U.S. patent application number 11/986376 was filed with the patent office on 2009-09-10 for interleukin-4 receptors.
Invention is credited to M. Patricia Beckmann, David J. Cosman, Rejean Idzerda, Carl J. March, Bruce A. Mosley, Linda S. Park.
Application Number | 20090226472 11/986376 |
Document ID | / |
Family ID | 27500837 |
Filed Date | 2009-09-10 |
United States Patent
Application |
20090226472 |
Kind Code |
A1 |
Mosley; Bruce A. ; et
al. |
September 10, 2009 |
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 A.; (Seattle,
WA) ; Cosman; David J.; (Bainbridge Island, WA)
; Park; Linda S.; (Seattle, WA) ; Beckmann; M.
Patricia; (Hansville, WA) ; March; Carl J.;
(Bainbridge Island, WA) ; Idzerda; Rejean;
(Seattle, WA) |
Correspondence
Address: |
IMMUNEX CORPORATION;LAW DEPARTMENT
1201 AMGEN COURT WEST
SEATTLE
WA
98119
US
|
Family ID: |
27500837 |
Appl. No.: |
11/986376 |
Filed: |
November 20, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10688495 |
Oct 17, 2003 |
7317090 |
|
|
11986376 |
|
|
|
|
10104590 |
Mar 22, 2002 |
6716587 |
|
|
10688495 |
|
|
|
|
09094917 |
Jun 15, 1998 |
6391581 |
|
|
10104590 |
|
|
|
|
07480694 |
Feb 14, 1990 |
5840869 |
|
|
09094917 |
|
|
|
|
07370924 |
Jun 23, 1989 |
|
|
|
07480694 |
|
|
|
|
07326156 |
Mar 20, 1989 |
|
|
|
07370924 |
|
|
|
|
07319438 |
Mar 2, 1989 |
|
|
|
07326156 |
|
|
|
|
07265047 |
|
|
|
|
07319438 |
|
|
|
|
Current U.S.
Class: |
424/185.1 ;
435/320.1; 435/325; 435/69.1; 436/501; 514/1.1; 530/350; 530/387.9;
536/23.5 |
Current CPC
Class: |
Y10S 514/885 20130101;
A61P 37/00 20180101; Y10S 514/826 20130101; C07K 14/7155 20130101;
Y10S 514/886 20130101; G01N 33/6869 20130101; A61K 38/00 20130101;
A61P 37/08 20180101; C07K 16/2866 20130101 |
Class at
Publication: |
424/185.1 ;
536/23.5; 435/320.1; 435/69.1; 435/325; 514/12; 530/350; 530/387.9;
436/501 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 15/11 20060101 C12N015/11; C12N 15/00 20060101
C12N015/00; C12P 21/00 20060101 C12P021/00; C12N 5/06 20060101
C12N005/06; A61K 38/16 20060101 A61K038/16; C07K 14/00 20060101
C07K014/00; C07K 16/18 20060101 C07K016/18; G01N 33/566 20060101
G01N033/566 |
Claims
1. An isolated DNA sequence encoding a mammalian IL-4 receptor
(IL-4R).
2. An isolated DNA sequence according to claim 1 which encodes a
human IL-4R comprising the sequence of human IL-4R (1-800).
3. An isolated DNA sequence according to claim 3 which encodes a
soluble human IL-4R.
4. An isolated DNA sequence according to claim 3 which encodes a
soluble human IL-4R comprising the sequence of human IL-4R
(1-207).
5. An isolated DNA sequence according to claim 2 which encodes a
soluble human IL-4R comprising the sequence of human IL-4R
(1-197).
6. A recombinant expression vector comprising a DNA sequence
according to claim 1.
7. A recombinant expression vector comprising a DNA sequence
according to claim 2.
8. A recombinant expression vector comprising a DNA sequence
according to claim 3.
9. A recombinant expression vector comprising a DNA sequence
according to claim 4.
10. A recombinant expression vector comprising a DNA sequence
according to claim 5.
11. A process for preparing a mammalian IL-4 receptor or an analog
thereof, comprising culturing a suitable host cell comprising a
vector according to claim 6 under conditions promoting
expression.
12. A process for preparing a human IL-4 receptor or an analog
thereof, comprising culturing a suitable host cell comprising a
vector according to claim 9 under conditions promoting
expression.
13. A process for preparing a human IL-4 receptor or an analog
thereof, comprising culturing a suitable host cell comprising a
vector according to claim 10 under conditions promoting
expression.
14. A population of eukaryotic cells which express more than
10.sup.4 surface IL-4 receptors per cell.
15. A population of eukaryotic cells according to claim 14, which
express more than 10.sup.5 surface IL-4 receptors per cell.
16. A substantially homogeneous biologically active mammalian IL-4
receptor composition.
17. A substantially homogeneous biologically active mammalian IL-4
receptor composition according to claim 16, consisting essentially
of murine IL-4 receptor.
18. A homogeneous biologically active mammalian IL-4 receptor
composition according to claim 16, consisting essentially of human
IL-4 receptor.
19. A composition for regulating immune responses in a mammal,
comprising administering an effective amount of a composition
according to claim 16, and a suitable diluent or carrier.
20. A composition accordingly to claim 19 having a specific binding
activity of at least about 0.01 nanomole IL-4/nanomole IL-4
receptor.
21. A composition according to claim 19, consisting essentially of
a substantially homogeneous protein composition comprising human
IL-4 receptor in the form of a glycoprotein having a binding
affinity (K.sub.a) for human IL-4 of about 1-8.times.10.sup.9
M.sup.-1, and the N-terminal amino acid sequence Lys Val Leu Gln
Glu Pro Thr Cys Val Ser Asp Tyr Met.
22. An assay method for detection of IL-4 or IL-4 receptor
molecules or the interaction thereof, comprising use of a protein
composition according to claim 19.
23. Antibodies immunoreactive with mammalian IL-4 receptors.
24. A homogeneous biologically active IL-4 receptor composition
according to claim 16, wherein said IL-4 receptor is capable of
retaining IL-4 binding activity when bound to a solid support.
25. A method for suppressing or inhibiting an IL-4-mediated immune
or inflammatory response in a mammal, including a human, which
comprises: administering an amount of soluble IL-4 receptor
(sIL-4R) and a suitable diluent or carrier effective to suppress or
inhibit the IL-4-mediated immune or inflammatory response.
26. A method for suppressing or inhibiting an alloantigen induced
IL-4-mediated immune or inflammatory response in a mammal,
including a human, which comprises: administering an amount of
soluble IL-4 receptor (sIL-4R) and a suitable diluent or carrier
effective to suppress or inhibit the alloantigen induced
IL-4-mediated immune or inflammatory response.
27. A method for suppressing IL-4-mediated proliferation of
lymphocytes in a mammal, including a human, which comprises:
administering an amount of soluble IL-4 receptor (sIL-4R) and a
suitable diluent or carrier effective to suppress the IL-4-mediated
proliferation of lymphocytes.
28. A method for suppressing allograft rejection in a mammal,
including a human, which comprises: administering an amount of
soluble IL-4 receptor (sIL-4R) and a suitable diluent or carrier
effective to suppress the allograft rejection.
29. A method for suppressing allograft rejection according to claim
28, wherein the allograft comprises tissue from an organ selected
from the group consisting of skin, kidney, heart, lung, liver and
pancreas.
30. A method for suppressing antibody production in a mammal,
including a human, which comprises: administering an amount of
soluble IL-4 receptor (sIL-4R) and a suitable diluent or carrier
effective to suppress the antibody production.
31. A method for suppressing antibody production in a mammal
according to claim 30, wherein the antibodies are IgE
antibodies.
32. A method for suppressing an allergic or asthmatic reaction in a
mammal, including a human, which comprises: administering an amount
of soluble IL-4 receptor (sIL-4R) and a suitable diluent or carrier
effective to suppress the allergic asthmatic reaction.
33. A method for suppressing bone marrow transplant failure in a
mammal, including a human, which comprises: administering an amount
of soluble IL-4 receptor (sIL-4R) and a suitable diluent or carrier
effective to suppress the bone marrow transplant failure.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of Ser. No. 10/688,495, filed Oct.
17, 2003, now allowed, which is a divisional of Ser. No.
10/104,590, filed Mar. 22, 2002, now U.S. Pat. No. 6,716,587, which
is a divisional of Ser. No. 09/094,917, filed Jun. 15, 1998, now
U.S. Pat. No. 6,391,581, which is a continuation of Ser. No.
07/480,694, filed Feb. 14, 1990, now U.S. Pat. No. 5,840,869, 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.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to cytokine
receptors and, more specifically, to Interleukin-4 receptors.
[0003] 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).
[0004] 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.
[0005] 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.125I-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.125I
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.125I-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.
[0006] 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
[0007] 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.
[0008] 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 7B9-4 from
the murine 7B9 library is 2.times.10.sup.9 to 1.times.10.sup.10
M.sup.-1. The mature murine IL-4 receptor molecule has an
N-terminal amino acid sequence as follows: I K V L G E P T C F S D
Y I R T S T C E W.
[0009] 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 acid sequence, predicted from the cDNA sequence
and by analogy to the biochemically determined N-terminal sequence
of the mature murine protein, as follows: M K V L Q E P T C V S D Y
M S I S T C E W.
[0010] 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.
[0011] 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.
[0012] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] 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 Sst I are
represented by the letters R, P, H and S, respectively.
[0014] 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 coding 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
peptide sequence), but is followed by a TAG terminator codon (not
shown) which ends the open 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.
[0015] FIG. 3 is a schematic illustration of the mammalian high
expression plasmid pCAV/NOT, which is described in greater detail
in Example 8.
[0016] 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.
[0017] 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.
[0018] FIG. 6 shows the inhibition of B cell proliferation with
IL-4 (panel A) or IL-1 (panel B) at various doses either alone ()
or in the presence of sIL-4R (), sIL-1R (.box-solid.) or anti-IL-4
antibody ( ) as described in Example 15.
[0019] FIG. 7 shows the inhibition of B cell proliferation with
fixed concentrations of 10 ( ), 1 (), 0.1 () or 0 () ng/ml of IL-4
(panels A-C) and IL-1 (panels D-F) at various doses of sIL-4R
(panels A & D), anti-IL-4 antibody (panels B & E) and
sIL-1R (panels C & F) as described in Example 15.
[0020] FIG. 8 shows the inhibition of immunoglobulin class
switching with various doses of IL-4 and with sIL-4R (), sIL-1R
(.box-solid.), or anti-IL-4 antibody ( ) or medium control () as
described in Example 15.
[0021] FIG. 9 shows the inhibition of IL-4-induced immunoglobulin
class switching with fixed concentration of IL-4 and various doses
of sIL-4R (), sIL-1R (.box-solid.), anti-IL-4 antibody ( ) or
medium control (.DELTA.) as described in Example 16.
[0022] FIG. 10 shows the inhibition of MHC class II antigen
expression with (dashed line) or without (solid line) IL-4 in the
presence of medium control (panel A), sIL-4R (panel B), anti-IL-4
antibody (panel C) or sIL-1R (panel D) as described in Example
17.
[0023] FIG. 11 shows the inhibition of Fc.epsilon.R (CD23)
expression with (dashed line) or without (solid line) IL-4 in the
presence of medium control (panel A), sIL-4R (panel B), anti-IL-4
antibody (panel C) or sIL-1R (panel D) as described in Example
17.
[0024] FIG. 12 shows the inhibition of antigen specific polyclonal
IgE levels by sIL-4R as described in Example 18.
[0025] FIG. 13 shows the inhibition of antigen specific
anti-TNP-KLH IgE levels by sIL-4R as described in Example 18.
[0026] 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
antigen specific anti-IgD IgE levels.
[0027] FIG. 15 shows the inhibition of contact hypersensitivity
responses to DNFB with sIL-4R as described in Example 20.
[0028] FIG. 16 shows the inhibition of delayed-type
hypersensitivity responses to SRBC with sIL-4R as described in
Example 21.
[0029] 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
[0030] "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 lymphoid 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.
[0031] As used herein, the terms "IL-4 receptor" or "IL-4R" refer
to proteins which bind interleukin-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.
[0032] "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.
[0033] "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 acid 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.
[0034] "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 FIG. 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 polypeptide 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.
[0035] "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 pattern different from that expressed in mammalian
cells.
[0036] "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).
[0037] "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.
[0038] "Nucleotide sequence" refers to a heteropolymer of
deoxyribonucleotides. 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.
[0039] "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.
[0040] "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
[0041] 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.
[0042] 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 .alpha.-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-4
receptor 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.
[0043] 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.
[0044] 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 bacteria 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.
[0045] 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
polypeptide 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.
[0046] 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.
[0047] 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 (10207) 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).
[0048] 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.
[0049] 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 loops 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.
[0050] 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 acid insertion,
substitution, or deletion.
[0051] 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
(BioTechniques, 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 IL-4R
[0052] The present invention provides recombinant expression
vectors which include synthetic or cDNA-derived DNA fragments
encoding mammalian IL-4R or bioequivalent 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.
[0053] 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.
[0054] 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 recombinant
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.
[0055] 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. coli or bacilli. Higher eukaryotic cells include
established cell lines of mammalian origin 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, N.Y., 1985), the relevant disclosure of which is hereby
incorporated by reference.
[0056] 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
subtilis, Salmonella typhimurium, and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0057] Useful expression vectors for bacterial use can comprise 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. coli is
typically transformed using derivatives of pBR322, a plasmid
derived from an E. coli 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.
[0058] 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 .lamda. P.sub.L
promoter and c1857ts thermolabile repressor. Plasmid vectors
available from the American Type Culture Collection which
incorporate derivatives of the .lamda. P.sub.L promoter include
plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and
pPLc28, resident in E. coli RR1 (ATCC 53082).
[0059] 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
origin 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. coli 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.
[0060] Suitable promoter sequences in yeast vectors include the
promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. 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.
[0061] Preferred yeast vectors can be assembled using DNA sequences
from pBR322 for selection and replication in E. coli (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.
[0062] 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.
[0063] 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.
[0064] 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 origin 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.
[0065] 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 origin 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).
[0066] 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).
[0067] 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
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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 origin 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
associated with IL-4R as it is found in nature in its species of
origin, e.g. in cells, cell exudates or body fluids.
Administration of Soluble IL-4 Receptor Compositions
[0074] 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.
[0075] 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.
[0076] 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 (IgG1 abd 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.
[0077] sIL-4R compositions may also be used to regulate the
function of T cells. Although T cell dependent functions were
formerly though 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
potentially effective in the clinical treatment of, for example,
rejection of allografts (such as skin, kidney, heart, lung liver
and pancreas transplants), and graft-versus-host reactions in
patients who have received bone marrow transplants.
[0078] 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.
[0079] 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 conjunction with other soluble cytokine
receptors, e.g., IL-1 receptor, is also contemplated.
[0080] The following examples are offered by way of illustration,
and not by way of limitation.
EXAMPLES
Example 1
Binding Assays for IL-4 Receptor
[0081] 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 pg rIL-4 in
50 .mu.l 0.2 M sodium phosphate, pH 7.2 are combined with 50 .mu.l
enzymobead reagent, 2 MCi of sodium iodide in 20 .mu.l of 0.05 M
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 Memorial
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.125I-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.
[0082] 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 cells 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.125I-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.125I-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.125I-IL-4 by cells at 37.degree. C.
[0083] For analysis of inhibition of binding by soluble IL-4R,
supernatants from COS cells transfected with recombinant IL-4R
constructs were harvested three days after transfection. Serial
two-fold dilutions of conditioned media were pre-incubated with
3.times.10.sup.-10 M .sup.125I-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.125I-IL-4.
[0084] 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 Schuell, Keene, N.H.) and allowed to dry. The
membranes are incubated in tissue culture dishes for 30 minutes in
Tris (0.05 M) buffered saline (0.15 M) 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.125I-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, dried 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)
[0085] 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 pg in 300 .mu.l of 0.1 M citrate-phosphate buffer, pH 5.5)
with 30 .mu.l of 10 mM sodium m-periodiate (Sigma), freshly
prepared in 0.1 M 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 0.1 M glycerol and dialyzed for 18
hours at 4.degree. C. against 0.1 M 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.
[0086] 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.
[0087] Progress was monitored by doing binding assays with
.sup.125I-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.125I-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
[0088] 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 debris. 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
[0089] In order to obtain sufficient quantities of murine IL-4R to
determine its N-terminal sequence or to further characterize human
IL-4R, protein 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 0.1 M 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 manufacturer's 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 0.05
M 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.35S-cysteine/methionine-labeled cell extracts for small-scale
affinity purifications and gel electrophoresis.
[0090] 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 (0.05 M Tris, 0.15 M 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 1 M Hepes, pH 7.4. The presence of receptor in the
fractions was detected by the solid phase binding assay as
described above, using .sup.125I-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 (0.125 M 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).
[0091] 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.125I-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
Sequencing of IL-4 Receptor Protein
[0092] CTLL 19.4 mL-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.2O 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.2O for 20 minutes post injection. The HPLC column
containing the bound protein was then developed with a gradient as
follows:
TABLE-US-00001 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.
[0093] 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.
[0094] 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-Ile-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 internal methionine residues. Sequencing
of the resulting cleavage products yielded the following data,
indicating that the CNBr cleaved the protein after two internal
methionine residues:
TABLE-US-00002 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:
TABLE-US-00003 1 5 10 15 Sequence 1:
(Met)-Val-Asn-Ile-Ser-Arg-Glu-Asp-Asn-Pro-Ala-Glu-Phe-Ile-Val-Tyr-Asn-Val-
-Thr 1 5 10 15 18 Sequence 2:
(Met)-Ser-Gly-Val-Tyr-Tyr-Thr-Ala-Arg-Val-Arg-Val-Arg-Ser-Gln-Ile-Leu-Thr-
-Gly
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.
[0095] In addition, the amino terminal sequence matched a sequence
derived from the clone with position 9 being defined as a Cys.
[0096] 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
[0097] In order to screen a library for clones encoding a murine
IL-4 receptor, a highly enriched 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 sorted 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
Manual (Cold Spring 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.32P-labeling of the cDNA, 100 .mu.Ci of .sup.32P-dCTP
(s.a.=3000 Ci/mmol) was used in a 50 .mu.l reaction with
non-radioactive dCTP at 10 .mu.M. 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 0.2 M 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 0.5 M. 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.
[0098] 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 pg of polyA.sup.+ mRNA isolated
from unsorted CTLL cells, resuspending in 16 .mu.l of 0.25 M
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 0.02 M
NaPO.sub.4, pH 6.8, bound to hydroxyapatite at room temperature,
and single-stranded cDNA was then eluted from the resin with 0.12 M
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 enriched for radiolabeled IL-4 receptor
cDNA which can be used to probe a cDNA library (as described
below).
Example 7
Synthesis of cDNA Library and Plaque Screening
[0099] 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
.lamda.ZAP.RTM. arms and the resulting ligation mix was packaged in
vitro (Gigapack.RTM.) according to the manufacturer's instructions.
Other suitable methods and reagents for generating cDNA libraries
in .lamda. 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. .lamda.ZAP.RTM. is a phage A cloning vector
similar to .lamda.gt11 (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 .lamda. phage DNA. .lamda.ZAP.RTM.), Bluescript.RTM.,
and Gigapack.RTM. are registered trademarks of Stratagene, San
Diego, Calif., USA.
[0100] 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 .lamda.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. USA76:3683, 1979. Filters were then washed
at 68.degree. C. in 0.2.times.SSC. Sixteen positive plaques were
purified for further analysis.
[0101] 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.
[0102] Restriction mapping (shown in FIG. 1) and DNA sequencing of
the isolated CTLL clones indicated the existence of at least two
distinct mRNA populations. Both mRNA types have homologous 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 acid
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 adds the 3' nucleotide sequence CCAAGTAATGAAAATCTG which
encodes the C-terminal 6 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
[0103] A. Expression in COS-7 Cells. A eukaryotic expression vector
pCAV/NOT, shown in FIG. 3, was derived from the mammalian high
expression vector pDC201, 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 Bgl 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 VAII 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.
[0104] 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 blunt-ended
with T4 polymerase followed by ligation into the vector pCAV/NOT
cut with Sma I and dephosphorylated.
[0105] 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. (Nuc. 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.
[0106] B. Expression in CHO Cells. IL-4R was also expressed in the
mammalian CHO cell line by first ligating an Asp718/NotI
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.
[0107] C. Expression 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 origin 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.
Appl. 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.
[0108] A 760 bp IL-4R fragment of clone C-18 from the CTLL 19.4
library was released from the Bluescript.RTM. plasmid of the
.lamda.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 polymerase 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.
[0109] 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
nitrocellulose 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.125I-IL-4 (4.times.10.sup.-11M, 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.
[0110] 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.125I-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.125I-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.125I-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).
[0111] 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.RTM.-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 0.1 M 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
manufacturer's 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 0.05 M 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.35S-cysteine/methionine-labeled cell extracts for small-scale
affinity purifications and gel electrophoresis.
[0112] Aliquots (25 ml) of HeLa E3C3 culture supernatants
(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 material except the bound mIL-4R. The column was
then eluted with 0.01 M acetic acid, 0.15 M 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 1 M Hepes, pH 7.4. The presence of
receptor in the fractions was detected by the inhibition binding
assay described above.
[0113] 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 -25,000 daltons on
SDS-PAGE. In addition, amino acid sequencing confirmed that the
bands have the same N-terminal sequence. Purity and protein
concentrations were also confirmed by amino acid analysis.
Example 9
Expression of IL-4R in Yeast Cells
[0114] For expression of mIL-4R, a yeast expression vector derived
from pIXY120 was constructed as follows. pIXY120 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 .alpha.-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, BioTechniques, 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 SpeI
site in the 2.mu. sequence:
TABLE-US-00004 Asp 718 Stu I Nco I BamH ISma I Spe I
GTACCTTTGGATAAAAGAGACTACAAGGACGACGATGACAAGAGGCCTCCATGGATCCCCCGGGACA
GAAACCTATTTTCTCTGATGTTCCTGCTGCTACTGTTCTCCGGAGGTACCTAGGGGGCCCTGTGATC
|<-----------Polylinker------- --->|
pBC120 also varies from pY.alpha.HuGM by the presence of a 514 bp
DNA fragment derived from the single-stranded phage f1 containing
the origin 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, pIXY120 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.
[0115] 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 Ile and Lys.
pIXY120 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.
TABLE-US-00005 .alpha.-factor processing ---->| GTA CCT CTA GAT
AAA AGA ATC AAG GA CAT CTA TTT TCT TAG TTC CAG Val Pro Leu Asp Lys
Arg Ile Lys |<---mIL-4R
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 acid sequence.
[0116] 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
165,654, 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. To 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
[0117] 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 .lamda.ZAP (Stratagene, San
Diego), as described in Example 7. The .lamda.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.32P-labeled 700 bp EcoR I fragment isolated
from CTLL 19.4 clone 16. Thirteen clones were isolated and
characterized by restriction analysis.
[0118] 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.
[0119] 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 acid 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
[0120] 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 .lamda.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
polymerase, followed by .sup.32P-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/0.4 M 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 portion 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.32P-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 Ile. 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 Ile.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.
[0121] 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 Asp 718 plus
Sma I, respectively; these are called soluble hIL-4R-5 and soluble
hIL-4R-11, respectively.
[0122] 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 acid mature external 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
[0123] 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.125I-IL-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.
[0124] Inhibition of .sup.125I-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.125I-IL-4 binding to the
IL-4 receptor on CTLL cells. .sup.125I-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.125I-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.
[0125] 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 0.1 M glycine, 0.15 M NaCl pH 3.0. Immediately following
elution, samples were neutralized with 80 .mu.l of 1 M Hepes pH
7.4. Samples were tested for their ability to inhibit binding of
.sup.125I-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 colorimetrically 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.
[0126] Purified recombinant soluble murine IL-4 receptor protein
may also be tested for its ability to inhibit IL-4 induced
.sup.3H-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.
[0127] 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.35S-cysteine
and .sup.35S-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.125IL-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.
[0128] 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 itself 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.125I-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.
[0129] 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.35S-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 support migrated at
about 140,000 daltons on SDS-PAGE, agreeing well with previous
estimates of hIL-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.
[0130] 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.125I-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.35S-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
[0131] 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.
[0132] 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.125I-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.
[0133] 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.125I-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.125I-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.35S-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.
[0134] 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
[0135] 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.
[0136] 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, Ark.). Cells were then passed over Sephadex
G-10 (Pharmacia Uppsala, Sweden) to remove adherent cells. B
lymphocytes were positively selected by panning on petri dishes
coated with affinity purified goat anti-mouse IgM (Organon Teknika
Corp., West Chester, Pa.). The resultant preparations were >98%
B cells as determined by flow cytometry.
[0137] 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.
[0138] Human rIL-1.beta. was produced in Escherichia coli and
purified to homogeneity as described by Kronheim et al.,
Bio/Technology 4:1708, 1986. Recombinant murine IL-4 was produced
in yeast, purified to homogeneity, and radiolabeled as described by
Mosley et al., Cell 59:355, 1989, and Park et al., Proc. Natl.
Acad. Sci. USA 84:1669, 1987.
[0139] 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
IgG1 anti-murine IL-4 antibody produced as described by Ohara et
al., Nature 315:333, 1985) or medium control with 50 ul
.sup.125I-labeled IL-4 (1.65.times.10.sup.-10 M) for thirty minutes
in 10% CO.sub.2 at 37.degree. C. in binding medium (RPMI/2.5%
BSA/0.2% sodium azide/0.2 M 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.125I-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 carried out in 96-well round bottom plates
(Linbro, Hamden, Conn.).
[0140] 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-US-00006 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
[0141] 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.
[0142] 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 (panel A) or
IL-1 (panel B), either alone () or in the presence of 1000 ng/ml
sIL-4R (), 1000 ng/ml sIL-1R (.box-solid.), or 555 ng/ml 11B11 ( )
as inhibitors. After 2 days, cultures received 2 uCi/well of
[.sup.3H]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 [cite reference]. 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.
[0143] 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 .mu.g/ml; Zymed
Laboratories, Inc., So. San Francisco, Calif.) and fixed
concentrations of 10 (.box-solid.), 1 (), 0.1 (), or 0 () ng/ml of
IL-4 (panels A-C) or IL-1 (panels D-F). Culture wells also included
three-fold dilutions of sIL-4R (panels A, D), 11B11 (panels B,E),
or sIL-1R (panels C,F). After 2 days, cultures received 2 uCi/well
of [.sup.3H]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.
[0144] FIGS. 6 and 7 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
[0145] 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.
[0146] 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
Laboratories, Detroit, Mich.) and three-fold dilutions of IL-4 with
sIL-4R (), sIL-1R (.box-solid.) or 11B11 ( ), each at 555 ng/ml or
medium control () (see FIG. 8). Six days after initiation of
culture, cells were pelleted by centrifugation at 750.times.g and
culture supernatant fluids were harvested.
[0147] 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 Macintosh (Biometallics, Inc., Princeton,
N.J.).
[0148] For the IgG1 and IgG3 assays, unconjugated and horseradish
peroxidase-conjugated affinity purified goat anti-mouse isotype
specific reagents (Southern Biotechnology Associates, Inc.,
Birmingham, AB) 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.
[0149] The effect of various doses of inhibitor on the inhibition
of IgG1, IgG3 and IgE secretion is shown in FIG. 9. 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 (), sIL-1R (.box-solid.) or 11B11 (
). 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.
[0150] FIG. 8 (panels A and B) shows 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, panel C 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.
[0151] FIG. 9 shows 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
[0152] 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.
[0153] 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.
[0154] FIG. 10 shows that in medium control (panel A) 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 (panel B)
or 11B11 (panel C) at the onset of culture returned Ia expression
to constitutive levels, whereas addition of sIL-1R (panel D) had no
effect. The results shown in FIG. 10 are representative of 3
separate experiments.
[0155] 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.
[0156] FIG. 11 shows that in medium control (panel A) 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 (panel B) or 11B11 (panel C) returned cell surface CD23
expression to constitutive levels, whereas addition of sIL-1R
(panel D) 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 FIG. 10 are also
representative of 3 separate experiments.
[0157] 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-4R In Vivo
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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 titre. 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
[0163] 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.
[0164] In order to determine the effect of sIL-4R administration on
the IgD-induced polyclonal 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.
[0165] 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
[0166] 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 elicited 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. FIG. 15 shows the results of these
experiments. The data are presented as mean footpad swelling
.+-.SEM.
[0167] 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 period (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
[0168] 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.
[0169] 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
[0170] 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.
[0171] 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 10.sup.7
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 10.sup.7 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).
[0172] Seven days after antigen administration, the mice were
sacrificed and the popliteal lymph nodes (PLN) were removed from
the right and left popliteal fossa by surgical dissection. Lymph
nodes were weighed and the results expressed as the difference ()
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 1 ug 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-US-00007 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
[0173] 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
[0174] Soluble murine IL-4 receptor also suppresses rejection of
organ grafts in vivo. In order to demonstrate this, neonatal
C57BL/6 (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.
[0175] 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-US-00008 TABLE C Effects of smuIL-1R Treatment on
Nonvascularized Heterotopic Cardiac Allograft Survival Treatment
Survival Time Median Survival Group (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
[0176] 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.
* * * * *