment
excised from plasmid pBR322 (ATCC 37017), including the origin of
replication and the ampicillin resistance marker for selection in
E. coli; (2) S. cerevisiae DNA including the TRP-1 marker, 2.mu.
origin of replication, ADH2 promoter; and (3) DNA encoding an 85
amino acid signal peptide derived from the gene encoding the
secreted peptide .alpha.-factor (See Kurjan et al., U.S. Pat. No.
4,546,082). An Asp 718 restriction site was introduced at position
237 in the G-factor signal peptide to facilitate fusion to
heterologous genes. This was achieved by changing the thymidine
residue at nucleotide 241 to a cytosine residue by
oligonucleotide-directed in vitro mutagenesis as described by
Craik, Bio Techniques, January 1985, pp. 12-19. A synthetic
oligonucleotide containing multiple cloning sites and having the
following sequence was inserted from the Asp718 site at amino acid
79 near the 3' end of the .alpha.-factor signal peptide to a Spel
site in the 2.mu. sequence: ##STR2## pBC120 also varies from
pY.alpha.HuGM by the presence of a 514 bp DNA fragment derived from
the single-stranded phage fl containing the odgin of replication
and intergenic region, which has been inserted at the Nru I site in
the pBR322 sequence. The presence of an f1 origin of replication
permits generation of single-stranded DNA copies of the vector when
transformed into appropriate strains of E. coli and superinfected
with bacteriophage f1, which facilitates DNA sequencing of the
vector and provides a basis for in vitro mutagenesis. To insert a
cDNA, plXY120 is digested with Asp 718 which cleaves near the 3'
end of the .alpha.-factor leader peptide (nucleotide 237) and, for
example, BamH I which cleaves in the polylinker. The large vector
fragment is then purified and ligated to a DNA fragment encoding
the protein to be expressed.
To create a secretion vector for expressing mIL-4R, a cDNA fragment
encoding mIL-4R was excised from the Bluescript.RTM. plasmid of
Example 8 by digestion with Ppum I and Bgl II to release an 831 bp
fragment from the Ppum I site (see FIGURE ) to an Bgl II site
located 3' to the open reading frame containing the mIL-4R sequence
minus the first two 5' codons encoding lie and Lys. plXY120 was
digested with Asp 718 near the 3' end of the .alpha.-factor leader
and BamH I. The vector fragment was ligated to the Ppum I/Bgl II
hIL-4R cDNA fragment and the following fragment created by
annealing a pair of synthetic oligonucleotides to recreate the last
6 amino acids of the .alpha.-factor leader and the first two amino
acids of mature mIL-4R. ##STR3## The oligonucleotide also included
a change from the nucleotide sequence TGG ATA to CTA GAT which
introduces a Xba I restriction site, without altering the encoded
amino add sequence.
The foregoing expression vector was then purified and employed to
transform a diploid yeast strain of S. cerevisiae (XV2181) by
standard techniques, such as those disclosed in EPA 1 65,654,
selecting for tryptophan prototrophs. The resulting transformants
were cultured for expression of a secreted mIL-4R protein. Cultures
to be assayed for biological activity were grown in 20-50 ml of YPD
medium (1% yeast extract, 2% peptone, 1% glucose) at 37.degree. C.
to a cell density of 1-5.times.10.sup.8 cells/ml. separate cells
from medium, cells were removed by centrifugation and the medium
filtered through a 0.45.mu. cellulose acetate filter prior to
assay. Supernatants produced by the transformed yeast strain, or
crude extracts prepared from disrupted yeast cells transformed the
plasmid, were assayed to verify expression of a biologically active
protein.
Example 10
Isolation of Full-Length and Truncated Forms of Murine IL-4
Receptor cDNAs from Unsorted 7B9 Cells
Polyadenylated RNA was isolated from 7B9 cells, an
antigen-dependent helper T cell clone derived from C57BL/6 mice,
and used to construct a cDNA library in .lambda.ZAP (Stratagene,
San Diego), as described in Example 7. The .lambda.ZAP library was
amplified once and a total of 300,000 plaques were screened as
described in Example 7, with the exception that the probe was a
randomly primed .sup.32 p-labeled 700 bp EcoR I fragment isolated
from CTLL 19.4 clone 16. Thirteen clones were isolated and
characterized by restriction analysis.
Nucleic acid sequence analysis of clone 7B9-2 revealed that it
contains a polyadenylated tail, a putative polyadenylation signal,
and an open reading frame of 810 amino acids (shown in FIG. 2), the
first 258 of which are identical to those encoded by CTLL 19.4
clone 16, including the 25 amino acid putative signal peptide
sequence. The 7B9-2 cDNA was subcloned into the eukaryotic
expression vector, pCAV/NOT, and the resulting plasmid was
transfected into COS-7 cells as described in Example 8. COS-7
transfectants were analyzed as set forth in Example 12.
A second cDNA form, similar to clone 18 in the CTLL 19.4 library,
was isolated from the 7B9 library and subjected to sequence
analysis. This cDNA, clone 7B9-4, is 376 bp shorter than clone
7B9-2 at the 5' end, and lacks the first 47 amino acids encoded by
7B9-2, but encodes the remaining N-terminal amino acids 23-199 (in
FIG. 2). At position 200, clone 7B9-4 (like clone 18 from CTLL
19.4) has a 114 bp insert which changes the amino acid sequence to
Pro Ser Asn Glu Asn Leu followed by a termination codon. The 114 bp
inserts, found in both clone 7B9-4 and CTLL 19.4 clone 18 are
identical in nucleic add sequence. The fact that this cDNA form,
which produces a secreted form of the IL-4 receptor when expressed
in COS-7 cells, was isolated from these two different cell lines
indicates that it is neither a cloning artifact nor a mutant form
peculiar to the sorted CTLL cells.
Example 11
Isolation of Human IL-4 Receptor cDNAs from PBL and T22 Libraries
by Cross-Species Hybridization
Polyadenylated RNA was isolated from pooled human peripheral blood
lymphocytes (PBL) that were obtained by standard Ficoll
purification and were cultured in IL-2 for six days followed by
stimulation with PMA and Con-A for eight hours. An oligo dT primed
cDNA library was constructed in .lambda.gt10 using techniques
described in example 7. A probe was produced by synthesizing an
unlabeled RNA transcript of the 7B9-4 cDNA insert using T7 RNA
pelymerase, followed by .sup.32 P-labeled cDNA synthesis with
reverse transcriptase using random primers (Boehringer-Mannheim).
This murine single-stranded cDNA probe was used to screen 50,000
plaques from the human cDNA library in 50% formamide/0.4M NaCl at
42.degree. C., followed by washing in 2.times.SSC at 55.degree. C.
Three positive plaques were purified, and the EcoR I inserts
subcloned into the Bluescript.RTM. plasmid vector. Nucleic acid
sequencing of a potion of clone PBL-1, a 3.4 kb cDNA, indicated the
clone was approximately 67% homologous to the corresponding
sequence of the murine IL-4 receptor. However, an insert of 68 bp,
containing a termination codon and bearing no homology to the mouse
IL-4 receptor clones, was found 45 amino acids downstream of the
predicted N-terminus of the mature protein, suggesting that clone
PBL-1 encodes a non-functional truncated form of the receptor. Nine
additional human PBL clones were obtained by screening the same
library (under stringent conditions) with a .sup.32 P-labeled
random-primed probe made from the clone PBL-1 (the 3.4 kb EcoR I
cDNA insert). Two of these clones, PBL-11 and PBL-5, span the 5'
region that contains the 68 bp insert in PBL-1, but lack the 68 bp
insert and do not extend fully 3', as evidenced by their size, thus
precluding functional analysis by mammalian expression. In order to
obtain a construct expressible in COS-7 cells, the 5' Not I-Hinc II
fragment of clones PBL-11 and PBL-5 were separately ligated to the
3' Hinc II-BamH I end of clone PBL-1, and subcloned into the
pCAV/NOT expression vector cut with Not I and Bgl II described in
Example 8. These chimeric human IL-4R cDNAs containing PBL-11/PBL-1
and PBL-5/PBL-1 DNA sequences have been termed clones A5 and B4,
respectively, as further described in Example 12. These constructs
were transfected into COS-7 cells, and assayed for IL-4 binding in
a plate binding assay substantially as described in Sims et al.
(Science 241:585, 1988). Both composite constructs encoded protein
which exhibited IL-4 binding activity. The nucleotide sequence and
predicted amino acid sequence of the composite A5 construct
correspond to the sequence information set forth in FIGS. 4A-4C,
with the exception that a GTC codon encodes the amino acid Val at
position 50, instead of lie. No other clones that were sequenced
contained this change. The consensus codon from clones PBL-1, PBL-5
and T22-8, however, is ATC and encodes lie.sup.50, as set forth in
FIG. 4A. The nucleotide and predicted amino acid sequence of the
composite B4 construct also shows that the 25 amino acid leader
sequence of PBL-11 is replaced with the sequence
Met-Gln-Lys-Asp-Ala-Arg-Arg-Glu-Gly-Asn.
Constructs expressing a soluble form of the human IL-4 receptor
were made by excising a 5'-terminal 0.8 kb Sma I-Dra III fragment
from PBL-5 and the corresponding 0.8 kb Asp718-Dra III fragment
from PBL-11, of which the Dra III overhangs were blunt-ended with
T4 polymerase. The PBL-5 and PBL-11 fragments were separately
subcloned into CAV/NOT cut with Sma I or Asp718 plus Sma I,
respectively; these are called soluble hIL-4R-5 and soluble
hIL-4R-11, respectively.
A second library made from a CD4+/CD8- human T cell clone, T22,
(Acres et al., J. Immunol. 138:2132, 1987) was screened (using
duplicate filters) with two different probes synthesized as
described above. The first probe was obtained from a 220 bp Pvu II
fragment from the 5' end of clone PBL-1 and the second probe was
obtained from a 300 bp Pvu II -EcoR I fragment from the 3' end of
clone PBL-1. Five additional cDNA clones were identified using
these two probes. Two of these clones span the 5' region containing
the 68 bp insert, but neither contain the insert. The third of
these clones, T22-8, was approximately 3.6 kb in size and contained
an open reading frame of 825 amino acids, including a 25 amino acid
leader sequence, a 207 amino add-mature extemal domain, a 24 amino
acid transmembrane region and a 569 amino acid cytoplasmic domain.
The sequence of clone T22-8 is set forth in FIGS. 4A-4C. FIGS.
5A-5B compare the predicted human IL-4R amino acid sequence with
the predicted murine IL-4R sequence and show approximately 53%
sequence identity between the two proteins.
Example 12
Analysis and Purification of IL-4 Receptor in COS Transfectants
Equilibrium binding studies were conducted for COS cells
transfected with murine IL-4 receptor clones 16 and 18 from the
CTLL 19.4 library. In all cases analysis of the data in the
Scatchard coordinate system (Scatchard, Ann. N.Y. Acad. Sci.
51:660-672, 1949) yielded a straight line, indicating a single
class of high-affinity receptors for murine IL-4. For COS pCAV-16
cells the calculated apparent K.sub.a was 3.6.times.10.sup.9
M.sup.-1 with 5.9.times.10.sup.5 specific binding sites per cell. A
similar apparent K.sub.a was calculated for COS pCAV-18 cells at
1.5.times.10.sup.9 M.sup.-1 but receptor number expressed at the
cell surface was 4.2.times.10.sup.4. Equilibrium binding studies
performed on COS cells transfected with IL-4R DNA clones isolated
from the 7B9 cell library also showed high affinity binding of the
receptor to IL-4. Specifically, studies using COS cells transfected
with pCAV-7B9-2 demonstrated that the full length murine IL-4
receptor bound .sup.125 l-IL-.sub.4 with an apparent K.sub.a of
about 1.4.times.10.sup.10 M.sup.-1 with 4.5.times.10.sup.4 specific
binding sites per cell. The apparent K.sub.a of CAV-7B9-4 IL-4R was
calculated to be about 1.7.times.10.sup.9 M.sup.-1. Although
absolute values for K.sub.a and binding sites per cell varied
between transfections, the binding affinities were generally
similar (1.times.10.sup.9 -1.times.10.sup.10 M.sup.-1) and matched
well with previously published affinity constants for IL-4
binding.
Inhibition of .sup.125 l-mIL-4 binding to CTLL cells by conditioned
media from COS cells transfected with plasmid pCAV, pCAV-18, or
pCAV-7B9-4 was used to determine if these cDNAs encoded functional
soluble receptor molecules. Approximately 1.5 .mu.l of COS pCAV-18
conditioned media in a final assay volume of 150 .mu.l gives
approximately 50% inhibition of .sup.125 l-IL-4 binding to the IL-4
receptor on CTLL cells. .sup.125 l-IL-4 receptor competing activity
is not detected in control pCAV transfected COS supernatants. From
quantitative analysis of the dilution of pCAV-18 supernatant
required to inhibit .sup.125 l-IL-4 binding by 50%, it is estimated
that approximately 60-100 ng/ml of soluble IL-4 receptor has been
secreted by COS cells when harvested three days after transfection.
Similar results were obtained utilizing supernatants from COS cells
transfected with pCAV-7B9-4.
Conditioned medium from COS cells transfected with pCAV-18 or
pCAV-7B9-4 (see Example 8) and grown in DMEM containing 3% FBS was
harvested three days after transfection. Supernatants were
centrifuged at 3,000 cpm for 10 minutes, and frozen until needed.
Two hundred ml of conditioned media was loaded onto a column
containing 4 ml of muIL-4 Affigel prepared as described above. The
column was washed extensively with PBS and IL-4 receptor eluted
with 0.1M glycine, 0.15M NaCl pH 3.0. Immediately following
elution, samples were neutralized with 80 .mu.l of 1M Hepes pH 7.4.
Samples were tested for their ability to inhibit binding of
.sup.125 l-muIL-4 to CTLL cells as set forth in Example 1B.
Additionally samples were tested for purity by analysis on SDS-PAGE
and silver staining as previously described. Alternative methods
for testing functional soluble receptor activity or IL-4 binding
inhibition include solid-phase binding assays, as described in
Example 1C, or other similar cell free assays which may utilize
either radio iodinated or colormetrically developed IL-4 binding,
such as RIA or ELISA. The protein analyzed by SDS-PAGE under
reducing conditions has a molecular weight of approximately 37,500,
and appears approximately 90% pure by silver stain analysis of
gels.
Purified recombinant soluble murine IL-4 receptor protein may also
be tested for its ability to inhibit IL-4 induced .sup.3
H-thymidine incorporation in CTLL cells. Pursuant to such methods,
soluble IL-4 receptor has been found to block IL-4 stimulated
proliferation, but does not affect IL-2 driven mitogenic
response.
Molecular weight estimates were performed on mIL-4 receptor clones
transfected into COS cells. Utilizing M2 monoclonal antibody
prepared against murine CTLL 19.4 cells (see Example 13), IL-4
receptor is immunoprecipitated from COS cells transfected with
CAV-16, CAV-7B9-2 and CAV-7B9-4 and labeled with .sup.35 S-cysteine
and .sup.35 S-methionine. Cell associated receptor from CAV-7B9-4
shows molecular weight heterogeneity ranging from 32-39 kDa.
Secreted CAV-7B9-4 receptor has molecular weight between 36 and 41
kDa. Cell associated receptor from CAV-16 transfected COS cells is
about 40-41 kDa. This is significantly smaller than molecular
weight estimations from crosslinking studies described by Park et
al., J. Exp. Med. 166:476, 1987; J. Cell. Biol., Suppl. 12A, 1988.
Immunoprecipitation of COS CAV-7B9-2 cell-associated receptor
showed a molecular weight of 130-140 kDa, similar to the estimates
of Park et al., J. Cell. Biol., Suppl. 12A, 1988, estimated to be
the full length IL-4 receptor. Similar molecular weight estimates
of cell-associated CAV-16 and CAV-7B9-2 IL-4 receptor have also
been made based on cross-linking .sup.125 IL-4 to COS cells
transfected with these cDNAs. Heterogeneity of molecular weight of
the individual clones can be partially attributed to glycosylation.
This data, together with DNA sequence analysis, suggests that the
7B9-2 cDNA encodes the full length cell-surface IL-4 receptor,
whereas both 7B9-4 and clone 18 represent soluble forms of murine
IL-4 receptor.
Receptor characterization studies were also done on COS cells
transfected with hIL-4R containing expression plasmids. The two
chimeric human IL-4R molecules A5 and B4 (defined in Example 11)
were transfected into COS cells and equilibrium binding studies
undertaken. The COS monkey cell itseft has receptors capable of
binding hIL-4; therefore the binding calculations performed on COS
cells transfected with and overexpressing hIL-4R cDNAs represent
background binding from endogenous monkey IL-4R molecules
subtracted from the total binding. COS cells transfected with
hIL-4R A5 had 5.3.times.10.sup.4 hIL-4 binding sites with a
calculated K.sub.a of 3.48.times.10.sup.9 M.sup.-1. Similarly, the
hIL-4R B4 expressed in COS cells bound .sup.125 I-hIL-4 with an
affinity of 3.94.times.10.sup.9 M.sup.-1 exhibiting
3.2.times.10.sup.4 receptors per cell.
Molecular weight estimates of human IL-4R expressed in COS cells
were also performed. COS cells transfected with clones A5 or B4 in
pCAV/NOT were labeled with .sup.35 S-cysteine/methionine and lysed.
Human IL-4R was affinity purified from the resulting lysates with
hIL-4-coupled Affigel.RTM. (as described in Example 4). The hIL-4R
A5 and B4 eluted from this affinity suppert migrated at about
140,000 daltons on SDS-PAGE, agreeing well with previous estimates
of hIIL-4R molecular weight by cross-linking (Park et al., J. Exp.
Med. 166:476, 1987), as well as with estimates of full-length
mIL-4R presented here.
Because no soluble human IL-4R cDNA has thus far been found
occurring naturally, as was the case for the murine receptor
(clones 18 and 7B9-4), a truncated form was constructed as
described in Example 11. Following expression in COS cells,
supernatants were harvested three days after transfection with
soluble hIL-4R-11 and soluble hIL-4R-5 and tested for inhibition of
.sup.125 I-hIL-4 binding to the human B cell line Raji.
Supernatants from two of the soluble hIL-4R-11 and one of the
soluble hIL-4R-5 transfected plates contained 29-149 ng/ml of IL-4R
competing activity into the medium. In addition, the truncated
protein could be detected in .sup.35 S-methionine/cysteine-labeled
COS cell transfectants by affinity purification on hIL-4-coupled
Affigel.RTM. as approximately a 44 kDa protein by SDS-PAGE.
Example 13
Preparation of Monoclonal Antibodies to IL-4R
Preparations of purified recombinant IL-4 receptor, for example,
human or murine IL-4 receptor, transfected COS cells expressing
high levels of IL-4 receptor or CTLL 19.4 cells are employed to
generate monoclonal antibodies against IL-4 receptor using
conventional techniques, such as those disclosed in U.S. Pat. No.
4,411,993. Such antibodies are likely to be useful in interfering
with IL-4 binding to IL-4 receptors, for example, in ameliorating
toxic or other undesired effects of IL-4.
To immunize rats, IL-4 receptor bearing CTLL 19.4 cells were used
as immunogen emulsified in complete Freund's adjuvant and injected
in amounts ranging from 10-100 .mu.l subcutaneously into Lewis
rats. Three weeks later, the immunized animals were boosted with
additional immunogen emulsified in incomplete Freund's adjuvant and
boosted every three weeks thereafter. Serum samples are
periodically taken by retro-orbital bleeding or tail-tip excision
for testing by dot-blot assay, ELISA (enzyme-linked immunosorbent
assay), or inhibition of binding of .sup.125 I-IL-4 to extracts of
CTLL cells (as described in Example 1). Other assay procedures are
also suitable. Following detection of an appropriate antibody
titer, positive animals were given a final intravenous injection of
antigen in saline. Three to four days later, the animals were
sacrificed, splenocytes harvested, and fused to the murine myeloma
cell line AG8653. Hybridoma cell lines generated by this procedure
were plated in multiple microtiter plates in a HAT selective medium
(hypoxanthine, aminopterin, and thymidine) to inhibit proliferation
of non-fused cells, myeloma hybrids, and spleen cell hybrids.
Hybridoma clones thus generated were screened for reactivity with
IL-4 receptor. Initial screening of hybridoma supernatants utilized
an antibody capture and binding of partially purified .sup.125
I-mIL-4 receptor. Two of over 400 hybridomas screened were positive
by this method. These two monoclonal antibodies, M1 and M2, were
tested by a modified antibody capture to detect blocking antibody.
Only M1 was able to inhibit .sup.125 I-mIL-4 binding to intact CTLL
cells. Both antibodies are capable of immunoprecipitating native
mIL-4R protein from CTLL cells or COS-7 cells transfected with
IL-4R clones labelled with .sup.35 S-cysteine/methionine. M1 and M2
were then injected into the peritoneal cavities of nude mice to
produce ascites containing high concentrations (>1 mg/ml) of
anti-IL-4R monoclonal antibody. The resulting monoclonal antibody
was purified by ammonium sulfate precipitation followed by gel
exclusion chromatography, and/or affinity chromatography based on
binding of antibody to Protein G.
A series of experiments (Examples 14-19) was conducted to show that
sIL-4R inhibits IL-4 mediated B cell growth, differentiation and
function in vitro. Each of these experiments utilized soluble IL-4R
produced as described in Example 8C. Example 14 shows that sIL-4R
inhibits the proliferation of stimulated B cells. Examples 15, 16
and 17 show, respectively, that sIL-4R inhibits IL-4 dependent B
cell differentiation as measured by induction of IgG1 and IgE
secretion by LPS activated B cells, down regulation of IgG3
secretion by LPS activated B cells, and increased Ia and
Fc.epsilon.R (CD23) expression, respectively. In the following
experiments, the activity of sIL-4R is compared with sIL-1R to show
that the inhibitory effects of the soluble receptors are specific
in that sIL-4R has no effect on IL-1 induced B cell activity and
sIL-1R has no effect on IL-4 activity, thus demonstrating two
independent pathways of B cell activation directed by IL-1 and
IL-4.
Example 14
Inhibition of IL-4 Binding to B cells in vitro by Soluble IL-4R
Untreated B cells express low, but detectable levels of IL-4
receptors. Upon stimulation with the B cell mitogen LPS, these
cells show enhanced cell surface IL-4 receptor expression. The
following experiments were conducted to show that sIL-4R inhibits
radiolabeled IL-4 binding to LPS activated B lymphocytes.
B lymphocytes were first purified from spleens of 8 to 12 week-old
C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me. and Simonson,
Gilroy, Calif.) as described by Grabstein, et al., J. Exp. Med.
163:1405, 1986. Briefly, murine splenocytes were depleted of T
cells by incubation in a cocktail containing T24 rat anti-Thy 1 mAb
(Dennert et al., J. Immunol. 131:2445 (1983), GK1.5 rat anti-mouse
L3T4 mAb (Dialynas et al., Cell. Immunol. 53:350, (1980), rabbit
anti-mouse thymocyte serum (absorbed with C57BL/6 liver and bone
marrow), and rabbit complement (Pel-Freeze Biologicals, Rogers.
Ariz.). Cells were then passed over Sephadex G-10 (Pharmacia
Uppsala, Sweden) to remove adherent cells. B iymptsurfacetocyles
were positively selected by panting on petri dishes coated with
affinity purified goat anti-muse IgM (Organon Teknika Corp., West
Chester, Pa.). The resultant preparations were >98% B cells as
determined by flow cytometry.
The purified B cells were then cultured in RPMI 1640 supplemented
with 5% fetal calf serum (Hazelton), sodium pyruvate (1 mM),
nonessential amino acids (0.1 mM), penicillin (100 U/ml),
streptomycin (100 ug/ml). L-glutamine (2 mM), and 2-mercaptoethanol
(50 uM), as well as Salmonella typhimurium LPS (10 ug/ml; Difco
Laboratories, Detroit, Mich.) to produce activated B cells.
Human rIL-1.beta.was produced in Escherichia coli and purified to
homegeneity as described by Kronheim et al., Bio/Technology 4:1708,
1986. Recombinant murine IL-4 was produced in yeast, puurfied to
homogeneity, and radio labeled as described by Mosley et al., Cell
59:355, 1989, and Park et al., Proc. Natl. Acad. Sci. USA 84:1669,
1987.
Inhibition assays were performed by first incubating various
concentrations of unlabeled cytokines (IL-1 or IL-4), soluble
receptors (sIL-1R or sIL-4R), monoclonal antibody (11B11, a rat
IgGI anti-murine IL-4 antibody produced as described by Ohara et
al., Nature 315:333, 1985) or medium control with 50 ul .sup.125
I-labeled IL-4 (1.65.times.10.sup.-1 M) for thirty minutes in 10%
CO.sub.2 at 37.degree. C. in binding medium (RPMI/2.5%
BSA/0.2%sodium azide/0.2M Hepes, pH 7.4). To these 2.times.10.sup.6
murine B cells were added in 50 ul of binding medium for 30 min at
37.degree. C. Cells were then separated by the phthalate oil method
as described by Dower et al., J. Immunol. 132:751, 1984. Percent
specific inhibition was calculated using incubation of .sup.125
I-IL-4 with excess unlabeled IL-4 (4.times.10.sup.-9 M) as positive
control and 50 ul of binding medium as negative control. Each assay
was performed with 3-fold dilutions in duplicate of each competitor
compound through binding medium, and incubations carded out in
96-well round bottom plates (Linbro, Hamden, Conn.).
Results of the inhibition assays indicate that IL-4 binding was
inhibited by unlabeled sIL-4R, unlabeled IL-4, and 11B11, an
anti-IL-4 specific mAb. The blocking effect was cytokine specific,
and cross-competition between IL-4 and either 11B11 or sIL-4R
generated similar inhibition constants as shown in Table A below.
No competition of IL-4 binding was detected by IL-1 or sIL-1R.
TABLE A ______________________________________ Inhibition of
Radiolabeled IL-4 Binding to LPS Blasts Inhibitor Inhibition
Constant (M.sup.-1) ______________________________________ IL-4 4.9
.times. 10.sup.10 sIL-4R 4.8 .times. 10.sup.9 11B11 7.9 .times.
10.sup.9 IL-1 No Inhibition sIL-1R No Inhibition
______________________________________
Example 15
Inhibition of Lymphokine Induced B Cell Proliferation in vitro by
sIL-4R
Murine B cell proliferation is stimulated by treatment of
anti-immunoglobulin and either IL-1 (Howard et al., J. Exp. Med.
157:1529, 1983; Booth et al., J. Immunol. 33:1346, 1984) or IL-4
(Grabstein et al., J. Mol. Cell. Immunol. 2:199, 1986; Howard et
al, J. Exp. Med. 155:914, 1982). The ability of sIL-1R and sIL-4R
to inhibit these B cell mitogenic responses was tested in a B cell
proliferation assay as follows.
B cells were purified and cultured as described in Example 14
above. In order to determine the effect of various doses of
inhibitors on B cell proliferation, the purified B cells were
seeded at 1.times.10.sup.5 cells/well in 96-well flat-bottom tissue
culture plates (Costar) in the presence of affinity purified goat
anti-mouse IgM (2.5 ug/ml; Zymed Laboratories, Inc., So. San
Francisco, Calif.) and various concentrations of IL-4 (FIG. 6A) or
IL-1 (FIG. 6B), either alone (.smallcircle.) or in the presence of
1000 ng/ml sIL-4R (.quadrature.), 1000 ng/ml sIL-1R (.box-solid.),
or 555 ng/ml 11B11 (.circle-solid.) as inhibitors. After 2 days,
cultures received 2 uCi/well of [.sup.3 H]thymidine (25 Ci/mmol;
Amersham, Arlington Heights., Ill.) for 16 hours, and were then
harvested onto glass fiber filters. Incorporation of radioactivity
was measured by liquid scintillation spectrophotometry. Tritiated
thymidine incorporation for triplicate wells was determined for the
final 16 hours of a three day culture period. Results are presented
as mean cpm.+-.SEM.
In order to determine the effect of various doses of inhibitor on
the inhibition of B cell proliferation by cytokines, purified B
cells were seeded at 1.times.10.sup.5 cells/well in 96-well
flat-bottom tissue culture plates (Costar) in the presence of
affinity purified goat anti-mouse IgM (25 ug/ml; Zymed
Laboratories, Inc., So. San Francisco, Calif.) and fixed
concentrations of 10 (.circle-solid.), 1 (.quadrature.),0.1 (602 ),
or 0 (.DELTA.) ng/ml of IL-4 (FIGS. 7A-7C) or IL-1 (FIGS. 7D-7F).
Culture wells also included three-fold dilutions of sIL-4R (FIGS.
7A and 7D), 11B11 (FIGS. 7B and 7E), or sIL-1R (FIGS. 7C and 7F).
After 2 days, cultures received 2 uCi/well of [.sup.3 H]thymidine
(25 Ci/mmol; Amersham, Arlington Heights., Ill.) for 16 hours, and
were then harvested onto glass fiber filters. Incorporation of
radioactivity was measured by liquid scintillation
spectrophotometry as indicated above. Tritiated thymidine
incorporation for triplicate wells was determined for the final 15
hours of a three day culture period. Results are presented as mean
cpm.+-.SEM.
FIGS. 6A-6B and 7A-7F show that sIL-4R and sIL-1R inhibitory
activity was dose dependent and specific for the respective
ligands. The inhibitory effects of the sIL-4R and 11B11 were
virtually equivalent on a molar basis, with half-maximal inhibition
of IL-4-induced proliferation requiring a 100-200 fold molar excess
of either inhibitor. Half-maximal inhibition of IL-1 activity was
achieved with a 300-400 fold molar excess of sIL-1R.
Example 16
Inhibition of IL-4 Induced Immunoglobulin Secretion In Vitro by
sIL-4R
IL-4 augments LPS-induced secretion of IgG1 and IgE and inhibits
IgG3 production, possibly by a mechanism involving class switching
from one isotype of an antibody to another isotype. The ability of
sIL-4R to inhibit IL-4 induced class switching in LPS-stimulated B
cells was tested in the following assay that measures
immunoglobulin secretion from LPS treated B cells.
B cells were purified and cultured as described in Example 14
above. In order to determine the effect of various doses of IL-4 on
IgG1, IgE and IgG3 secretion, the purified B cells
(1.times.10.sup.5 cells/well) were grown in 96-well flat bottom
plates in the presence of Salmonella typhimurium LPS (Difco
Laberatodes, Detroit, Mich.) and three-fold dilutions of IL-4 with
sIL-4R (.quadrature.), sIL-1R (.box-solid.) or 11B11
(.circle-solid.), each at 555 ng/ml or medium control (.DELTA.)
(see FIGS. 8A-8C). Six days after initiation of culture, cells were
pelleted by centrifugation at 750.times.g and culture supernatant
fluids were harvested.
Immunoglobulin (IgG1, IgG3, and IgE) levels were determined by an
isotype specific sandwich ELISA technique as follows. 96-well
flat-bottom Linbro plates (Flow Laboratories, Inc., McLean, Va.)
were coated overnight with the appropriate (see below) first step
isotype specific antibody (100 ul) and washed. This and all
subsequent washing steps were done with phosphate buffered saline
containing 0.05% Tween 20, 6 rinses per cycle. Nonspecific sites
were blocked by incubation for one hour with 150 ul of 5% nonfat
dry milk. Test material (100 ul), either culture supernatant or
isotype standard curve solutions (all sample and antibody dilutions
in PBS/3% BSA), was added to each well, incubated for 1 hour, then
washed. 100 ul of the appropriate (see below) horseradish
peroxidase-conjugated second step antibody was added and plates
were incubated for 1 hour and washed. The presence of
peroxidase-conjugated antibody was determined by using the TMB
Microwell peroxidase substrate system (Kirkegaard & Perry
Laboratories, Inc., Gaithersburg, Md.). Plates were read on a
Dynatech ELISA reader. Immunoglobulin concentrations in test
samples were determined by comparing triplicate test values with
isotype control standard curves, using the DeltaSoft 1.8 ELISA
analysis program for the Madntosh (Biometallics, Inc., Princeton,
N.J.).
For the IgG1 and IgG3 assays, unconjugated and horseradish
peroxidase-conjugated affinity purified goat anti-mouse isotype
specific reagents (Southern Biotechnology Associates, Inc.,
Birmingham, Ala.) were used as plate coating and second step
reagents, respectively. Standard curves for IgG1 and IgG3 were run
with isotype matched murine myeloma proteins (Southern). For the
IgE assay, the EM95 IgG2a anti-mouse IgE mAb (Baniyash et al., Eur.
J. Immunol. 14:797, 1984) (provided by Dr. Fred Finkelman,
Uniformed Services, Bethesda, Md.) was used as plate coating step
reagent and biotinylated rat anti-mouse IgE (Bioproducts for
Science, Inc., Indianapolis, Ind.) was used as second step reagent,
and horse radish peroxidase-conjugated streptavidin (Zymed) was
used in the third step. Standard curves were established with a
murine anti-dinitrophenol specific IgE myeloma antibody (ATCC No.
TIB 141). All three ELISA assays were determined to be specific
based upon cross-reactivity experiments using all individual murine
antibody isotypes as controls.
The effect of various doses of inhibitor on the inhibition of IgG1,
IgG3 and IgE secretion is shown in FIGS. 9A-9C. In this experiment,
purified B cells (1.times.10.sup.5 cells/well) were grown in
96-well flat bottom plates in the presence of Salmonella
typhimurium LPS (Difco Laboratories, Detroit, Mich.) and IL-4 (30
ng/ml for IgE; 3 ng/ml for IgG1 and IgG3) in the presence of
three-fold dilutions of sIL-4R (.quadrature.), sIL-1R (.box-solid.)
or 11B11 (.circle-solid.). Six days after initiation of culture,
cells were pelleted by centrifugation at 750.times.g and culture
supernatant fluids were harvested. The supernatants were analyzed
for IgG1, IgG3 and IgE secretion using the isotype specific
sandwich ELISA technique described above.
FIGS. 8A and 8B show that IgG1 and IgE secretion from LPS treated B
cells was induced by IL-4 and that these activities were inhibited
by both the sIL-4R as well as 11B11. In contrast, FIG. 8C shows
that IgG3 secretion was induced by LPS directly in the absence of
exogenous cytokines. When IL-4 was present at concentrations of 10
ng/ml or less, LPS induced IgG3 secretion was ablated. sIL-4R
blocked this inhibitory effect of IL-4, shifting the IL-4 dose
response curve and effectively permitting induction of IgG3
secretion in the presence of otherwise inhibitory doses of
IL-4.
FIGS. 9A-9C show that the inhibition of IL-4 induced class
switching by sIL-4R was dose dependent: with increasing
concentrations of inhibitors, progressively lower levels of IgG1
and IgE and progressively higher levels of IgG3 were secreted.
sIL-1R had no such effect, even at the higher concentrations (>1
ug/ml).
Example 17
Inhibition of IL-4 Induced Cell Surface Antigen Expression on B
Cells In Vitro by sIL-4R
IL-4 induces increased expression of various cell surface antigens
on resting murine B cells. In order to determine the effect of
sIL-4R on the inhibition of cell surface antigen expression,
fluorescent-labeled antibodies to two specific cell surface
antigens, MHC class II (Ia) antigens and Fc.epsilon.R (CD23), were
used to measure the level of antigen expressed on B cells as
follows.
Inhibition of MHC class II (Ia) Antigens
Purified B cells (5.times.10.sup.5 cells/ml) were cultured for 16 h
in 24-well plates (Costar) with or without IL-4 (0.1 ng/ml) in the
presence of sIL-4R, 11B11 or sIL-1R each at 500 ng/ml or medium
control with or without inhibitors. Cells were washed and
preincubated for 20 min on ice with the rat IgG2b anti-murine
Fc.gamma.R mAb 2.4G2 (Unkeless, et al., J. Exp. Med. 150:580, 1979)
to block IgG Fc receptors. Fluorescinated mAbs (25-9-17, a murine
IgG2a anti-murine I-A.sup.b antibody, described by Ozato et al., J.
Immunol. 126:317, 1981; or control murine IgG1) were added directly
and cells were incubated for 30 min at 4.degree. C. and washed. The
antibody diluent and wash solution was PBS/1% fetal calf
serum/0.01% NAN.sub.3. Stained cells were analyzed on a FACScan
flow cytometer (Beckton-Dickinson, San Jose, Calif.) using a
logarithmic fluorescence intensity scale.
FIGS. 10A-10D show that in medium control (FIG. 10A) Ia expression
in B cells is significantly greater in the presence of IL-4 (dashed
line) than without IL-4 (solid line). Addition of sIL-4R (FIG. 10B)
or 11B11 (FIG. 10C) at the onset of culture returned Ia expression
to constitutive levels, whereas addition of sIL-1R (FIG. 10D) had
no effect. The results shown in FIGS. 10A-10D are representative of
3 separate experiments.
Inhibition of Fc.epsilon.R (CD23)
IL-4 also induces expression of CD23 on murine and human B cells. B
cells were cultured without or without IL-4 in the presence of
sIL-4R, 11B11, sIL-1R or medium control as described above. Cells
were stained with FITC-labeled anti-CD23 antibody (B3B4, a rat
IgG2a anti-murine Fc.epsilon.R [CD23], described by Rao et al., J.
Immunol. 138:1845, 1987) as described above.
FIGS. 11A-11D shows that in medium control (FIG. 11A) CD23
expression in B cells is significantly greater in the presence of
IL-4 (dashed line) than without IL-4 (solid line). Addition of
sIL-4R (FIG. 11B) or 11B11 (FIG. 11C) returned cell surface CD23
expression to constitutive levels, whereas addition of sIL-1R (FIG.
11D) had no effect. As with Ia expression, the sIL-4R and 11B11
inhibitors did not diminish the constitutive expression of CD23,
indicating that the inhibition was limited to the IL-4 dependent
increase. The results shown in FIGS. 11A-11D are also
representative of 3 separate experiments.
Examples 18 and 19 are experiments which show the effect of sIL-4R
on the inhibition of IgE responses in vivo. Example 18 shows that
sIL-4R inhibits an IgE response to a specific antigen. Example 19
shows that administration of sIL-4R in doses ranging from 1-25 ug
twice daily on days -1,0 and +1 do not inhibit an IgE response to a
cocktail of anti-IgD antibodies.
Example 18
Inhibition of IL-4 Dependent Antigen-Specific IgE Response of B
Cells by sIL-4In Vivo
Animals immunized with the hapten-carrier conjugate TNP-KLH
(trinitrophenol-keyhole limpet hemocyanin) in alum generate a
strong anti-TNP IgE antibody response. In order to determine the
effect of sIL-4R administration on the IgE response, the following
experiment was conducted.
Balb/c mice (3 mice/group, 7 groups) were immunized i.p. with 1 ug
of TNP-KLH in alum on day 0. On day 21, the mice were boosted with
the same amount of TNP-KLH, then bled 5 days later. Serum was then
assayed by immunoglobulin isotype-specific ELISA for levels of both
polyclonal and antigen-specific (TNP-specific)immunoglobulin. This
secondary antibody response is characterized by, although not
restricted to, the generation of a strong IgE response, most of
which is anti-TNP specific.
On days -1, 0, and +1 of the secondary immunization, mice were
given twice-daily injections of sIL-4R in total daily doses of 25,
5, and 1 ug/mouse. Thus, each mouse received a total of 75, 15, or
3 ug of sIL-4R over the three day treatment period. Serum was
prepared from each animal, and analyzed for polyclonal and anti-TNP
IgE concentrations.
The results of these experiments, shown in FIG. 12, indicate that
untreated mice (saline control), primary-immunized mice (1-no
boost), and primed and boosted mice (1-2) displayed polyclonal IgE
levels of approximately 1 ug/ml, 5 ug/ml, and 25 ug/ml,
respectively. Treatment of boosted mice with 11B11 anti-IL-4
antibody lowered IgE levels to approximately 2 ug/ml. Treatment of
boosted mice with sIL-4R lowered IgE levels significantly, with the
highest concentration of sIL-4R (25 ug/hit) resulting in greater
than 80% reduction in IgE. Lower doses of sIL-4R inhibited the
polyclonal IgE response, although less dramatically. Thus, the
sIL-4R acts as an inhibitor of an antigen-induced polyclonal IgE
response.
FIG. 13 shows that priming of mice with TNP-KLH (1-no boost)
resulted in a detectable anti-TNP response of the IgE isotype.
Boosting with TNP-KLH caused a significant increase in the anti-TNP
IgE tire. Treatment of primed and boosted mice with 11B11 at the
time of secondary immunization diminished the antigen-specific IgE
levels to less than levels seen in primed-only mice. The highest
concentration of sIL-4R also dramatically decreased anti-TNP
specific levels. Lower concentrations of sIL-4R had no discernible
effect upon the secondary IgE response to TNP-KLH.
Example 19
In Vivo Inhibition of IL-4-Dependent Polyclonal IgE Response of B
Cells by sIL-4R
Animals immunized with a cocktail of monoclonal IgD antibodies
specific for murine IgD (allotype specific) generate a strong
polyclonal IgE antibody response. One likely mechanism of anti-IgD
action involves crosslinking of surface immunoglobulin on B cells,
internalization and processing of the anti-IgD, and presentation to
helper T cells. The Ig-allotype specific T cells are thus triggered
to provide signals (presumably cytokines) which induce
immunoglobulin class switching and secretion by B cells.
In order to determine the effect of sIL-4R administration on the
IgD-induced polycional IgE response, the following experiment was
conducted. BALB/c mice (3 mice/group) immunized i.v. with 800 ug of
anti-IgD were treated twice daily with three different doses of
sIL-4R (12.5, 2.5, or 0.5 ug/injection) on days -1, 0, and +1. Mice
were bled on day 9 and serum IgE levels were determined. FIG. 14
shows that anti-IgD treatment (MSA control) caused large increases
in levels of secreted IgE when compared with unimmunized controls
(saline control). This effect was blocked by anti-IL-4 (11B11)
administration, but not by any of the doses of sIL-4R. Anti-IgD
treatment also caused large increases in IgG1, IgG2a, and IgG3.
Whereas sIL-4R administration had no effect upon these isotypes,
11B11 administration resulted in increased IgG2a and IgG3
secretion.
The failure of sIL-4R to inhibit the IgE stimulatory effect of
anti-IgD may be due to the fact that the inhibitor must be present
for longer than one day after anti-IgD treatment, that the 11B11
antibody has a longer serum half-life than the sIL-4R, or that
higher doses of sIL-4R are required.
Example 20
Use of Soluble IL-4 Receptor to Inhibit Contact Hypersensitivity
Responses to DNFB
The effect of soluble IL-4 receptor (sIL-4R) and soluble IL-1
receptor (sIL-1R) on contact hypersensitivity (CHS) responses were
evaluated in a murine system using 2,4-dinitrofluorobenzene (DNFB)
as the contact sensitizer. Groups of female BALB/c mice (5 mice per
group) were treated with either sIL-4R or sIL-1R on either days -1
through +1, days 4 and 5 or days -1 through 5 with 500 ng, b.i.d.
via intraperitoneal injection. Control mice were treated with
equivalent doses of the carrier solution containing mouse serum
albumin (MSA). Epicutaneous sensitization with DNFB was performed
by application of 25 ul of a solution of 0.5% DNFB in 4:1 mixture
of acetone:olive oil to the shaved backs of mice on day 0. Negative
control mice were not sensitized to DNFB. CHS responses were
elidted by challenging all groups of mice on day 5 by application
of 10 ul of the DNFB solution to the right rear footpads of the
mice in each of the treatment groups. The extent of CHS induction
was determined by measuring the difference in thickness (in units
of 10.sup.-2 mm) between the challenged right and unchallenged left
rear footpads (as measured with a dial micrometer) 24 hours later.
FIGS. 15 shows the results of these experiments. The data are
presented as mean footpad swelling .+-. SEM.
As shown in FIG. 15, mice treated with sIL-1R developed CHS
responses that were not significantly different from control mice
treated with MSA regardless of the treatment regimen used. Mice
treated with sIL-4R on days -1 through 1 developed CHS responses
that were not significantly different from the MSA control group.
Although there appeared to be a slight increase in the CHS
responses to DNFB induced in mice treated with sIL-4R on days 4 and
5, inspection of the data indicated that this was primarily due to
the enhanced response of only one out of five of the mice in the
group. No significant effect of treatment (either enhancement or
inhibition) is observed if the response of this single mouse is
considered to be an outlier and is not considered in the evaluation
of the data (FIG. 15). However, mice treated with sIL-4R during the
entire pedod (days -1 through 5) were significantly (p<0.001)
inhibited relative to MSA-treated control mice, and not
significantly different from mice which had not been sensitized,
but only challenged, with DNFB. This data thus indicates that
sIL-4R is effective in inhibiting contact hypersensitivity
responses to DNFB.
Example 21
Use of Soluble IL-4 Receptor to Inhibit Delayed-Type
Hypersensitivity Responses to SRBC
The effect of soluble IL-4 receptor (sIL-4R) and soluble IL-1
receptor (sIL-1R) on delayed-type hypersensitivity (DTH) responses
to sheep red blood cells (SRBC) were evaluated in a murine system
as described by Kitamura, J. Immunol. Meth. 39:277, 1980. Three
groups of female BALB/c mice (4 mice per group) were sensitized on
day 0 by i.v. injection of 2.times.10.sup.5 SRBC. A fourth group of
negative control mice were not sensitized to SRBC. DTH responses
were elicited in the sensitized mice by challenging the mice on day
4 with 1.times.10.sup.8 SRBC via intracutaneous injection in the
right rear footpads of the mice and 100 ul normal saline in the
contralateral footpad as a control. The mice were treated with
either 0.1 ug or 1.0 ug of sIL-4R in 100 ul MSA via intraperitoneal
injection, on the day of challenge and the day of immunization.
Control mice were treated with equivalent doses of the carrier
solution (100 ul) containing mouse serum albumin (MSA). The fourth
group of negative control mice not sensitized to SRBC were treated
with 100 ul of MSA. The extent of the DTH response induced was
determined by excising the footpads at the tarsus and measuring the
difference in weight between SRBC challenged and saline challenged
footpads. FIG. 16 shows the results of these experiments. The data
are presented as mean footpad swelling .+-. SEM.
As shown in FIG. 16, the DTH response was almost totally blocked by
intraperitoneal injection with 1 ug sIL-4R on the day of challenge
and the day of immunization. Treatment with 0.1 ug sIL-4R was less
effective. Treatment with 0.2 ug or 2 ug of sIL-1R inhibited the
response (not shown). These data thus indicate that sIL-4R is
effective in inhibiting delayed-type hypersensitivity
responses.
Example 22
Use of Soluble IL-4R to Suppress Immune Response to Alloantigen In
Vivo
Experiments were conducted to show that systemic administration of
sIL-4R suppresses a localized, T cell-dependent, immune response to
alloantigen presented by allogeneic cells. The response to
allogeneic cells in vivo was quantified using the popliteal lymph
node enlargement assay described by Twist et al., Transplantation
15: 182, 1973, which is used as a measure of allograft transplant
immunity (see Grebe et al., Adv. Immunol. 22:119, 1976). In this
assay mice are injected in the footpad with irradiated, allogeneic
spleen cells. The mice are then injected in the contralateral
footpad with irradiated, syngeneic spleen cells. An alloreactive
response (marked by proliferation of lymphocytes and inflammation)
occurs in the footpad receiving the allogeneic cells, which can be
measured by determining the increase in size and weight of the
popliteal lymph node draining the site of antigen deposition
relative to controls or by an increase in cellularity.
Specific pathogen free 8-12 week old BALB/c (H-2.sup.d) and C57BL/6
(H-2.sup.b) mice (Jackson Laboratory, Bar Harbor, Me.) were used in
this experiment. 9 BALB/c mice were divided into 3 groups, each
having 3 mice. Each group of mice received a different mode of
treatment as indicated below in Tables B. On day 0 the left
footpads of all mice were injected intracutaneously with 107
irradiated (2500R), allogeneic spleen cells from C57BL/6 mice in 50
ul of RPMI-1640 (Gibco) as antigen and the right contralateral
footpads of the same mice were injected with 107 irradiated
(2500R), syngeneic spleen cells from BALB/c mice. All doses of
soluble murine IL-4 receptor (sIL-4R) were diluted in phosphate
buffered saline (PBS). On days -1, 0 and +1 three mice were
injected (intravenously on days -1 and 0, and subcutaneously on day
+1) with 100 ng of purified smuIL-4R, three mice were injected
intravenously with 1 ug of smuIL-4R, three mice were injected with
2 ug of smuIL-4R and three mice were injected with MSA
(control).
Seven days after antigen administration, the mice were sacrificed
and the popliteal lymph nodes (PLN) were removed from the dght and
left popliteal fossa by surgical dissection. Lymph nodes were
weighed and the results expressed as the difference (.DELTA.) in
weight (mg) of the lymph node draining the site of allogeneic cell
injection and the weight of the node draining the syngeneic cell
injection site (Table B; FIG. 17). The mean difference in weight of
the lymph nodes from the sites of allogeneic and syngeneic spleen
cells was approximately 2.5 mg for the mice treated with MSA, 1 mg
for the mice treated with 100 ng of sIL-4R, and 0.5 mg for mice
treated with Iug sIL-4R. No detectable difference in weight of
lymph nodes was ascertainable for the mice treated with 2 ug
sIL-4R. Lymph nodes draining the syngeneic cell injection site
weighed approximately 1 mg, regardless of whether they were
obtained from mice treated with MSA or smuIL-4R, and did not differ
significantly in weight from nodes obtained from mice given no cell
injection. Values for statistical significance were calculated
using the two-tailed Student's t-test. Thus, IL-4R significantly
(p<0.1 in all groups, using a two-tailed T test) suppressed the
in vivo lymphoproliferative response in a dose dependent fashion
relative to control mice.
TABLE B ______________________________________ Effect of smuIL-4R
Administration on Proliferation of Lymph Node Cells Treatment
Weight (mg) of Lymph Node Group Allogeneic Syngeneic .DELTA.
______________________________________ MSA 3.9 .+-. 0.06 1.27 .+-.
0.07 2.63 .+-. 0.1 100 ng smuIL-1R 2.3 .+-. 0.03 1.3 .+-. 0.03 1.0
.+-. 0.06 1 ug smuIL-1R 2.1 .+-. 0.9 1.9 .+-. 0.3 0.23 .+-. 0.6 2
ug smuIL-1R 1.6 .+-. 0.3 1.5 .+-. 0.1 0.0 .+-. 0.4
______________________________________
Table B (illustrated graphically in FIG. 17) shows that systemic
administration of sIL-4R for 3 days beginning on day -1 relative to
alloantigenic challenge resulted in a dramatic decrease in the size
of lymph nodes, indicating that the lymphoproliferative response is
inhibited. The effect was dose dependent and, in some cases, the
response was virtually eliminated.
Example 23
Use of Soluble IL-4 Receptor to Suppress Allograft Rejection
Soluble murine IL-4 receptor also suppresses rejection of organ
grafts in vivo. In order to demonstrate this, neonatal C57BL/6
surface(H-2.sup.b) hearts were transplanted into the ear pinnae of
adult BALB/c (H-2.sup.d) recipients utilizing the method of Fulmer
et al., Am. J. Anat. 113:273, 1963, modified as described by Trager
et al., Transplantation 47:587, 1989, and Van Buren et al.,
Transplant. Proc. 15:2967, 1983. Survival of the transplanted
hearts was assessed by visually inspecting the grafts for pulsatile
activity. Pulsatile activity was determined by examining the
ear-heart grafts of anesthetized recipients under a dissecting
microscope with soft reflected light beginning on day 5 or 6 post
transplant. The time of graft rejection was defined as the day
after transplantation on which contractile activity ceases.
Recipient mice were divided into two groups, a primary treatment
group and a secondary treatment group. The primary treatment group
were not exposed to antigen from C57BL/6 mice previous to being
treated, while the secondary treatment group had been exposed to
antigen. All mice were transplanted on day 0 and injected with
either smuIL-4R (1000 ng/day) plus MSA (mouse serum albumin, 100
ng) or with MSA alone on days 0 through 2, i.p. The results of this
experiment are reported below in Table C. The probability (p value)
that the survival time for the group treated with smuIL-4R differs
by chance alone from the group treated with MSA is less than 0.04
when analyzed by the Student's t-test for the primary treatment
group. The corresponding p values for secondary treatment group are
not significant.
TABLE C ______________________________________ Effects of smuIL-1R
Treatment on Nonvascularized Heterotopic Cardiac Allograft Survival
Treatment Median Survival Group Survival Time (days) Time .+-. S.D.
______________________________________ Primary Treatment MSA (100
ng) 9, 10, 12, 14 11.3 .+-. 1.1 smuIL-1R (100 ng) 10, 10, 10, 12
10.5 .+-. 0.5 smuIL-4R (1000 ng) 12, 14, 14, 16, 17, 19 15.3 .+-.
1.0 Secondary Treatment MSA (100 ng) 8, 8, 8 8 .+-. 0.0 smuIL-1R
(1000 ng) 8, 10, 12 10 .+-. 1.2
______________________________________
Table C shows that heart allografts survived 10-14 days in
individual control mice treated with MSA. When primary allograft
recipients were given 3 daily injections of 1000 ng smuIL-4R, graft
survival was prolonged. The median graft survival time in smuIL-4R
treated mice (14-19 days) was approximately four days longer than
the median graft survival time of identical grafts in control mice.
A subtle increase in graft survival following secondary
transplantation suggests that acute rejection episodes are
influenced by smuIL-4R administration as well. This data is
evidence of the therapeutic potential of soluble human IL-4
receptor in humans for the suppression of heart allograft
rejection.
* * * * *