Note: Descriptions are shown in the official language in which they were submitted.
_~ _ 1341299
MAMMALIAN INTERLEUKIN-4
The present invention relates generally to
protein and mutein factors of the mammalian immune system
and to nucleic acids coding therefor. More particularly,
the invention relates to protein and mutein factors
(along with their encoding nucleic acids) which exhibit
both T cell growth factor activity and B cell growth
factor activity.
Recombinant DNA technology refers generally to
the technique of integrating genetic information from a
donor source into vectors for subsequent processing, such
as through introduction into a host, whereby the
transferred genetic information is copied and/or
expressed in the new environment. Commonly, the genetic
information exists in the form of complementary DNA
(cDNA) derived from messenger RNA (mRNA) coding for a
desired protein product. The carrier is frequently a
plasmid having the capacity to incorporate cDNA for later
replication in a host and, in some cases, actually to
control expression of the cDNA and thereby direct
synthesis of the encoded product in the host.
For some time, it has been known that the
mammalian immune response is based on a series of complex
cellular interactions, called the "immune network." _
Recent research has provided new insights into the inner
workings of this network. While it remains clear that
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much of the response does, in fact, revolve around the
network-like interactions of lymphocytes, macrophages,
granulocytes and other cells, immunologists now generally
hold the opinion that soluble proteins (e.g., the so-
called "lymphokines" or "monokines") play a critical role
in controlling these cellular interactions. Thus, there
is considerable interest in the isolation,
characterization, and mechanisms of action of cell
modulatory factors, an understanding of which should
yield significant breakthroughs in the diagnosis and
therapy of numerous disease states.
Lymphokines apparently mediate cellular
activities in a variety of ways. They have been shown to
support the proliferation, growth and differentiation of
the pluripotential hematopoietic stem cells into the vast
number of progenitors composing the diverse cellular
lineages responsible for the immune response. These
lineages often respond in a different manner when
lymphokines are used in conjunction with other agents.
Cell lineages that are especially important to
the immune response include two classes of lymphocytes:
B-cells, which can produce and secrete immunoglobulins
(proteins with the capability of recognizing and binding
to foreign matter to effect its removal), and T-cells of
various subsets that secrete lymphokines and induce or
suppress the B-cells and some of the other cells
(including other T-cells) making up the immune network.
Another important cell lineage is the mast cell
(which has not been positively identified in all
mammalian species)--a granule-containing connective
tissue cell located proximal to capillaries throughout
the body, with especially high concentrations in the
lungs, skin, and gastrointestinal and genitourinary
tracts. Mast cells play a central role in allergy-
related disorders, particularly anaphylaxis, as
follows: when selected antigens crosslink one class of
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immunoglobulins bound to receptors on the mast cell
surface, the mast cell degranulates and releases the
mediators (e. g., histamine, serotonin, heparin,
prostaglandins, etc.) which cause allergic reactions,
e.g., anaphylaxis.
Research to better understand (and thus
potentially treat therapeutically) various immune
disorders has been hampered by the general inability to
maintain cells of the immune system in vitro.
Immunologists have discovered that culturing these cells
can be accomplished through the use of T-cell and other
cell supernatants, which contain various growth factors,
such as some of the lymphokines.
The detection, isolation and purification of
factors such as lymphokines and other soluble mediators
of immune reactions is extremely difficult, and is
frequently complicated by the very nature of the
supernatants they are typically located in, the
divergencies and cross-overs of activities of the various
components in the mixtures, the sensitivity (or lack
thereof) of the assays utilized to ascertain the factors'
properties, the frequent similarity in the range of
molecular weights and other characteristics of the
factors, and the very low concentration of the factors in
their natural setting.
As more lymphokines become available, primarily
through molecular cloning, interest has heightened in
finding clinical applications for them. Because of
physiological similarities to hormones (e. g., soluble
factors, growth mediators, action via cell receptors),
potential uses of lymphokines have been analogized to the
current uses of hormones, e.g. Dexter, Nature, Vol. 321,
pg. 198 (1986). One hope is that the levels of
lymphokines in a patient can be manipulated directly or
indirectly to bring about a beneficial immune response,
e.g. suppression of inflammation, allergy, or tissue
X341299
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rejection, or stimulation or potentiation against
infection or malignant growth. Other potential clinical
uses of lymphokines include maintaining and expanding _in
vitro populations of certain immune system cells of one
person for eventual reintroduction into the same or
another person for a beneficial effect. For example,
investigations are currently underway to determine
whether populations of lymphokine-activated killer T
cells of a patient can be expanded outside his or her
body~and then reinjected to bring about an enhanced
antitumor response. Another potential clinical use of
lymphokines, particularly colony stimulating factors,
such as granulocyte-macrophage colony stimulating factor
(GM-CSF), and factors which enhance their activities, is
stimulating blood cell generation, for example in pre- or
post-chemotherapy or radiation therapy against tumors, in
treatment of myeloid hypoplasias, or in treatment of
neutrophil deficiency syndromes: Dexter, Nature, Vol.
321, pg. 198 (198b). Such factors would also be useful
in bone marrow transplant therapy, which is being used
increasingly to treat aplastic anemia and certain
leukemias.
There are two properties of lymphokines that
have important consequences for such clinical
applications: individual lymphokines are frequently
pleiotropic; and the biological effects of one lymphokine
can usually be modulated by at least one other
lymphokine, either by inhibition or by potentiation. For
example, tumor necrosis factor, which synergizes with
gamma-interferon, stimulates interleukin-1 (IL-1)
production and can activate the phagocytic activity of
neutrophils. IL-1, a protein produced by activated
macrophages, mediates a wide range of biological
activities, including stimulation of thymocyte
proliferation via induction of interleu~Cin-2 (IL-2)
release, stimulation of B-lymphocyte maturation and
~ 34~ 299
proliferation, fibroblast growth factor activity and
induction of acute-phase protein synthesis by
hepatocytes. IL-1 has also been reported to stimulate
prostaglandin and collagenase release from synovial
cells, and to be identical to endogenous pyrogen:
Krampschmidt, J. Leuk. Biol., Vol. 36, pgs. 341-355
(1984) .
Interleukin-2, formerly referred to as T-cell
growth factor, is a lymphokine which is produced by
lectin- or antigen-activated T cells. The reported
biological activities of IL-2 include stimulation of the
long-term in vitro growth of activated T-cell clones,
enhancement of thymocyte mitogenesis, and induction of
cytotoxic T-cell reactivity and plaque-forming cell
responses in cultures of nude mouse-spleen cells. In
addition, like interferons (IFNs), IL-2 has been shown to
augment natural killer cell activity, suggesting a
potential use in the treatment of neoplastic diseases:
Henney et al., Nature, Vol, 291, pgs. 335-338 (1981).
Some success has been reported in such therapy, e.g.
Lotze and Rosenberg, "Treatment of Tumor Patients with
Purified Human Interleukin-2," pgs. 711-7.19, in Sorg et
al., Eds. Cellular and Molecular Biology of Lymphokines
(Academic Press, Inc., New York, 1985); and Rosenberg and
Lotze, "Cancer Immunotherapy Using Interleukin-2 and
Interleukin-2-Activated Lymphocytes," Ann. Rev. Immunol.,
Vol. 4, pgs. 681-709 (1986). However, IL-2 toxicity has
limited the dosages which can be delivered to cancer
patients for taking advantage of these properties: Lotze
and Rosenberg, pgs. 711-719; and Welte et al., pgs. 755-
759, in Sorg et al. Eds. (cited above).
Metcalf, D., The Hematopoietic Colony
Stimulating Factors, (Elsevier, Amsterdam, 1984),
provides an overview of research concerning lymphokines
and various growth factors involved in the mammalian
immune response. Yung, Y.-P., et al., J. Immunol. Vol.
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127 pg. 794 (1981), describe the partial purification of
the protein of approximately 35 kd exhibiting mast cell
growth factor (MCGF) activity and its separation from
interleukin-2 (IL-2), also known as T-cell growth factor
(TCGF). Nabel, G., et al., Nature, Vol. 291, pg. 332
(1981) report an MCGF exhibiting a molecular weight of
about 45 kd and a pI of about 6Ø Clark-Lewis, I. and
Schrader, J., J. Immunol., Vol. 127, pg. 1941 (1981),
describe a protein having mast cell like growth factor
activity that exhibits a molecular weight of about 29 kd
in phosphate-buffered saline and about 23 kd in 6M
guanidine hydrochloride, with a pI of about 4-8 but of
about 6-8 after neuraminidase treatment. Murine IL-2 and
interleukin-3 (IL-3) have been partially characterized
biochemically by Gillis, S., et al., J. Immunol., Vol.
124, pgs. 1954-1962 (1980), and Ihle, J., et al., _J.
Immunol., Vol. 129, pgs. 2431-2436 (1982), respectively,
with IL-2 having an apparent molecular weight (probably
as a dimer) of about 30-35 kd and IL-3 having a molecular
weight of about 28 kd. Human IL-2 apparently has a
molecular weight of about l5.kd and is described by
Gillis, S., et al., Immu. Rev., Vol. 63, pgs. 167-209
(1982). Comparison between IL-3 and MCGF activities of
T-cell supernatants have been reported by Yung Y. and
Moore, M., Contemp. Top. Mol. Immunol., Vol. 10, pgs.
147-179 (1985), and Rennick, D., et al., J. Immunol.,
Vol. 134, pgs. 910-919 (1985).
An extensive literature exists concerning the
regulation of B-cell growth and differentiation by
soluble factors: e.g. for reviews see Howard and Paul,
Ann. Rev. Immunol., Vol. 1, pgs. 307-333 (1983); Howard
et al., Immunol. Rev., 1984, No. 78, pgs. 185-210;
Kishimoto et al., Immunol. Rev., 1984, No. 78 pgs. 97-
118; and Kishimoto, Ann. Rev. Immunol., Vol. 3, pgs. 133-
157 (1985). Some confusion has existed over the
nomenclature used for labeling the various factors
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_7_
because of differences in source materials, difficulties
in purification, and differences in the assays used to
define their biological activities. Consensus, in regard
to nomenclature apparently has been reached in some
cases: Paul, Immunology Today, Vol. 4, pg.. 322 (1983);
and Paul, Molecular Immunol., Vol. 21, pg. 343 (1984).
B-cell growth factor (BCGF) activity is characterized by
a capacity to cause DNA synthesis in B cells co-
stimulated by exposure to anti-IgM, or like antigens. It
is believed that interleukin-1 (IL-1) is also required
for BCGF activity to be manifested, at least when the
assay is conducted with low densities of B cells.
Alternative assays for human BCGF have been described:
e.g. Maizel et al, Proc. Natl. Acad. Sci., Vol. 80, pgs.
5047-5051 (1983) (support of long-term growth of human B
cells in culture). The activity associated with the
former assay has also been labelled B cell stimulatory
factor-1 (BSF-1) activity and BCGF I, to distinguish it
from similar and/or related activities. In particular,
an activity designated BCGF II has been described. It is
characterized by a capacity to cause DNA synthesis in
mitogen-stimulated B cells or in transformed B cell
lines. Mitogens associated with BCGF II activity include
dextran sulfate, lipopolysaccharide, and Staphylococcus
extracts. BCGF I registers no response in these
assays. In humans it is believed that BCGF II is a
molecule having a molecular weight of about 50
kilodaltons (kD), and that it acts synergistically with
BCGF I (i.e. BSF-1) in promoting B cell proliferation in
an immune response: Yoshizaka et al., J. Immunol., Vol.
130, pgs. 1241-1246 (1983). Howard et al., J. Exp. Med.,
Vol. 155, pgs. 914-923 (1982) were the first to show the
existence of a murine BCGF (later to be called variously
BCGF I, BSF-1, or IgGl induction factor) distinct from
interleukin-2. Similar observations were reported almost
simultaneously for a human system by Yoshizaki et al., J.
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Immunol., Vol. 128, pgs. 1296-1301 (1x82), and later by
Okada et al., J. Exp. Med., Vol. 157, pgs. 583-590
(1983).
Biochemical and biological characterization of
molecules exhibiting BCGF, or BSF-1, activity has
progressed steadily since these initial discoveries.
Maizel et al., Proc. Natl. Acad. Sci., Vol. 79, pgs.
5998-6002 (1982), have reported a trypsin-sensitive human
BCGF having a molecular weight of 12-13 kD and an
isoelectric point (pI) of about 6.3-6.6. Farrar et al.,
J. Immunol., vol. 131, pgs. 1838-1842 (1983) reported
partial purification of a heterogeneous murine BCGF
having molecular weights of 11 and 15 kD by SDS-PAGE and
pIs of 6.4-8.7. Ohara and Paul, in Nature, Vol. 315,
pgs. 333-336 (1985) describe a monoclonal antibody
specific for murine BSF-1, and give molecular weights for
BSF-1 of 14 kD and 19-20 kD with pI of 6.7. Butler et
al., J. Immunol., Vol. 133, pgs. 251-255 (1984), report a
human BCGF having a molecular weight of 18-20 kD and a pI
of 6.3-6.6. Rubin et al., Proc. Natl. Acad. Sci., vol.
82, pgs. 2935-2939 (1985), report that pre-incubation of
resting B cells with BSF-1 prior to exposure to anti-IgM
antibodies increases cell volume, and later speeds entry
to S phase upon exposure to anti-IgM antibodies. Vitetta
et al, J. Exp. Med., Vol. 162, pgs. 1726-1731 (1985),
describe partial purification of murine BSF-1 by reverse
phase HPLC of serum-free supernatants of EL-4 cells.
SDS-PAGE indicated a protein of about 20-22kD. Ohara et
al., J. Immunol., Vol. 135, pgs. 2518-2523 (1985) also
report partial purification of murine BSF-l by a similar
procedure, and report the factor to be a protein of about
18-21.7 kD. Sideras et al., in Eur. J. Immunol., Vol.
15, pgs. 586-593, and 593-598 (1985), report partial
purification of a murine IgGl-inducing factor, that is a
BSF-1, and report. the factor to be a protein of about 20
kD having pIs of 7.2-7.41and 6.2-6.4; and Smith and
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Rennick, in Proc. Natl. Acad. Sci., Vol. 83, ngs. 1857-
1861 (1986), report the separation of a factor from IL-2
and IL-3 which exhibits T cell growth factor activity and
mast cell growth factor activity. Later, Noma et al.,
Nature, Vol. 319, pgs. 640-646 (1986), cloned and
sequenced a nucleic acid coding for the Sideras et al.
factor, and Lee et al., Proc. Natl. Acad. Sci., Vol. 83,
pgs. 2061-2065 (1986) cloned and sequenced a nucleic acid
coding for the Smith and Rennick factor. More recently,
Grabstein et al., J. Exp. Med., vol. 163, pgs. 1405-1414
(1986), report purifying and sequencing murine BSF-1.
Milanese, et al, in Science, Vol. 231, pgs.
1118-1122 (1986), report a lymphokine unrelated to BSF-1
which they provisionally designate IL-4A. Their IL-4A is
a 10-12 kD protein secreted from helper T cells after
cross-linking of T3-Ti receptors. It stimulates resting
lymphocytes via interaction with T11 receptors and
subsequent induction of interleukin-2 (IL-2) receptors.
Sanderson et al., in Proc. Natl. Acad. Sci.,
Vol. 83, pgs. 437-440 (1986), proposed that the name
interleukin 4 be given to eosinophil differentiation
factor based on evidence that it is apparently the same
as B cell growth factor II.
From the foregoing it is evident that the
discovery and development of new lymphokines could
contribute to the development of therapies for a wide
range of degenerative conditions which directly or
indirectly involve the immune system and/or hematopoietic
cells. In particular, the discovery and development of
lymphokines which enhance or potentiate the beneficial
activities of known lymphokines would be highly
advantageous. For example, the dose-limiting toxicity of
IL-2 in tumor therapy could be reduced by the
availability of a lymphokine or cofactor with
potentiating effects; or the efficacy of bone marrow
transplants could be increased by the availability of
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factors which potentiate the activities of the colony
stimulating factors.
The present invention is directed to mammalian
interleukin-4 (IL-4). It includes nucleic acids coding
for polypeptides exhibiting IL-4 activity, as well as the
polypeptides themselves and methods f.or their
production. The nucleic acids of the invention are
defined (1) by their homology to cloned complementary DNa
(cDNA) sequences disclosed herein, and (2) by functional
assays for IL-4 activity applied to the polypeptides
encoded by the nucleic acids. As used herein, the term
"IL-4 activity" in reference to a protein or a
polypeptide means that the protein or polypeptide
exhibits both B-cell growth factor (BCGF) activity and T
cell growth factor (TCGF) activity. For a given mammal,
IL-4 activity is determined by species-specific TCGF and
BCGF assays. As explained more fully below, specific
embodiments of IL-4 can be further characterized by
additional assays. For example, some forms of murine IL-
4 exhibit mast cell growth factor (MCGF) activity; some
forms of both human and murine IL-4 potentiate the TCGF
activity of IL-2; some forms of both murine and human IL-
4 potentiate GM-CSF stimulated proliferation in certain
cell types; some forms of both human and murine IL-4 can
induce Fc-epsilon receptor expression on B cells; and
some forms of both human and murine IL-4 can induce the
expression of major histocompatibility complex (MHC)
antigens on B cells: the class II DR antigen on human B
cells, and the Ia antigen on mouse B cells.
The invention is based in part on the discovery
and cloning of cDNAS which are capable of expressing
proteins having IL-4 activity. cDNA clones of the
invention include human cDNA inserts of plasmid vectors
"clone 46" (also referred to herein as pcD-2F1-13 or pcD-
46) and "clone 125" (also referred to herein as pcD-125) ;
and mouse cDNA insert of plasmid vector pcD-2A-E3. The
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three vectors are deposited with the American Type
Culture Collection (ATCC), Rockville, MD, under ATCC
accession numbers 53337, 67029, and 53330, respectively.
The invention includes nucleic acids having
nucleotide sequences which are effectively homologous to
the cDNA clones of the invention and which express IL-4
activity. Nucleic acids and proteins of the invention
can be derived from the above-mentioned cDNAs by standard
techniques for mutating nucleic,acid sequences. They can
be prepared de novo from immune system-derived cell
lines, such as T cell hybridomas, which contain or can be
induced to contain messenger RNA (mRNA) sequences coding
for IL-4; and they can be obtained by probing DNA or RNA
extracts or libraries with probes derived from the cDNA
clones of the invention.
The term "effectively homologous" as used
herein means that the nucleotide sequence is capable of
being detected by a hybridization probe derived from a
cDNA clone of the invention. The exact numerical measure
of homology necessary to detect nucleic acids coding for
IL-4 activity depends on several factors including (1)
the homology of the probe to non-IL-4 coding sequences ,
associated with the target nucleic acids, (2) the
stringency of the hybridization conditions, (3) whether
single stranded or double stranded probes are employed,
(4) whether RNA or DNA probes are employed, (5) the
measures taken to reduce nonspecific binding of the
probe, (6) the nature of the label used to detect the
probe, (7) the fraction of guanidine and cytosine bases
in the probe, (8) the distribution of mismatches between
probe and target, (9) the size of the probe, and the
like.
Preferably, an effectively homologous probe
derived from the cDNA of the invention is at least fifty
percent (50~) homologous to the sequence to be
isolated. More preferably, the effectively homologous
1 3~1 299
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probe is at least seventy-five to eighty percent (75-80~)
homologous to the sequence to be isolated. Most
preferably, the effectively homologous probe is at least
ninety percent (90$) homologous to the sequence to be
isolated.
Homology as the term is used herein is a
measure of similarity between two nucleotide (or amino
acid) sequences. Homology is expressed as the fraction
or percentage of matching bases (or amino acids) after
two sequences (possibly. of unequal length ) have been
aligned. The term alignment is used in the sense defined
by Sankoff and Kruskal in chapter one of Time Warps,
String Edits, and Macromolecules~ The Theory and
Practice of Sequence Comparison (Addison-Wesley, Reading,
MA, 1983). Roughly, two sequences are aligned by
maximizing the number of matching bases (or amino acids)
between the two sequences with the insertion of a minimal
number of "blank" or "null" bases into either sequence to
bring about the maximum overlap. Given two sequences,
algorithms are available for computing their homology:
e.g. ~Teedleham and Wunsch, J. Mol. Biol., Vol. 48, pqs.
443-453 (1970); and Sankof.f and Kruskal (cited above)
pqs. 23-29. Also, commercial services are availahle f_or
performing such comparisons, e.g. Intelligenetics, Inc.
(Palo Alto, CA).
The present invention also relates to the
natural mammalian growth factors (polypeptides)
exhibiting a broad activity spectrum for a variety of
cells integral to the immune response. Methods of
producing such factors and their use for in vivo
treatment of mammals are provided. Such polypeptide
compositions include a mammalian factor in substantially
r~ure form capable of exhibiting 8-cell, T-cell and mast
cell stimulatory activity.
These factors, which were originally isolated
from the supernatant fluid of various T cells, include
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natural polypeptides exhibiting, under non-reducing
conditions, a molecular weight of about 20 kd or 15 kd,
or active fragments of such polypeptides. The factors
may be used alone or in conjunction with other compounds
for various diagnostic and therapeutic purposes,
including the production of specific antibodies.
A preferred embodiment of the invention is the
set of glycosylated or unglycosylated human IL-4 proteins
and muteins defined by the following formula:
X(His) - X(Lys)- X(Cys) - X(Asp) - X(Ile) - X(Thr)
-
X (Leu) - X (Gln)- X (Glu)- X (Ile)- X (Ile)- X (Lys)
-
X (Thr - X (Leu)- X (Asn)- X (Ser)- X (Leu)- X (Thr
) ) -
X(Glu) - X(Gln)- X(Lys) - X(Thr) - X(Leu) - X(Cys)
-
X (Thr) - X (Glu)- X (Leu)- X (Thr - X (Val)- X (Thr
) ) -
X (Asp) - X (Ile)- X (Phe - X (Ala)- X (Ala)- X (Ser
) ) -
X (Lys) - X (Asn)- X (Thr)- X (Thr)- X (Glu)- X (Lys)
-
X(Glu) - X(Thr)- X(Phe) - X(Cys) - X(Arg) - X(Ala)
-
X (Ala) - X (Thr)- X (Val)- X (Leu)- X (Arg)- X (Gln)
-
X(Phe) - X(Tyr)- X(Ser) - X(His) - X(His) - X(Glu)
-
X (Lys) - X (Asp)- X (Thr - X (Arg - X (Cys)- X (Leu)
) ) -
X (Gly) - X (Ala)- X (Thr - X (Ala)- X (Gln)- X (Gln)
) -
X (Phe) - X (His)- X (Arg)X (His) - X (Lys)- X (Gln)
- -
X(Leu) X(Ile) - X(Arg) X(Phe) - X(Leu) - X(Lys)
- - -
X (Arg X (Leu) - X (Asp)X (Arg) - X (ASn)X (Leu)
) - - - -
X (Trp) X (Gly) X (Leu) X (Ala) - X (Gly)X (Leu)
- - - - -
X (ASn) X (Ser X (Cys) X (Pro) - X (Val)X (Lys)
- ) - - - -
X (Glu) X (Ala) X (ASn) X (Gln) X (Ser X (Thr
- - - - ) - ) -
X (Leu) X (Glu) X (ASn) X (Phe) X (Leu) X (Glu)
- - - - - -
X(Arg) X(Leu) X(Lys) X(Thr) X(Ile) X(Met)
- - - - - -
X(Arg) X(Glu) X(Lys) X(Tyr) X(Ser) X(Lys)
- - - - - -
X (Cys X ( Ser X ( Ser
) - ) - )
Formula I
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wherein the term X(Xaa) represents the group of
synonymous amino acids to the amino acid Xaa. Synonymous
amino acids within a group have sufficiently similar
physicochemical properties for substitution between
members of the group to preserve the biological function
of the molecule: Grantham, Science, vol. 185, pgs. 862-
864 (1974). It is clear that insertions and deletions of
amino acids may also be made in the above-defined
sequence without altering biological function,
particularly if the insertions or deletions only involve
a few amino acids, e.g. under ten, and do not remove or
displace amino acids which are critical to a functional
conformation, e.g. cysteine residues, Anfinsen,
"Principles That Govern The Folding of Protein Chains",
Science, Vol. 181, pgs. 223-230 (1973). Proteins and
muteins produced by such deletions and/or insertions come
within the purview of the present invention. Whenever
amino acid residues of the protein of Formula I are
referred to herein by number, such number or numbers are
in reference to the N-terminus of the protein.
Preferably the synonymous amino acid groups are
those defined in Table I. More preferably, the
synonymous amino acid groups are those listed before the
second slash in each line in Tahle I; and most preferably
the synonymous amino acid Groups are those listed before
the first slash in each line in Table IT.
Table I. Preferred Grou s of Synonymous Amino Acids
Amino Acid Synonymous Grou
Ser Ser,//Thr, Gly, Asn
Arg Arg,/His, Lys,/Glu, Gln
Leu Leu, Ile, Met,/Phe,/Val, Tyr
Pro Pro,/Ala,/Thr, Gly
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Table I (continued)
Amino Acid Synonymous Group
Thr Thr,//Pro, Ser, Ala, Gly, His, Gln
Ala Ala,/Pro,/Gly, Thr
Val Val,/Met, Ile/Tyr, Phe,
Leu, Val
Gly Gly,//Ala, Thr, Pro, Ser
Ile Ile, Met, Leu,/Phe, Val,/Ile, Tyr
Phe Phe,/Met, Tyr, Ile, Leu,/Trp, Val
Tyr Tyr,/Phe,/Trp, Met, Ile, Val, Leu,
Cys Cys, Ser,//Thr
His His,/Gln, Arg,/Lys, Glu, Thr
Gln Gln,/Glu, His,/Lys, Asn, Thr, Arg
Asn Asn,/Asp,/Ser, Gln
Lys Lys,/Arg,/Glu, Gln, His
Asp Asp,/Asn,/Glu
Glu Glu,/Gln,/ Asp, Lys, Asn, His, Arg
Met Met, Ile, Leu,/Phe, Val/
The invention includes the polypeptides of
Formula I with amino acid substitutions (between an amino
acid of the native human IL-4 and a synonymous amino
acid) at a single position or at multiple positions. The
term "N-f old substituted" is used to describe a subset of
polypeptides defined by Formula I wherein the native
amino acids have been substituted by synonymous amino
acids at at most N positions. Thus, for example, the
group of 1-fold substituted polypeptides of Formula I
consists of 559 polypeptides for the preferred groups of
synonymous amino acids, 189 for the more preferred groups
of synonymous amino acids, and 56 for the most preferred
groups of synonymous amino acids. These numbers were
arrived at by summing the number of amino acids of each
kind in the native chain times the size of the synonymous
amino acid group for that amino acid. Preferably the
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group of human IL-4 polypeptides consists of the 10-fold
substituted polypeptides of Formula I; more preferably it
consists of 3-fold substituted polypeptides of Formula I;
and most preferably it consists of 1-fold substituted
polypeptides of Formula I, which in particular includes
the native human IL-4 polypeptide.
Likewise, the term "N-fold inserted" in
reference to the peptides of Formula I is used to
describe a set of polypeptides wherein from 1 to N amino
acids have been inserted into the sequence defined by
Formula I. Preferably, the inserted amino acids are
selected from the preferred groups of synonymous amino
acids (Table I) of the amino acids flanking the
insertion; more preferably they are selected from the
more preferred groups of synonymous amino acids (Table
II) of the amino acids flanking the insertion, and most
preferably they are selected from the most preferred
groups of synonymous amino acids (Table I:II) of the amino
acids flanking the insertion. Thins, for example, one
subgroup of the group of 1-fold inserted peptides
comprises an amino acid inserted between the N-terminal
X(His) and the adjacent X(Gly). The insertions defining
the members of this subgroup are preferably selected from
the group consisting of Pro, Ala, Gly, Thr, Ser, Gln,
Glu, Arg, His, and Lys; more preferably they are selected
from the group consisting of Gly, His, Gln and Arg, and
most preferably they are selected from the group
consisting of His and Gly. Insertions can be made
between any adjacent amino acids of Formula I. Since
there are 128 possible insertion locations, and since
multiple insertions can be made at the same location, a
2-fold inserted peptide of Formula I gives rise to 16,384
subgroups of peptides, and the size of each subgroup
depends on the sizes of the synonymous amino acid groups
of the amino acids flanking the insertions.
o...
1 341 299
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The term "N-fold deleted" in reference to the
polypeptides of Formula I is used to describe a set of
peptides having from 1 to N amino acids deleted from the
sequence defined by Formula I. Thus, the set of 1-fold
deleted polypeptides of Formula I consists of 129
subgroups of polypeptides each 128 amino acids in length
(128-mers). Each of the subgroups in turn consists of
all the 128-mers defined by the preferred, more
preferred, and most preferred synonymous amino acid
groups.
The above preferred embodiment of the invention
further includes nucleotide sequences effectively
homologous to or capable of encoding the polypeptides of
Formula I for the preferred, more preferred, and most
preferred groups of synonymous amino acids. More
preferably said nucleotide sequences are capable of
encoding the polypeptides of Formula I for the preferred,
more preferred, and most preferred groups of synonymous
amino acids.
In particular, the invention includes native
human IL-4, and all nucleotide sequences
capable of encoding it.
Throughout, standard abbreviations are used to
designate amino acids, nucleotides, restriction
endonucleases, and the like: e.g. Cohn, "Nomenclature and
Symbolism of a-Amino Acids, "Methods in Enzymology, Vol.
106, pgs. 3-17 (1984); Wood et al. Biochemistry: A
Problems Approach, 2nd ed. (Benjami.n, Menlo Park, 1981);
and Roberts, "Directory of Restriction Endonucleases",
Methods in Enzymology, Vol. 68, pgs. 27-40 (1979).
The present invention is addressed to problems
associated with the application of immunoregulatory
agents to treat medical and/or veterinary disorders. In
particular, it provides compounds which have beneficial
effects when used alone or which can act in concert with
1 341 299
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other lymphokines and immune-system mediators to produce
beneficial effects.
Preferred embodiments of the invention will now
be described, partly in conjunction with the accompanying
drawings (Figs. 1 to 19).
Figure lA illustrates the nucleotide sequence
and deduced amino acid sequence of the insert of vector
pcD-2A-E3, which expresses murine IL-4.
Figure 1B illustrates the nucleotide sequence
and deduced amino. acid sequence of the insert of vector
pcD-125, which expresses human IL-4.
Figure 1C illustrates the amino acid sequence
of purified native human IL-4 expressed and secreted by
COS 7 monkey cells transfected with pcD-125.
Figure 2A is a map of vector pcD-2A-E3, the
insert of which codes murine IL-4.
Figure 28 is a restriction endonuclease
cleavage map of the insert of vector pcD-2A-E3.
Figure 2C is a map of vector pcD-46, the insert
of which codes human IL-4.
Figure 2D is a restriction endonuclease
cleavage map of the insert of vector pcD-~6.
Figure 3H illustrates relative TCGF activities
(over a range of dilutions) of various culture
supernatants including one (curve 1) from pcD-2a-E3-
transfected COS 7 cells.
Figure 3B illustrates relative MCGF activities
(over a range of dilutions) of various culture
supernatants including one (curve 1) from pcD-2A-E3-
transfected COS 7 cells.
Figure 3C illustrates the relative degrees of
Ia induction produced by the indicated amounts of
supernatant from pcD-2A-E3-transfected COS 7 cells (curve-
1), Cl.Ly1+2-/9 cells (curve 2), and mock transfected COS
7 cells (curve 3) .
1341299
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Figure 3D graphically illustrates the extent of
IgE and IgGl induction by supernatants from pcD-2A-E3-
transfected COS 7 cells and various controls in T cell-
depleted mouse spleen cells.
Figure ~4A illustrates the TCGF activities of
several pcD-125 transfection supernatants and controls as
measured by a color~metric proliferation assay on the
factor-dependent human helper T cell line, JL-EBV.
Figure 4B illustrates the TCGF activities of a
pcD-125 transfection supernatant and controls as measured
by a colorimetric proliferation assay on PHa-stimulated
peripheral blood lymphocytes.
Figure 4C illustrates the TCGF activities of a
pcD-125 transfection supernatant and controls as measured
by tritiated thymidine incorporation by PHA-stimulated
peripheral blood lymphocytes.
Figure 5A is a histogram of cell frequency
versus fluorescence intensity for a control population of
stimulated human tonsilar B cells whose Fc-epsilon
receptors have been fluorescently labeled.
Figure 5B is a histogram of cell frequency
versus fluorescence intensity for a population of
stimulated human tonsilar B cells which had been exposed
to medium consisting of 0.1~ supernatant from pcD-125-
transfected COS 7 cells and whose Fc-epsilon receptors
have been fluorescently labeled.
Figure 5C is a histogram of cell frequency
versus fluorescence intensity for a population of
stimulated human tonsilar B cells which had been exposed
to medium consisting of 1$ supernatant from pcD-125-
transfected COS 7 cells and whose Fc-epsilon receptors
have been fluorescently labeled.
Figure 5D is a histogram of cell frequency
versus fluorescence intensity for a population of
stimulated human tonsilar B cells which had been exposed
to medium consisting of 10$ supernatant from pcD-125-
1341~gg
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transfected COS 7 cells and whose Fc-epsilon receptors
have been fluorescently labeled.
Figure 6A illustrates the nucleotide sequence
of a synthetic human IL-4 gene useful for expressing
native or mutant IL-4s in E. coli.
Figure 6B is a restriction endonuclease
cleavage map of a synthetic human IL-4 gene inserted in.
plasmid pUCl8.
Figures 7A-7F respectively illustrate the
double stranded DNA fragments lA/B through 6A/B used to
construct a synthetic human IL-4 gene.
Figure 8 illustrates nucleotide sequences
adjacent to the initiator ATG codon in the E. coli
expression vector TAC-RBS. The sequences commence at an
EcoRI restriction site and end with a HindIII site. The
ribosome binding sequence (RBS) showing complementarity
to the 3' end of 16S ribosomal RNA is underlined, and the
ATG initiator codon is underlined.
Figure 9 illustrates histograms of cell
frequency versus fluorescence intensity for populations
of cells derived from a patient with bare lymphocyte
syndrome. The cells were stained with fluorescently-
labeled anti-DR monoclonal antibodies.
Figure 10 illustrates the 215 nm absorption
profile in the final human IL-4 purification step, which
consisted of reversed-phase HPLC on a C-4 column.
Figure 11 is a construction map of plasmid
pEBV-178 containing human IL-4 cDNA.
Figure 12 is a construction map of plasmid
T RPC 11.
Figure 13A is a construction map of plasmid
pMF-alpha8.
Figure 13B illustrates the TCGF activities of
several transfection supernatants from yeast cultures
expressing native human IL-4 and various muteins thereof.
1 341 29 9
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Figure 14 depicts growth curves of factor-
dependent cell lines; MC/9 mast cells (lA), DX-2 mast
cells (1B), NFS-60 cells (1C) and HT2 T cells (1D). 5 x
103 cells were cultured with varying concentrations of
C1.1 supernatant (closed circles), IL-2R (closed
squares), IL-3R (open circles), GM-CSFR (open triangles),
IFN-yR (closed triangles), or P388D1 supernatant (open
diamonds). MC/9 cells (lA) were also cultured with
varying concentrations of purified IL-3 (closed circles)
or IL-3R mixed with 200 units of IL-2R (closed squares),
GM-CSFR (open triangles), IFN-YR (closed triangles), or
P388D1 supernatant (open diamonds). Growth factor
activity was measured after 24 hours by colorimetric
assay. The absorbance at 570 nm ( reference 630 nm) was
measured on a Dynatek Micro Elisa reader.
Figure 15 illustrates the FPLC cation exchange
chromatography of C1.1 supernatant. 7 ml (35 mg protein)
of concentrated supernatant was dialyzed into 50 nM Na
phosphate, 1 mm EDTA, pH 7.0 (7.8 mS/cm) and applied to
Pharmacia Mono S column (0.5 x 5 cm) equilibrated with
the same buffer (Buffer A). Buffer B = A + 1M NaCl.
Elution conditions: 0.5 ml/min flow rate; 0.5 ml/
fraction; 0-40~ B in 40 min, 40-100 R in 10 minutes.
Aliquots of_ each fraction were assayed for proliferation
activity on NFS-60 (IL-3, closed circles), HT2 (TCGF,
open circles) and MC/9 (MCGF) cells. The MC/9 response
is not shown; arrows denote positions were MC/9
proliferation levels reached those of C1.1 supernatant.
Figure 16A illustrates reverse phase C8
chromatography of IL-3 depleted C1.1 supernatant. 100 ul
of 3X mono-S passed supernatant (fractions 59-61, Fig.
15) in 0.1~ TFA was loaded onto Pharmacia C8 reversed
phase column (0.5 x 2 cm). Buffer A = 0.1$ TEA in H20,
Buffer B = 0.1$ TFA in acetonitrile. Elution
conditions: 0.5 ml/min, 0.5 ml/fraction, 0-25~ B in 4
minutes, 25-60$ B in 50 minutes, 60-100$ B in 4
1 341 X99
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minutes. Aliquots of each fraction were assayed for
proliferation activity on ~)FS-60 (IL-3, closed circles),
HT2, (TCGF, open circles) and MC/9 cells (MCGF, closed
triangles). Arrow denotes fraction containing peak TCGF
activity, and which produced MC/9 proliferation levels
similar to those of C1.1 supernatant after addition of
saturating IL-3R levels to each fraction.
Figure 16B illustrates reverse phase C8
chromatography of GK15-1 murine T cell supernatant. 2 mg
(0.5 ml) of concentrated GK15-1 supernatant in 0.1~ TFA
was applied to a Pharmacia C8 reverse phase column and
eluted as above. Aliquots of each fraction were assayed
for TCGF activity on HT2 cells (open circles). Arrow
marks the position where murine IL-2R eluted under
identical conditions. INSERT: titrations of C1.1
(closed circles) and IL-2R (closed squares) and GK15-1
supernatant (open circles) TCGF activities directly
compared on the same plate.
Figure 17 depicts the MCGF and TCGF activity of
partially purified factor from reverse phase column (RP
factory. Proliferation was rqpasured by [3H]-thymidine
incorporation after a 24 hour culture period. MC/9 mast
cells and HT2 T cells were cultured with. varying
concentration of C1.1 supernatant (closed circles), IL-3R
(open circles), IL-2R (open triangles), RP factor (open
squares) and dilutions of RP factor in the presence of
200 units of IL-3R (closed squares) or dilutions of_ IL-2
in the presence of saturating levels of RP factor (closed
triangles).
Figure 18 illustrates chromatofocussing of Cl.l
supernatant. 1.5 ml (3.5 mq) of supernatant in 25 nM
bis-tris, pH 7.1 was loaded onto a Pharmacia Mono P
column (0.5 x 20 cm) and eluted with Polybuffer 74
(1:10), pH 4.0, flow rate 0.5 ml/min, 1 ml/fraction.
Aliquots of each fraction were assayed by proliferation
on NFS-60 (IL-3, open circles) HT2 (TCGF, closed circles)
1341298
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and MC/9 (MCGF, not shown, except that peak MCGF activity
is indicated by arrow). The TCGF peak (fraction 12,
pI=6.2) coincides with a minimum IL-3 activity; MCGF
proliferation levels are highest at the arrow
designation.
Figure 19 depicts an SDS PAGE of C1.1 super-
natant and partially purified MCGF/TCGF.
A) Non-reducing SDS PAGE of unfractionated
supernatant. Prior reduction with 50 mM DTT (60° x
5 minutes) shifts TCGF peaks to slightly higher
molecular weights (21 Kd and 16 Kd), and is
accompanied by a drastic loss in activity.
B) Non-reducing SDS PAGE of peak MCGF/TCGF
fractions from cation exchange chromatography
(Fractions 59-61, Fig. 15). Prior reduction with 50
mM DTT (at 60°C for 5 minutes) shifts MCGF and TCGF
peaks to slightly higher molecular weights (21 Kd
and 16 Kd), and is accompanied by a drastic loss in
activity. Arrows mark the only fractions to which
addition of saturating amounts of IL-3 raises MCGF
activity to supernatant levels: IL-3 (open
circles), TCGF (closed circles), MCGF (open
triangles).
The present invention includes glycosylated or
unglycosylated mammalian polypeptides which exhibit IL-4
activity, and which are derivable from the IL-4
polypeptides disclosed herein using standard protein
engineering techniaues. The invention also includes
nucleic acids having sequences capable of coding for the
polypeptides of the invention, and nucleic acids whose
seauences are effectively homologous to the cDNA clones
of the invention. Finally, the invention includes
methods of making the qlycosylated or unqlycosylated
polypeptides of the invention which utilize the
nucleotides seauences disclosed herein, and methods of
using the polypeptides of the invention.
134199
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Techniques for making, using, and identifying
the polypeptides and nucleic acids of the invention are
discussed below in general terms. Afterwards several
specific examples are provided wherein the general
techniques are applied using specific cell types,
vectors, reagents, and the like. Their isolation from
natural sources will first be briefly described.
These polypeptides can be purified to apparent
homogenity from supernatants of readily available
cellular sources, and can thus be economically
manufactured in pure form, enabling various therapeutic
and other utilities.
These polypeptides are characterized by
including a polypeptide exhibiting B-cell-, T-cell- and
mast-cell-stimulatory activity. In natural form in one
species, the polypeptides exhibit a molecular weight on
SDS PAGE of about 20 kd and 15 kd under non-reducing
conditions and 21 kd and 16 kd under reducing
conditions. A composition containing the subject
polypeptides in substantially pure form exhibits an
isoelectric point (p~) of about 6.2 by chromato-
focussinq. The growth factors are extremely potent
agents, exhibiting significant activity at extremely low
concentrations (e.q., less than 1 nanogram per ml). The
polypeptides can be distinguished from hormones and other
proteins produced naturally by T cells (e.g., IL-2, IL-3,
GM-CSF and IFN-Y) through cation exchange chromatography
or unique elution conditions from reverse-phase columns.
In murine species, some polypeptides of the
present invention support only low levels of
proliferation of certain cells, such as IL-3-dependent
mast cell lines, but synergistically enhance the growth
of such cells (e.q., mast cells) in the presence of a
second factor (e.q., IL-3). The polypeptides can
stimulate the proliferation o.f several T-cell lines, but
generally to a lesser extent than purified IL-2. Also,
1 341 29 9
-25-
such polypeptides can induce Ia expression on resting B-
cells and enhance IqGl and IqE secretion by B-cells,
similar to BSE-1. These activities are not separable
despite multiple biochemical fractionations. However,
most of the activities are destroyed after reduction,
e.g., in the presence of SDS.
These factors can be obtained from a number of
natural sources in a variety of ways. One suitable
source is any of_ a number of T-cell lines that produce
the subject polypeptides. One such T-cell line was
derived from C57GL6 mice (Noble, G. et al., Proc. Natl.
Acad. Sci. U.S.A., 78: 1157-1161 (1981), which line can
be maintained in modified supplemental DME (Noble, G. et
al., Cell, 23: 19-28 (1982). This cell line, originally
designated Cl.Ly1+2-/9, was deposited at the American
Type Culture Collection and designated accession number
CRL 8179. Other suitable cellular sources of these
polypeptides include almost any cells that secrete the
various antibodies ascribed to the polypeptides, such as
human peripheral blood lymphocytes as well as any of the
well known T-cell sources, such as tonsils, spleens, etc.
The supernatant from such cellular sources or,
in some cases, fluid from the disrupted cells, may be
subjected to a variety of well-known purification
procedures in order to separate the proteins of the
present invention. Preferably, the purification
techniques are used in combination to ensure high purity
levels. Typically, the purifications will utilize gel
filtration chromatography, SP cation exchange chroma-
tography, reverse phase chromatography, group-specific
chromatography (e. g., on Heparin Sepharose), or other
common separation methods providing effective protein
fractionation. High pressure liquid chromatography
(HPLC), isoelectric focussing, and SDS polyacrylamide gel
electrophoresis may also be utilized.
1 341 299
-26-
For example, a supernatant from an appropriate
cell line may he first fractionated by strong cation
exchange chromatography. Typically, the polypeptides of
the present invention remain bound to the column, while
about 98$ of the loaded protein passes through. The
remaining bound protein may be eluted with a salt
gradient. In one embodiment of the present invention, a.
sodium chloride gradient released the subject polypeptide
at about 0.19 M. This fractionation was repeated twice
more (primarily to remove residual IL-3), providing
greater than about 95$ purity. Thereafter, the material
was successively fractionated by Heparin-Sepharose and
reverse phase (c4 or c18) chromatography. In the latter
case, these polypeptides eluted at 42$ acetonitrile.
These pooled fractions contain the subject polypeptide in
extremely high purity, essentially to homogeneity (a
single band at about 20 kd on silver-stained SDS-PAGE).
This purified material is at least 65,000 times more pure
than the material as it exists in natural form.
These polypeptides may be used as antigens for
the production of antibodies, which in turn may be used
as antigens for the production of anti-idiotypic
antibodies. Either polyclonal or monoclonal antibodies
may be prepared in conventional ways. These polypeptides
or fragments thereof, generally fragments having at least
about 15 amino acids, may be used by themselves, but are
preferably bound or linked to an adjuvant or antigen
which activates the immune system. Various antigens may
be used, such as serum albumins, keyhole limpet
hemocyanin, globulins, or the like. A wide variety of
techniques are available for linking adjuvants to poly-
peptides, such as glutaraldehyde, maleimidobenzoic acid,
diazobenzoic acid, or the like. Adjuvants include
Freund's adjuvant, aluminum hydroxide, or the like. The
antigen is injected into an appropriate host in
conventional amounts, and one or more booster injections
1341299
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may be made at from 2 to 4 week intervals. Where
monoclonal antibodies are employed, normally a mouse is
injected with the original and booster injections and the
spleen is isolated, and then the splenacytes are fused
with an appropriate fusion partner in accordance with
conventional techniques. See, for example, Galfre et
al., Nature (1977) 266:550; Kennett et al., Current
Topics in Microbiology and Immunolocly (1978) 81:77; and
U.S. Patent Nos. 4,381,292 and 4,363,799. However, for
special purposes, other mammals may be employed, such as
primates, e.g., humans, for production of antibodies
having human Fc chains.
The polypeptides may be used by themselves or
in combination with the antibodies in diagnosis for the
polypeptides. Either or both may be labeled or unlabeled
for use in diagnostic assays. A large number of
diagnostic assays are described in the literature and
including binding, either directly or indirectly, these
polypeptides or antibodies to a variety of labels, such
as enzymes, radionuclides, fluorescers, substrates,
coenzymes, particles, e.g., magnetic particles, or the
like. As illustrative of. these assays see for example
U.S. Patents Nos. 3,817,837; 3,850,752; 4,174,384;
4,177,437 and 4,374,925.
Various assays are divided arbitrarily into
homogeneous and heterogeneous immunoassays, where the
distinction is based on whether the complex between the
polypeptide and its antibody must be separated from the
uncomplexed members of the specific binding pair.
Various assays are referred to as EI, EhISA, RIA,
homogeneous EIA, dot-blot, Westerns, or the like.
Antibodies to these polypeptides may be used in
themselves as antigens to produce anti-idiotypes, which
may serve as competitive antigens, having epitopic sites
competitive with epitopic sites of these polypeptides.
These anti-idiotypes may therefore serve as tumor
1341299
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inhibitors, as substitutes for the subject polypeptides
or as antagonists for the subject polypeptides.
These polypeptides form a family of naturally-
occurring polypeptides which may be derived from natural
sources, as well as non-naturally occurring polypeptides
which share physiological properties, such as binding
specificity and B-cell or mast cell stimulatory
activity. Minor differences between species or origin
may require modification in the purification protocols,
as known to those skilled in the art. Alternatively,
these polypeptides may be sequenced and then fragments
synthesized in accordance with well-known techniques.
See, for example, Barany and Merrifield, Solid-Phase
Peptide Synthesis, "The Peptides, Analysis Synthesis
Biology," Special Methods in Peptide Synthesis, Part A,
Vol. 2, Gross and Merenhofer, eds., Academic Press, N.Y.
1980, pp. 1-284.
The polypeptide compositions of the present
invention will find utility in a variety of ways. For
example, these polypeptides can indue the growth of T-
cells and mast cells, either in,.culture or in vivo. Mast
cells, as noted previously, are important sources of
heparin, histamines, prostaglanc~ins, and other
physiologically important materials. Similarly, the
growth factors may be utilized to stimulate B- and T-
cells, especially those known to secrete proteins (e. g.,
lymphokines and immunoqlobulins) useful in the immune
system. Typically, the factors will be incorporated into
culture medium added to cell cultures. A suitable
concentration of the factors will vary depending upon the
type of cell line, but will generally be present in about
0.1 ug/ml to about 1 mg/ml, more preferably about 1 ug/ml
to about 10 ug/ml, in cultures. The use of the growth
factors of the present invention can eliminate the
requirement of utilizing feeder cells to maintain the
1 ~4~ 299
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desired cell lines, resulting in substantial increases in
the purity of the products from the cell line.
The polypeptide compositions of the present
invention will also find use in vivo in the treatment of
various immune deficiencies, or to increase the natural
immune response. For example, such polypeptide
compositions may aid in the treatment or prevention of
infection by stimulating the maturation and/or growth of
B-cells, T-cells and mast cells, thus decreasing the
animal's susceptibility to infection.
I. De PJovo Preparation of IL-4 cDNA
A variety of methods are now available for de
novo preparation and cloning of cDNAs, and for the
construction of cDNA libraries: e.g. recent reviews are
given by Doherty, "Cloning and Expression of cDNA",
Chapter 10 in Gottesman, Ed. Molecular Cell Genetics
(John Wiley & Sons, New York, 1985); and Brandis et al.,
"Preparation of cDNA Libraries and the Detection of
Specific Gene Sequences", in Setlow et al., Eds. Genetic
Engineering, Vol. 8, pgs. 299-316 (Plenum Press, New
York, 1986).
By way of example, total mRNA is extracted
(e. q., as reported by Berqer, S. et al., Biochemistry _18:
5143-5149 [1979]) from cells (e. g., a nontransformed
human T-cell source) producing polypeptic~es exhibiting
the desired activity. The double-stranded cDNAs from
this total mRNA can be constructed by using primer-
initiated reverse transcription (Verme, I., Biochem.
Biophys. Acta, Vol. 473, pqs. 1-38 [1977]) to make first
the complement of each mRNA sequence, and then by priming
for second strand synthesis (Land, H, et al., Nucleic
Acids Res., 9: 2251-2266 [1981]). Subsequently, the
cDNAs can be cloned by joining them to suitable ~lasmid
or bacteriophaqe vectors (Rougeon, F. et al., Nucleic
Acids Res., 2, 2365-2378 [1975]) or Scherer, G. et al.,
1341299
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Dev. Biol. 86, 438-447 [1981]) through complementary
homopolymeric tails (Efstratiadis, A. et al., Cell, 10,
571-585 (1977]) or cohesive ends created with linker
segments containing appropriate restriction sites
(Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, N.Y. 1982), and then
transforming a suitable host. (See .generally,
Efstratiadis, A., and Villa-Kormaroff, L., "Cloning of
double stranded cDNA" in Setlow, J. and Hollaender, A.
(eds.) Genetic Engineering, Vol, 1, Plenum Publishing
Corp., N.Y., U.S.A. [1982].)
A preferred source of mRNA encoding the desired
polypeptides is cells whose supernatants contain the
stimulating activities for the B-cell, T-cell and/or mast
cells, or other activities associated with the
polypeptides of the present invention. One such line is
the mouse T-cell line Cl.Ly1+2-/9 (A.T.C.C. Accession No.
CRL8179) (Nabel, G. et al., Nature 291: 332-334
(1981)). In general, suitable T-cells can be obtained
from a variety of sources, such as mammalian (e. g. human)
spleen, tonsils and peripheral blood. T-cell clones,
such as those isolated from peripheral blood T-
lymphocytes, may also be used (see Research Monographs in
Immunology, eds. yon Doehmer, H. and Haaf, V.; Section
D: "Human T-Cell Clones", vol.8, pgs. 243-333; Elsevier
Science Publishers, N.Y. [1985]).
Production of mRNAS capable of coding for IL-4
by such cells can be confirmed by microinjection of the
extracted mRNA into oocytes of Xenopus laevis. This
microinjection technique is described more fully below,
and is disclosed generally in Colman et al., "Export of
Proteins from Oocytes of Xenopus Laevis", Cell, Vol. 17,
pgs. 517-526 (1979); and Maniatis et al. Molecular
Cloning: A Laboratory Manual, pgs. 350-352 (Cold Spring
Harbor Laboratory, New York, 1982).
1 341 29 g
-31-
If the mRNAs coding for a desired IL-4 make up
a very small fraction of the total mRNA steps may be
necessary to enrich the fractional concentration in order
to make practical the screening procedure for detecting
cDNA clones of interest. Such procedures are standard in
the art and are disclosed in the examples below and in
several papers and references, such as Maniati~ et al.,
pgs. 225-228, cited above; Suggs et al., Proc. Natl.
Acad. Sci., Vol. 78, pgs. 6613-6617 (1981); Parnes et
al., Proc. Natl. Acad. Sci., Vol. 78, pgs. 2253-2257
(1981), Davis et al., Proc. Natl. Acad. Sci., Vol. 81,
pgs. 2194-2198 (1984) .
A preferred method of de novo preparation of
IL-4 cDNAS relies on functional expression of the cDNAs
in pcD expression system developed by Okayama and Berg,
disclosed in Mol. Cell. Biol., Vol. 2, pgs. 161-170
(1982); and Vol. 3. pgs. 280-289 (1983), and available
from Pharmacia (Piscataway, N.J.). The pcD expression
vector contains the SV40 early promoter, late splicing
junction, and the replication origin. This vector
permits expression of cDNA inserts in COS 7~monkey cells
which provide T antigen for replication of the pcD
plasmid. Screening of cDNA libraries includes
transfection of pools of plasmid DNA into COS 7 cells
using DEAF-Dextran. Since lymphokines,, and in particular
IL-4s, are secreted proteins, the supernatants from the
transfected cells can be assayed for biological activity
after incubation for several days. Positive pools are
further divided to identify single cDNA clones which give
biological activity after transfection.
Briefly, the Okayama and Berg expression vector
is constructed as follows. Polyadenylated mRNA is
annealed to a polydeoxythymidylic acid (oligo dT) tail
attached to the protruding strand of a KpnI-digested
pBR322 plasmid containing the SV40 early promoter
region. That is, the entire vector serves as a primer
1 X41 299
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for cDNA synthesis. After cDNA synthesis, 3'
polydeoxycytidylate (oliqo dC) tails are attached,
followed by HindIII digestion, which lops off (at a
unique HindIII site) a fragment of the SV40 DNA to which
one of the oligo dC tails is attached. The SV40 early
promoter remains intact, and fortuitously occurring
HindIII, sites of the insert are affected minimally
because the hybrid cDNA/RNA is resistant to HindIII
digestion. A separately constructed HindIII fragment
having a 3' polyquanidylated (oliqo dG) tail is annealed
to the sticky end left by the Hind III digestion. The
vector is circularized and treated with E, coli RNase H,
DNA polymerase I, and DNA liqase to replace the RNA
strand with DNA. The vectors are cloned in E. coli to
form the cDNA library. The SV40 elements permit the
vectors to be expressed in eucaryotic cells as well as
procaryotic cells, and particularly fn mammalian cells,
such as COS7 monkey cells or Chinese hamster ovary (CHO)
cells.
Once the cDNA library in the Okayama/Berg
plasmid vector has been completed, the cDNA clones are
collected, and random pools checked for the presence of
the desired cDNAs by standard procedures, e.g. hybrid
selection, detection of antigenic determinants on
expressed products, and/or functional assays. Positive
pools can then be probed with a cDNA from an induced T
cell line. Thereafter, the positive, probed pools are
divided into individual clones which are further tested
by transfection into a suitable host (such as a mammalian
cell culture), and the host supernatant is assayed for
activity.
II. Preparation of IL-4 cDNAs Via Hybridization Probes
Derived from Disclosed cDNAs
The cDNAs disclosed herein can be used as
probes to identify homologous sequences in different cell
1341299
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types, as an alternative to de novo isolation and cloning
of the Iy-4 coding nucleotides. Standard techniques are
employed, e.g. Beltz et al., "Isolation of Multigene
Families and Determination of Homologies by Filter
Hybridization Methods," Methods in Enzymology, Vol. 100,
pgs. 266-285 (1983); and Callahan et al., "Detection and
Cloning of Human DNA Sequences Related to the Mouse
Mammary Tumor Virus Genome," Proc. Natl. Acad. Sci., Vol.
79, pgs. 5503-5507 (1982). Basically, the cDNAS of the
invention are used to construct probes (using standard
techniques, e.g. see Maniatis et al., cited above) for
screening, at low hybridization stringencies, genomic or
cDNA libraries (again, constructed by standard
techniques) of a cell type suspected of producing IL-4.
Standard screening procedures are employed, e.g.
Grunstein et al., Proc. Natl. Acad. Sci., Vol. 72, pgs.
3961-3965 (1975); or Benton et al., Science, Vol. 196,
pgs. 180-183 (1977) .
As described more fully below, human IL-4 was
isolated by a murine IL-4 probe. Subsequent analysis
indicated about 70$ homology between selected regions of ,.
the human and mouse cDNAS. Given the evolutionary
distance between mice and humans it is believed that
most, if not all, mammalian IL-4 genes are detectable by
probes constructed from one or more cDNAS of the
invention: Wilson et. al. "Biochemical Evolution", Ann.
Rev. Biochem., Vol. 46, pgs. 573-639 (1977); Kimura, "The
Neutral Theory of Molecular Evolution," Chapter 11 in Nei
and Koehn, Eds. Evolution of Genes and Proteins (Sinauer
Associates, Sunderland, MA, 1983).
III. Preparation of Mutant IL-4s by Protein Engineerin
Once nucleic acid sequence and/ar amino acid
sequence information is available for a native protein a
' variety of techniques become available for producing
virtually any mutation in the native sequence. Shortle,
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in Science, Vol. 229, pgs. 1193-1201 (1985), reviews
techniques for .mutating nucleic acids which are
applicable to the present invention. Preferably, mutants
of the native IL-4s, i.e. IL-4 muteins, are produced by
site-specific oligonucleotide-directed mutagenesis, e.g.
Zoller and Smith, Methods in Enzymoloqy, Vol. 100, pgs.
468-500 (1983); Mark et al., U.S. Patent 4,518,584
entitled "Human Recombinant Interleukin-2 Muteins"; or by
so-called "cassette" mutaqenesis described by Wells et
al., in Gene, Vol. 34, pgs. 315-323 (1985); and Estell et
al., Science, Vol. 233, pqs. 659-663 (1986). In sections
below, the notation used by Estell et al. (cited above)
to identify muteins is followed and generalized. For
example, "human IL-4 mutein Leu82" (or simply "Leu82" if
the native protein is understood from the context)
indicates a polypeptide whose amino acid sequence is
identical to that of the native protein except for
position 82 with respect to the N-terminus. At that
position Leu has been substituted for Phe. More than one
substitution can be similarly indicated; e.g., a mutein
having Leu substituted for Phe at position 82 and Asp f_or
Asn at position 111 is referred to as human IL-4 mutein
(Leu82, Asplll). Deletions are indicated by "o's". For
example, a mu~ein lacking Gln at position 71 is referred
to as human IL-4 mutein e71. An insertion is indicated
by "IS(Xaa)". For example, a mutein with a Leu inserted
after Gln at position 71 is referred to as human IL-4
mutein IS71(Leu). Thus, human IL-4 mutein (Serl3, 071
IS94(Gly)) represents the native human IL-4 sequence
which has been modified by replacing Thr by Ser at
position 13, deleting Gln at position 71, and inserting
Gly immediately after Ala at position 94. Insertion of
multiple amino acids at the same site is indicated by
IS1(Xaal-Xaa2-Xaa3-...), where Xaal-Xaa2-Xaa3... is the
sequence inserted after position i. ~1-terminal additions
are indicated by superscript "0", e.q. ISO(Xaa), and a
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seQUence of deletions, for example of amino acids 6-10,
is designated either as ~6-1~ or as (05, 4~, ,'g, ~9,
010),
Most preferably cassette mutagenesis is
employed to generate human IL-4 muteins. As described
more fully below, a synthetic human IL-4 gene has been
constructed with a sequence of unique restriction
endonuclease sites spaced approximately uniformly along
the gene. The uniqueness of_ the restriction sites should
be retained when the gene is inserted into an appropriate
vector, so that segments of the gene can be conveniently
excised and replaced with synthetic oligonucleotides
(i.e. "cassettes") which code for desired muteins.
Determination of the number and distribution of
unique restriction sites entails the consideration of_
several factors including (1) preexisting restriction
sites in the expression vector, (2) whether species-
specific or genera-specific codon usage is desired, and
(3) the convenience and reliability of synthesizing
and/or sequencing the segments between the unique
restriction sites.
IV. Biological Properties and Assays f_or IL-4
Actiyity.
Mammalian IL-4 of the invention is defined in
terms of biological activities and/or homology with the
disclosed embodiments. Mammalian IL-4s of the invention
include proteins and muteins (of the disclosed native
~olypeptides) which are homologous to the disclosed
native polypeptides and which exhibit both BCGF activity
and TCGF activity. Mammalian IL-4s of the invention are
alternatively defined by their biological activities
(defined more fully below) which include BCGF activity
and TCGF activity (which is referred to herein as IL-4
activity) as well as at least one or more activities
selected from the group of activities consisting of MHC
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antigen induction activity, Fc-epsilon receptor induction
activity, GM-CSF stimulated granulocyte colony growth
potentiating activity, interleukin-2 TCGF potentiating
activity, and IgGl- and IgE-induction activity.
It is believed that IL-4s are species-specific
in their activities. That is, for example, human IL-4
exhibits TCGF activity as assayed by human T cell lines,
but not as assayed by murine T cell lines. And
conversely, murine IL-4 exhibits TCGF activity as assayed
by murine T cell lines, but not as assayed by human T
cell lines.
A. TCGF Activity
Several standard assays have been described for
TCGF activity, e.g. Devos et al., Nucleic Acids Research,
Vol. 11, pgs. 4307-4323 (1983); Thurman et al., J. Biol.
Response Modifiers, Vol. 5, pgs 85-107 (1986) ; and
Robert-Guroff et al., Chapter 9 in Guroff, Ed. Growth and
Maturation Factors (John Wiley, New York, 1984).
Generally, the TCGF assays are based on the ability of a
factor to promote the proliferation of peripheral T
lymphocytes or IL-2 dependent T cell lines, e.g. Gillis
et al. J. Immunol., Vol. 120, pg. 2027 (1'978).
Proliferation can be determined by standard techniques,
e.g. tritiated thymidine incorporation, or by
colorimetric methods, Mosmann, J. Immunol. Meth., Vol.
65, pgs. 55-63 (1983).
By way of example, human TCGF activity can be
assayed by the following steps: (1) washed human
peripheral blood lymphocytes (about 2 x 105 in 50
microliters) previously stimulated with phytohemag-
glutinin (PHA) for 7 days and subsequently cultured for 7
days with IL-2 are added to a microtiter well; (2)
dilutions (50 microliter) of the TCGF-containing material
are added to each well; (3) the lymphocytes are incubated
72 hours at 37°C; (4) tritiated thymidine (about 20
9 341 299
-37-
microliters, 20 microcuries/ml) is added to each well;
and (5) cells are harvested onto filter paper strips,
washed, and counted in a scintillation counter.
As described more fully in the examples, some
forms of IL-4 have the capability of potentiating the
TCGF activity of IL-2. "Potentiation" as used herein in
reference to such activity means that the maximal level
of proliferation in a TCGF assay system caused by~IL-2 is
increased by the addition of IL-4.
B. BCGF Activity
BCGF activity is defined by an assay disclosed
by Howard et al., J. Exp. Med, Vol. 155, pqs. 914-923
(1982). Assays for BCGF are reviewed generally by Howard
and Paul, in Ann. Rev. Immunol., Vol. 1, pgs. 307-333
(1983). Briefly, BCGF activity is measured by the degree
to which purified resting B cells are stimulated to
proliferate in the presence of a submitogenic concentra-
tion of anti-IgM, or like antigen. By way of example,
assay of human BCGF activity can be carried out by the
following steps:
Enriched B cell populations are obtained from
peripheral blood, spleen, tonsils, or other standard
sources by Ficoll/Hypaque density gradient centrifugation
(e.q. Pharmacia) and two cycles of rosetting with 2-
aminoethylisothiouronium bromide-treated sheep
erythrocytes to eliminate T cells. Such B cell
preparations should contain more than 95$ surface Ig+
cells and more than 95$ cells positive for human B-cell
specific antigen, as determined by the anti-human B-cell
specific monoclonal antibody B1 available from Coulter
(Hialeah, FL). T cell contamination should be less than
1$ as determined by staining with anti-Leu-1 monoclonal
antibodies (Becton-Dickinson, Mountain View, CA) or OKT
11 antibodies (~rtho Diagnostics, t4estwood, MA). 3
milliliter cultures of such B lymphocytes (about S x 105
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per ml in Yssel's medium, Yssel et al., J, Immunol.
Meth., Vol. 65, pgs. 55-63 (1984)), are activated by
either Staphylococcus aureus Cowan I strain (SAC) (e. g.
0.01$ solution of SAC, which is available from Calbiochem
under the tradename Pansorbin, or which can be prepared
as described by Falkoff et al., J. Immunol., Vol. 129,
pg. 97-102 (1982)) or anti-IgM antibodies (e. g. BRL,
Gaithersburg, MD) coupled to beads, e.g. 5 microgram/ml
of Immunobeads available from Bio-Rad (Richmond, CA).
The B cells are cultured either for 24 hours (in the case
~r
of SAC) or 72 hours (for anti-IgM beads) and then
repurified by Ficoll/Hypaque density centrifugation to
remove SAC particles, beads, nonviable cells, and the
like. B cell proliferation is measured by plating about
x 104 B lymphocytes in 50 microliters of medium in 0.2
ml flat-bottomed microtiter wells. Various dilutions of
the materials suspected of having BCGF activity are added
in a final volume of 50 microliters. Tritiated thymidine
incorporation is determined after 48 hours (anti-IgM
cultures) or 72 hours (SAC cultures). Similar assays are
also disclosed by Muraguchi et al., J. Immunol., Vol.
129, pgs. 1104-1108 (1982); and Yoshizaki et al., _J.
Immunol., Vol. 128, pgs. 1296-1301 (1981).
C. MHC Antigen Induction.
It has been demonstrated that IL-4 can induce
the expression of MHC antigens (e.g., Ia in mice) in
various cell types of the immune system, particularly B
cells. Roehm et al., in J. Exp. Med., Vol. 160, pgs.
679-6,94, presented evidence that a factor exhibiting BCGF
activity was also capable of inducing the expression of
MHC antigens on normal resting B cells. Assays for MHC
antigen induction are generalizations of the assays for
murine B cells presented in that reference. Briefly,
immune system cells are exposed to IL-4, and then
expression of particular MHC antigens on the cells'
r~-C CJ~2/y'~ c7 r lC'
13412gg
-39-
surfaces are determined by labeled antibodies specific
for that antigen. The degree of induction is determined
by comparison of the induced cells with controls.
Several different antibodies can be employed for any
given species. Several hybridomas are available from the
ATCC which produce monoclonal anti-MHC antigen
antibodies, and several are available commercially (for
example, anti-HLA-DR produced by hybridomas under ATCC
accession numbers HB103, HB109, HB110, or HB151; anti-
I-Abed produced by hybridoma under ATCC accession number
HB35; anti-HLA-DR L243 available from Becton Dickinson
(Mountain View, CA); or the like). Some routine
experimentation may be required to adapt the assay to a
particular species, and to optimize conditions to give
the most sensitive read-out of MHC antigen levels. For
the human MHC antigen induction assay, purified B cells
can be prepared as described above, or by similar
techniques. Alternatively, MHC induction can be assayed
on unpurified preparations of spleen cells. Antibody-
labeled cells are preferably detected by flow cytometry,
e.g. on a Becton Dickinson FACS-type instrument, or the
equivalent.
D. MCGF Activity
It is believed that IL-4s generally exhibit
MCGF activity. However, because of_ the lack of adequate
assay techniques, MCGF activity has only been
demonstrated for rodent IL-4. Murine IL-4 MCGF assays
are based on the proliferation of factor-dependent murine
mast or basophil cell lines. In particular, MCGF
activity can be assayed with the murine mast cell line
MC/9, which is deposited with the ATCC under accession
number CRL 8306 and is described in U.S. Patent 4,559,310
and in Nabel et al., Cell, Vol. 23, pg. 19 (1981).
Murine MCGF assays are also described by Ihle et al., in
J. Immunol., Vol. 127, pg. 794 (1981).
~ ~~~ zs9
-40-
Preferably MCGF activity is determined by the
colorimetric assay of Mosmann (cited above) with the use
of MC/9 cells. Briefly, MC/9 cells are cultured in flat-
bottom Falcon microtiter trays (104 cells/well) in
Dulbecco's modified medium supplemented with 4$ fetal
calf serum, 50 uM 2-mercaptoethanol, 2 mM glutamine,
nonessential amino acids, essential vitamins, and varied
concentrations of test supernatants in a final volume of_
0.1 ml. Fifty micrograms of 3-(4,5-dimethylthiazol-2-
yl)-2,5-Biphenyl tetrazolium bromide (Sigma) in 10 ul of
phosphate-buffered saline were added to each cell culture
after a 20-hr incubation. Four hours later, 0.1 ml of_
0.04 M HC1 in isopropanol was added to solubilize the
colored formazan reaction product. The absorbance at 570
nm (reference 630 nm) is measured on a Dynatek Microelisa
Autoreader (MR580), or similar instrument.
E. Fc-epsilon Receptor Induction.
It has been discovered that IL-4 induces Fc-
epsilon expression on B cells and on T cells, but
particularly on human B cells stimulated by anti-I_qM
antibodies, or like antigen. It has also been discovered
that gamma interferon specifically inhibits IL-4-induced
Fc-epsilon expression on B cells.
Preferably, the assay for Fc-epsilon receptor
induction proceeds initially as for the BCGF assay. That
is, purified B cells are obtained which are then
stimulated with anti-IgM antibody (or like antigen) and
are exposed to IL-4. Finally the cells are assayed for
Fc-epsilon receptors.
Several assays are available for quantifying
Fc-epsilon receptors on cell surfaces, e.g. Yodoi and
Ishizaka, J. Immunol., Vol. .122, pgs. 2577-2583 (1979);
Hudak et al.. J. Immunol Meth., Vol. 84, pas. 11-24
(1985); and Bonnefoy et al., J. Immunol. Meth., Vol. 88,
pc~s. 25-32 (1986). In particular, Fc-epsilon receptors
~ ~4~ 299
-41-
can be measured by flow cytometry with labeled monoclonal
antibodies specific for the receptors, e.g. using a
Becton Dickinson FRCS-IV, or like instrument. Fc-epsilon
receptor specific monoclonals can be constructed using
conventional techniques.
F. IgGl- and IgE-Induction.
IL-4 induces the secretion of IgE and IgGl
isotypes in lipopolysaccharide (LPS)-activated B cells,
e.g. Coff man et al., J. Immunol., Vol. 136, pgs.~ 4538-
4541 (1986); Sideras, et al., Eur. J. Immunol., Vol. 15,
pgs. 586-593 (1985). These activities can be measured by
standard immunoassays for antibody isotype, as described
by Coffman et al., J. Immunvl., Vol. 136, pgs. 949-954
(1986). Briefly, B cells are LPS-activated by culturing
them with, for example, about 4 micrograms/ml of
Salmonella typhimurium LPS (available from Sigma) or
about 50 microgram/ml LPS extracted from E. coli 055 (as
described by Sideras et al., cited above). After 4 - 8
days' culture, supernatants are harvested for assaying.
Standard isotype-specific ELISA-type assays can be used
to measure the various isotype concentrations. Anti-
isotype antibodies for the assay are available
commercially, or can be obtained from the ATCC.
G. Colony Stimulating Factor (CSF) Activity.
To determine CSF activity, hemopoietic cells,
e.g, bone marrow cells or fetal cord blood cells, are
made into a single cell suspension. The individual cells
are then "immobilized" in a semi-solid (agar) or viscous
(methylcellulose) medium containing nutrients and usually
fetal calf serum. In the presence of an appropriate
stimulating factor, individual cells will proliferate and
differentiate. Since the initial cells are immobilized,
colonies develop as the cells proliferate and mature.
These colonies can be scored after 7-14 days, Burgess,
1~41~99
-42-
A., Growth Factors and Stem Cells, pgs» 52-55, Academic
Press, New York [1984]. (For specific application to the
growth of qranulocytes and macrophages, see Bradely, T.
and Metcalf, D. , Aust. J. Exp. Biol. Med. Sci. Vol. 44,
pgs. 287-300 [1966], and see generally Metcalf, D.,
Hemopoietic Colonies, Springer-Verlag, Berlin [1977]).
If desired, individual colonies can be extracted, placed
on microscope slides, fixed and stained with
Wright/Geimsa (Todd-Sanford, Clinical Diagnosis by
Laboratory Methods, 15th Edition,. Eds. Davidson and Henry
[1974]). Morphological analysis of cell types present
per single colony can then be determined.
Bone marrow cells collected from patients with
nonhematologic disease are layered over Ficoll (type 400,
Sigma Chemical Co., St. Louis, MO), centrifuged (2,000
rpm, 20 min), and the cells at the interface removed.
These cells are washed twice in Iscove's Modified
Dulbecco's Medium containing 10% fetal calf serum (FCS),
resuspended in the same medium and the adherent cells
removed by adherence to plastic Petri dishes. The
nonadherent cells are added at 105 cells/ml to Iscove's
Medium containing 20% FCS, 50 uM 2-mercaptoethanol, 0.9~
methylcellulose and varied concentrations of either
supernatants known to contain colony stimulating activity
or test supernatants. One ml aliquots are plated in 35
mm petri dishes and cultured at 37°C in a fully
humidified atmosphere of 6$ C02 in air. Three days after
the initiation of_ the culture, 1 unit of erythropoietin
is added to each plate. Granulocyte-macrophage colonies
and erythroid bursts are scored at 10-14 days using an
inverted microscope.
Cord blood cells collected in heparin are spun
at 2,000 rpm f_or 6 min. The white blood cells at the
interface between the plasma and red blood cell peak are
transferred to a tube containing 0.17 N ammonium chloride
and 6% FCS. After 5 min on ice, the suspension is
1341299
-43-
underlaid with 4 ml FCS and centrifuged for 6 mins at
2,000 rpm. The cell pellet is washed with Dulpecco'~
phosphate buffered saline and put through the Ficoll and
plastic adherence steps as described above for bone
marrow cells. The low density nonadherent cells are
collected and placed at 105 cells/culture in the semi-
solid culture medium as described above.
at the end of the assays, the cellular
composition is determined after applying the individual
colonies to glass slides and staining with Wright-
Giemsa. Eosinophils are determined by staining with
Luxol Fast Blue (Johnson, G. and Metcalf, D., Exp.
Hematol. Vol. 8, pgs. 549-561 [1980]).
"Potentiation" as used herein in reference to
GM-CSF-stimulated granulocyte growth means that
granulocyte colonies in the assays described above are
larger when GM-CSF is used with IL-4 than when GM-CSF is
used alone to stimulate colony growth.
V. Purification and Pharmaceutical Compositions
The polypeptides of the present invention
expressed in E. coli, in yeast or in other cells can be
purified according to standard procedures of the art,
including ammonium sulfate precipitation, fractionation
column chromatography (e.g., ion exchange, gel
filtration, electrophoresis, affinity chromatography,
etc.) and ultimately crystallization (see generally
"Enzyme Purification and Related Techniques,"Methods in
Enzymology, 22: 233-577 [1977], and Scopes, R., Protein
Purification: Principles and Practice, Springer-Verlag,
New York [1982]). Once purified, partially or to
homogeneity, the polypeptides of the invention may be
used for research purposes, e.g., as a supplement to cell
growth media (e. g., minimum essential medium Eagle,
Iscove's modified Dulbecco Medium or RPMI 1640; available
from Sigma Chemical Company (St. Louis, MO) and GIBCO
~ ~4~ zss
-44-
Division (Chagrin Falls, OH)) and as an antigenic
substance for eliciting speciFic immunoglobulins useful
in immunoassays, immunofluorescent stainings, etc. (See
generally Immunological Methods, Vols. I & II, Eds.
Lefkovits, I. and Pernis, B., Academic Press, New York,
N.Y. [1979 & 1981]; and Handbook of Experimental
Immunology, ed. Weir, D., Blackwell Scientific
Publications, St. Louis, MO [1978].)
The polypeptides of the present invention may
also be used in pharmaceutical compositions, e.g., to
enhance natural defense against various infections.
Thus, patients with rheumatoid arthritis, in need of a
transplant, or with immunodeficiency caused by cancer
chemotherapy, advanced age, immunosuppressive agents,
etc., may be treated with such polypeptides. The
compositions can selectively stimulate various components
of the immune system, either alone or with other agents
well known to those skilled in the art. In particular,
the compositions may include other immune-reactive
agents, such as lymphokines (e.g. IL-1, IL-2, etc.), any
of the colony stimulating factors, immunoglobulins, etc.,
in view of the potentiating activities of the
polypeptides of the present invention. The polypeptides
will also find use in situations (in vivo or in vitro) in
which enhanced cellular proliferation or immunoglobulin
production is desired.
Pharmaceutical compositions of this invention
can be prepared by mixing these polypeptides with
preferably inert, pharmaceutically acceptable carriers.
Suitable carriers and processes for their preparation are
well known in the art (see, e.g., Remington's
Pharmaceutical Sciences and LJ.S. Pharmaco eia: National
Formulary, Mack Publishing Company, Easton, PA [1984]).
The preferred course of administration is parenteral and
can include use of mechanical delivery systems.
X341299
-45-
Preferably, the pharmaceutical composition is
in unit dosage form, each unit containing an appropriate
quantity of the active component. The quantity of active
compound in a unit dose of preparation may be varied or
adjusted from 1 ug to 100 mg, according to the particular
application and the potency of the active ingredient.
The composition can, if desired, also contain other
therapeutic agents.
The dosages may be varied depending upon the
requirement of the patient, the severity of the condition
being treated and the particular compound being
employed. The term "ef_fective amount" as used herein is
meant to take these factors into account when dosages are
considered. Determination of the proper dosage for a
particular situation is within the skill of the art.
Generally, treatment is initiated with smaller dosages
which are less than the optimum dose of the compound.
Thereafter, the dosage is increased by small amounts
until the optimum effect under the circumstances is
reached. For convenience, the total daily dosage may be
divided and administered in portions during the day.
VI. Expression Systems
A wide range of expression systems (i.e. host-
vector combinations) can be used to produce the proteins
and muteins of the present invention. Possible types of
host cells include but are not limited to cells from
bacteria, yeast, insects, mammals, and the like.
Optimizing the expression of a particular protein or
mutein depends on many factors, including (1) the nature
of the protein or mutein to be expressed, e.q. the
expressed product may be poisonous to some host
systems, (2) whether, and what type of, post-
translational modifications are desired, e.g. the extent
and kind of_ glycosylation desired may affect the choice
of host, ( 3 ) the nature of the 5' and 3' regions flanking
1341299
-46-
the coding region of the protein or mutein of interest,
e.q. selection of promoters and/or sequences involved in
the control of translation is crucial for efficient
expression, (4) whether transient or stahle expression
is sought, (5) the ease with which the expressed product
can be separated from the proteins and other materials of
the host cells and/or culture medium, (6)-the ease and
efficiency of transfecting hosts which transiently
express the protein or mutein of interest, (7) the scale
of cell culture employed to express the protein or mutein
of interest, (8) whether the protein or mutein of
interest is expressed fused to a fragment of protein
endogenous to the host, and like factors.
In general prokaryotes are preferred for
cloning the DNA sequences of the invention. General
guides for implementing prokaryotic expression systems
are provided by Maniatis et al., Molecular Clonin A
Laboratory Manual (Cold Spring Harbor Laboratory, N.Y.,
1982); Perbal. A Practical Guide to Molecular Cloning
(John Wiley & Sons, N.Y., 1984); Glover, DNA Cloning: A
Practical Approach, Vol. I and II (IRL Press, Oxford,
1985); and de Boer et al., "Strategies f_or Optimizing
Foreign Gene Expression in Escherichia coli," in Genes:
Structure and Expression, Kroon, ed. (John Wiley & Sons,
N.Y., 1983). For example, E. coli K12 strain 294 (ATCC
No. 31446) is particularly useful. Other microbial
strains which may be used include E, co:li strains such as
E, coli B, and E. coli X1776 (ATCC No. 31537). These
examples are, of course, intended to be illustrative
rather than limiting.
Prokaryotes may also be used for expression.
The aforementioned strains, as well as E. coli W3110
(Fs-, a-, prototrophic, ATCC No. 27325), bacilli such as
Bacillus subtilis, anc~ other enterobacteriaceae such as
Salmonella typhimurium or Serratia rnarcesans, and various
pseudomonas species may be used.
1341298
-47-
In general, plasmid vectors containing replicon
and control sequences which are derived from species
compatible with the host cell are used in connection with
these hosts. The vector ordinarily carries a replication
site, as well as marking sequences which are capable of
providing phenotypic selection in transformed cells. For
example, E. coli is frequently transformed using pBR322,
a plasmid derived from an E. coli species (Bolivar, et
al., Gene Vol. 2, pg. 95 (1977)). pBR322 contains genes
for ampicillin and tetracycline resistance and thus
provides easy means for identifying transformed cells.
The pBR322 plasmid or other microbial plasmid must also
contain, or be modified to contain, promoters which can
be used by the microbial organism for expression of. its
own proteins. Those promoters most commonly used in
recombinant DNA construction include the S-lactamase
(penicillinase) and lactose promoter systems (Chang et
al, Nature, Vol. 275, pg. 615 (1978); Itakura, et al,
Science, Vol. 198, pg. 1056 (1977); Goeddel, et al,
Nature Vol. 281, pg. 544 (1979)) and a tryptophan (trp)
promoter s~rstem (Goeddel, et al, Nucleic Acids Res., Vol.
8, pg. 4057 (1980); EPO Appl. Publ. No. 0036776). While
these are the most commonly used, other microbial
promoters have been discovered and utilized, and details
concerning their nucleotide sequences have been
published, enabling a skilled worker to ligate them
functionally with plasmid vectors (Siebenlist et al, Cell
Vol. 20, pq. 269 (1980)).
In addition to prokaryotes, eukaryotic
microbes, such as yeast cultures, may also be used.
Saccharomyces cerevisiae, or common haker's yeast, is the
most commonly used among eukaryotic microorganisms. For
expression in Saccharomyces, a commonly-used plasmid is
YRp7 (Stinchcomh, et al, Nature, Vol. 282, pq 39 (1979);
Kingsman et al, Gene, Vol. 7, pq. 141 (1979); Tschemper,
et al, Gene, Vol. 10, pg. 157 (1980)). This rlasmid
141299
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already contains the tr~l gene which provides a selection
marker for a mutant strain of yeast lacking the ability
to grow in tryptophan, for example ATCC No. 44076 or
PEP4-1 (Jones, Genetics, Vol. 85, pg. 12 (1977)). The
presence of the trgl lesion as a characteristic of the
yeast host cell genome then provides an effective
environment for detecting transformation by growth in the
absence of tryptophan.
Suitable promoting sequences in yeast vectors
include the promoters for 3-phosphoglycerate kinase
(Hitzeman, et al., J. Biol. Chem., Vol. 255, pg. 2073
(1980)) or other glycolytic enzymes (Hess, et al, J. Adv.
Enzyme Reg., Vol. 7, pg. 149 (1968); Holland, et al,
Biochemistry, Vol. 17, pg. 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. In constructing suitable
expression plasmids,.the termination sequences associated
with these genes are also ligated into the expression
vector at a position 3' of the sequence to be expressed,
to provide polyadenylation of the mRNA and termination.
Other promoters, which have the additional advantage of
transcription controlled by growth conditions, are the
promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes
associated with nitrogen metabolism, the aforementioned
glyceraldehyde-3-phosphate dehydrogenase, and enzymes
responsible for maltose and galactose utilization
(Holland, ibid.). Any plasmid vector containing a yeast-
compati;~le promoter, origin of replication and
termination sequences is suitable..
In addition to microorganisms, cultures of
cells derived from multicellular organisms may also be
used as hosts. In principle, any such cell culture is
1341299
-49-
workable, whether vertebrate or invertebrate. However
interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue
culture) has become a routine procedure in recent years
[Tissue Culture, Academic Press, Kruse and Patterson,
editors (1973)]. Examples of such useful host cell lines
are VERO and HeLa cells, Chinese hamster ovary (CHO) cell
lines, and W138, BHK, COS7, mouse myeloma (ATCC No. TIB
19 or TIB 20), and MDCK cell lines. Expression vectors
for such cells ordinarily include (if necessary) an
origin of replication and a promoter located in front of
the gene to be expressed, along with any necessary
ribosome binding sites, RNA splice sites, polyadenylation
site, and transcriptional terminator sequences.
For use in mammalian cells, the control
functions on the expression vectors are often provided by
viral material. For example, commonly used promoters are
derived from polyoma, Adenovirus 2, and most frequently
Simian Virus 40 (SV40). The early and late promoters of
SV40 virus are particularly useful because both are
obtained easily from the virus as a fragment which also
contains the SV40 viral origin of replication (H ers, et
al, Nature, Vol. 273, pg 113 (1978). Smaller or larger
SV40 fragments may also be used, provided that there is
included the approximately 250 by sequence extending from
the HindIII site toward the BglI site located in the
viral origin of replication. Further, it is also
possible, and often desirable, to utilize promoter or
control sequences normally associated with the desired
gene sequence, provided that such control sequences are
compatible with the host cell systems.
An origin of replication may be provided either
by an exogenous origin on the vector, e.g., on one
derived from SV40 or other viral (e. g. Polyoma, adeno,
VSV, BPV, etc.) source, or endogeneously by the host cell
chromosomal replication mechanism. It is often
141299
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sufficient to integrate the vector into the host cell
chromosome.
In selecting a preferred host cell for
transfection by the vectors of the invention which
comprise DNa sequences encoding both t-PA and DHFR
protein, it is appropriate to select the host according
to the type of DYFR protein employed. If wild type DHFa
protein is employed, it is preferable to select a host
cell which is deficient in DHFR, thus permitting the use
of the DHFR coding sequence as a marker for successful
transfection in selective medium which lacks
hypoxanthine, glycine, and thymidine. Such a appropriate
host cell is the Chinese hamster ovary (CHO) cell line
deficient in DHFR activity, prepared and propagated as
described by Urlaub and Chasin, Proc. Natl. Acad. Sci.
(USa) Vol. 77, pg. 4216 (1980) .
On the other hand, if DHFR protein with low
binding affinity for MTX is used as the controlling
sequence, it is not necessary to use DHFR-resistant
cells. Because the mutant DHFR is resistant to
methotrexate, MTX-containing media can be used as a means
of selection provided that the host cells are themselves
methotrexate-sensitive. Most eukaryotic cells which are
capable of absorbing MTX appear to be methotrexate-
sensitive. One such useful cell line is a CHO line, CHO-
K1 r~TCC No. CCL 61.
Invertebrate expression systems include the
larvae of silk worm, Bombyx mori, infected by a
baculovirus vector, BmNPV, described by Maeda et al. in
Nature, Vol. 315, pgs. 892-894 (1985), and in Saibo
Kv uku, Vol. 4, pgs. 767-779 (1985).
1 ~4~ 29 9
-51-
EXAMPLES
The following examples serve to illustrate the
present invention. Selection of_ vectors and hosts as
well as the concentration of reagents, temperatures, and
the values of other variables are only to exemplify
application of the present invention and are not to be
considered limitations thereof.
MATERIALS AND METHODS
T Cell lines Cl.Ly1+2-/9 (referred to as C1.1),
Nabel, G., Greenberqer, J.S., Sakakeeny, M.A. and Cantor,
H. (1981) Proc. Natl. Acad. Sci. USA 78, 1157-1161, and
GK15-1 (supplied by M. Giedlin, DNAX Research Institute
of Molecular and Cellular Biology, Inc.; DNAX) were
resuspended at 5 x 105 cells/ml in RPMI 1640, 50um 2ME,
1% FCS and 2ug/ml Con A for 24 hours. The supernatants
were collected and stored at -70°C.
A cloned mast cell line, MC/9 (Nable, G.,
Galli, S.J., Dvorak, H.F. and Cantor, H. (1981) Nature
291, 332-334), was obtained from G. Nabel (Dana.Farber
Cancer Institute; DFCI). The mast cell lines M~43
(provided by R. Coffman, DNAX) and Dx-2 (derived by D.
Rennick, DNAX) were characterized by the absence of
myelomonocytic-associated markers and by the presence of_
IqE receptors and histamine levels greater than 250
nq/106 cells. The myeloid NFS-60 cell line was provided
by J. Ihle (Frederick Cancer Research Facility; FCRF).
The NFS-60 cell line was subcloned to obtain an IL-3-
dependent clone. The T cell line HT2 was obtained from
S. Strober (Stanford University, Palo Alto, CA); CTLL-2
was supplied by W. Farrar (FCRF); and Ly 23/4 was
supplied by G. Nabel (DFCI). The mast cell and T cell
lines were grown in RPMI 1640, 10$ FCS, 50uM 2hlE
supplemented with recombinant IL-3 or IL-2.
1 341 29 9
-52-
Histamine levels of several IL-3-dependent cell
lines were assayed using the method of Shore, P.A.,
Burkhalter, A., and Conn, V.H. (1959) J. Pharm. Exp.
Ther. 127, 182-186. T Cell and mast cell growth factor
activities were determined by [3H]-thymidine
incorporation or by a colorimetric assay as described by
Mosmann, T. in J. Immunol. Methods 65: 55-63 (1983).
IL-3 purified from WEHI 3 supernatant was a
gift of J. Ihle (FCRF). Recombinant IL-2, IL-3, GM-CSF
and INF-Y were utilized in the form of supernatant from
COS-7 monkey kidney cells transfected with the
corresponding cDNA clones. One unit of IL-2, IL-3 or
GM-CSF was defined as that amount of factor which
stimulated 50$ maximum [3H]-thymidine incorporation of
factor-dependent cell lines (Rennick, D, et al. (1985) _J.
Immunol. 134: 910-919). One unit of IFN-Y protects 50$
of murine L cells from the cytopathic effect of vesicular
stomatitis virus (Schreiber, R. et al.(1982) J. Exp. Med.
156: 677-689).
B-cells were prepared from a single cell
suspension of mouse spleen cells by denletinq T-cells and
macrophages according to standard procedures (Howard, M.
et al., J. Exp. Med. 155: 914 (1982)). The purified
factor described herein was assessed for its ability to
induce Ia expression on B-cells using cytofluorometric
analysis and to stimulate B-cell proliferation using an
anti-Iq co-stimulation assay as described by Roehm, N. et
al., J. Exp. Med. 160: 679.
Protein determinations were based either on UV
absorption (280 nm or 220 nm) or standard curves
constructed using a dye binding assay (Bradford, M.
(1976) Annal. Biochem. 72: 248-254). Supernatants were
initially concentrated 20 fold with a Pellicon cassette
unit (Millipore, Bedford, MA), or (following reverse
phase chromatography) by solvent evaporation in a
Speedvac (Savant, Farminqdale, NY). SDS PAGE was
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performed usinc7 the Laemmli system (Laemmli, U. (1970)
Nature 227: 680-685) with a 12~ separating gel. darker
proteins were Pharmacia low MW standards (Pharmac~ia,
Uppsala, Sweden). Gels were silver-stained as described
by Merril, C. et al. (1981) Science 211: 1437-1438. To
assess directly MCGF and TCGF activity as a function of
molecular.weight, samples with or without prior reduction
with 50 mM DTT Were electrophoresed, the gels sliced to 1
mm sections and crushed, and protein was eluted overnight
at 4°C into 0.5 ml assay media supplemented with 5 mq/ml
ovalbumin (Sigma, St. Louis, MO).
Chromatography was performed at 18°C on a
Pharmacia FPLC system equipped with a Kratos Spectroflow
773 UV detector (Kratos, Ramsey, NJ). Cation exchange
chromatography utilized a Pharmacia Mono S column (0.5 x
cm) equilibrated with 50 mM sodium phosphate, 1 mM
EDTA, pH 7Ø Supernatants in the same buffer were
applied to the column and eluted with a NaCl gradient to
1M. For reverse phase chromatography, a Pharmacia C8
column (0.5 x 2 cm) was used. The sample, diluted with
0.1$ TFA to ~,H 2, was loaded onto the column and eluted
with acetonitrile gradients containing 0.1~ TFA.
Isoelectric points of MCGF and TCGF activities
were estimated by chromatofocussinq on a Pharmacia Mono P
column (0.5 x 20 cm). The sample, in 0.025 M bis tris,
pH 7.1, was loaded onto the "Mono P" column equilibrated
with the same buffer. Gradient elution was effected with
Pharmacia Polybuffer 74, pH 4.0 (1:10 dilution, pH
adjusted with 0.2 M aminodiacetic acid). Effluent pH was
continuously determined with a Pharmacia pH monitor.
a Prior to injections, all samples were filtered through a
Mi,llex GV *0.2u unit (Millipore).
Fractions from SDS PAGE or chromatographic runs
were assayed for their ability to support proliferation
of three cell lines, NFS-60 (IL-3), MC/9 (MCGF), and HT2
(TCGF).
I / Q~P/Y7 p r k
~~4a2ss
-54-
For purification of_ this factor to homogeneity,
8L of Con-A-activated Cl.l.supernatant was dialized into
20mM Hepes buffer, pH 7.0, then passed over a Pharmacia
Mono S 10/10 cation exchange column equilibrated with the
same buffer. Peak activity eluted with a linear salt
gradient at 0.2M NaCl. This material was pooled,
concentrated, re-dialized into 20 mM Hep~s buffer, pH
7.0, and loaded onto a Heparin-Sepharose column
equilibrated with the same buffer. Peak activity eluted
at 0.45M NaCl with a linear gradient to 2M NaCl. This
material was pooled, diluted lOx with 0.10/TFA/H20, pH 2,
and fractionated on a Vydac reverse phase column (c4).
Application of a linear gradient of acetonitrile, 0.1~
TFA, released the activity at 42$ acetonitrile.
Example I. de novo Preparation of Murine IL-4 cDNAs
from Cl.Ly1+2-/9 Cells and Transient
Expression in COS 7 Monkey Cells.
cDNA clones coding for IL-4 were isolated from
the murine helper T cell line Cl.Ly1+2-,i9, which is
deposited with the ATCC under accession number CRL 8179
and described by Nabel et al., in Cell, Vol. 23, pqs. 19-
28 (1981), and in Proc. Natl. Acad. Sci., Vol. 78, pqs.
1157-1161 (1981). Other murine cells known to produce
BCGF activity include the FL-4 line, available from the
ATCC under accession number TIB 39. The procedures used
in this example have been disclosed in Lee et al., Proc.
Natl. Acad. Sci., Vol. 83, pqs. 2061-2065 (1986).
Briefly, a pcD cDNA library was constructed with
messenger RNA (mRNA) from concanavalin A (ConA)-induced
Cl.Ly1+2-/9 cells following the procedure of Okayama and
Berg, described above. IL-3 and GM-CSF clones were
eliminated from a large sublihrary of randomly selected
clones by hybridization with 32P-labeled cDNA probes.
Pools and/or individual clones from the remainder of the
Tra ~P~~A~~~
1341 X99
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sublibrary were screened for IL-4 cDNA by transfecting
COS 7 monkey cells and testing culture supernatants Por
MCGF and TCGF activity.
A. Induction of IL-4 Production.
Cl.Ly1+2-/9 cells were induced to produce IL-4
mRNA by ConA as follows. The cells are cultured at 5 x
105/ml in Dulbecco's Modified Eagles medium (DME) with 4~
heat-inactivated fetal calf serum, 5 x 10-5 P9 2-
mercaptoethanol (2-ME), 2mM glutamine, non-essential
amino acids, essential vitamins and 2 uq/ml ConA. After
12-14 hrs' incubation at 37°C in 10~ C02, the cell
suspension is centrifuged at 1500 rpm for 10 minutes.
The cell pellets are collected and frozen immediately at
-70°C.
B. Isolation of mRNA
Total cellular DNA was isolated from cells
using the guanidine isothiocyanate procedure of Chirgwin,
J. et al. (Biochemistry, 18: 5294-5299 [1979]). Frozen
cell pellets from ConA-induced Cl.Ly1+2-/9 cells (12 hrs
after stimulation) were suspended in guanidine
isothiocyanate lysis solution. Twenty ml of lysis
solution was used for 1.5 x 108 cells. Pellets were
resuspended by pipettinq, and then DNA was sheared by 4
passes through a syringe using a 16 gauge needle. The
lysate was layered on top of 20 ml of 5.7 M CsCl, 10 mM
EDTA in 40 ml polyallomer centrifuge tube. This solution
was centrifuged at 25,000 rpm in a Reckman~SS,128 rotor
(Beckman Instruments, Inc., Palo Alto, CA) for 40 hrs at
15°C. The guanidine isothiocyanate phase containing DNA
was pipetted off from the top, down to the interface.
The walls of the tube and interface were washed with 2-3
ml of guanidine isothiocyanate lysis solution. The tube
was cut below the interface with scissors, and the CsC1
solution was decanted. RNA pellets were washed twice
I rGderna. i~
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with cold 70~ ethanol. Pellets were then resuspended in
500 ul of 10 mM Tris.HCl pH 7.4, 1 mM EDTA, 0.050 SDS.
50 ul of 3M sodium acetate was added and RNA was
precipitated with 1 ml ethanol. About 0.3 mg total RNA
was collected by centrifuging and the pellets washed once
with cold ethanol.
Washed and dried total RNA pellet was
resuspended in 900 ul of oligo (dT) elution buffer (10 mM
Tris.HCl, pH 7.4, 1 mM EDTA, 0.5~ SDS). RNA was heated
for 3 min. at 68°C and then chilled on ice. 100 ul of 5
M NaCl was added. The RNA sample was loaded onto a 1.0
ml oliqo (dT) cellulose column (Type 3, Collaborative
Research, Waltham, MA) equilibrated With binding buffer
(10 mM Tris.HCl pH 7.4, 1 mM EDTA, 0.5 M NaCl, 0.5$
SDS.) Flow-through from the column was passed over the
column twice more. The column was then washed with 20 ml
binding buffer. PolyA+ mRNA was collected by washing
with elution buffer. RNA usually eluted in the first 2
ml of elution buffer. RNA was precipitated with 0.1
volume 3 M sodium acetate (pH 6) and two volumes of
ethanol. The RNA pellet was collected by centrifugation,
washed twice with cold ethanol, and dried. The pellet
was then resuspended in water. Aliquots were diluted,
and absorbance at 260 nm was determined.
C. Construction of pcD cDNA Library
1) Preparation of Vector Primer and Oligo(dG)-Tailed
Linker DNAs.
The procedure of Okayama & Berg (P4ol. & Cell.
Biol. Vol. 2, pgs 161-170 [1982]) was used with only
minor modifications. The pcDVl and pLl plasmids are
described by Okayama & Berg (Mol. & Cell. Biol. 3: 380-
389 [1983]) and are available from Pharmacia (Piscataway,
N.J.). Specifically, a modified pcDVl plasmid was used
1 X41 299
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which contained an NsiI site at the previous location of
the KpnI site.
An 80 ug sample of pcDVl DNA was digested at
30°C with 20 U of KpnI endonuclease in a reaction mixture
of 450 ul containing 6 mM Tris.HCl (pH 7.5), 6 mM MgCl2,
6 mM NaCl, 6 mM 2-ME, and 0.1 mq of bovine serum albumin
( BSA ) per ml . Af ter 16 hr the digestion was terminated
with 40 ul of 0.25 M EDTA (pH 8.0) and 20 ul of 10~
sodium dodecyl sulfate (SDS ) ; the DNA was recovered after
extraction with water-saturated 1:1 phenol-CHC13
(hereafter referred to as phenol-CHC13) and then by
precipitation with ethanol. Homopolyme:r tails averaging
60, but not more than 80, deoxythymidylate (dT) residues
per end were added to the NsiI endonuclease-generated
termini with calf thymus terminal transferase as
follows: The reaction mixture (38 ul) contained sodium
cacodylate-30 mM Tris.HCl pH 6.8 as buffer, with 1 mM
CoCl2, 0.1 mM dithiothreitol, 0.25 mM dTTP, the NsiI
endonuclease-digested DNA, and 68 U of the terminal
deoxynucleotidyl transferase (P-L Biochemicals, Inc.,
Milwaukee, WI). After 30 min. at 37°C the reaction was
stopped with 20 ul of 0.25 M EDTA (pH 8.0) and 10 ul of_
10$ SDS, and the DNA was recovered after several
extractions with phenol-CHC13 by ethanol precipitation.
The DNA was then digested with 15 U of EcoRI endonuclease
in 50 ul containin4 10 mM Tris.HCl pH 7.4, 10 mM MQC12, 1
mM dithiothreitol, and 0.1 mg of BSA per ml for 5 hr at
37°C. The large fragment, containing the SV40
polyadenylation site and the pBR322 origin of_ replication
and am~icillin-resistance gene, was purified by aqarose
(1~) gel electrophoresis and recovered from the gel by a
modification of the glass powder method (Vogelstein, ~3. &
Gillespie, D., Proc. Natl. Acad. Sci. 76: 615-519
[1979J). The dT-tailed DNA was further purified by
absorption and elution from an oligo (dA)-cellulose
column as follows: The DNA was dissolved in 1 ml of 10
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mM Tris.HCl pH 7.3 buffer containing 1 mM EDTA and 1 M
NaCl, cooled at 0°C, and applied to an oliqo (dA)-
cellulose column (0.6 by 2.5 cm) equilibrated with the
same buffer at 0°C and eluted with water at room
temperature. The eluted DNA was precipitated with
ethanol and dissolved in 10 mM Tris.HCl pH 7.3 with 1 mM
EDTA.
The oliqo (dG)-tailed linked DNA was prepared
by digesting 75 uq of pLl DNA with 20 U of PstI
endonuclease in 450 ul containing 6 mM Tris.HCl pH 7.4, 6
mM MgCl2, 6 mM 2-ME, 50 mM NaCl, and 0.01 mg of BSA per
ml. After 16 hr at 30°C the reaction mixture was
extracted with phenol-CHC13 and the DNA was precipitated
with alcohol. Tails of 10 to 15 deoxyguanylate (dG)
residues were then added per end with 46 U of terminal
deoxynucleotidyl transferase in the same reaction mixture
(38 ul) as described above, except that 0.1 mM dGTP
replaced dTTP. After 20 min. at 37°C the mixture was
extracted with phenol-CHC13, and after the DNA was
precipitated with ethanol it was digested with 35 U of.
HindIII endonuclease in 50 ul containing 20 mM Tris.HCl
pH 7. 4, 7 mM MgCl2, 60 mM NaCl , and 0. 1 mq of BSA at 37 °C
for 4 hr. The small oliqo (dG)-tailed linker DNA was
purified by aqarose gel (1.8~) electrophoresis and
recovered as described above. .
2) cDNA Library Preparation:
Step 1: cDNA synthesis. The reaction mixture
(10 ul) contained 50 mM Tris.HCl pH 8.3, 8 mM MqCl2, 30
mM KC1, 0.3 mM dithiothreitol, 2 mM each dATP, dTTP,
dGTP, and dCTP, 20 uCi 32P-dCTP (3000 Ci/mmole), 3 uq
polyA+ RNA from Con-A induced T-cells, 60 units RNasin (a
tradenamed ribonuclease inhibitor from Promeqa Biotec,
Inc. , Madison, LdI ) , and 2 uq of_ the vector-primer DNA ( 15
pmol of primer end), and 45 U of reverse transcriptase.
The reaction was incubated 60 min at 42°C and then
~ 341 299
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stopped by the addition of_ 1 ul of 0.25 M ETDA (pH 8.0)
and 0.5 ul of 10~ SDS; 40 ul of phenol-CHC13 was added,
and the solution was blended vigorously in a Vortex mixer
and then centrifuged. 40 ul of 4 M ammonium acetate and
160 ul of ethanol were added to the aqueous phase, and
the solution was chilled with dry ice for 15 min., warmed
to room temperature with gentle shaking to dissolve
unreacted deoxynucleoside triphosphates that had
precipitated during chilling, and centrifuged for 10 min.
in an Eppendorf microfuge. The pellet was dissolved in
10 ul of 10 mM Tris.HCl pH 7.3 and 1 mM EDTA, mixed with
10 ul of 4 M ammonium acetate, and reprecipitated with 40
ul of ethanol, a procedure which removes more than 99$ of
unreacted deoxynucleotide triphosphates. The pellet was
rinsed with ethanol.
Step 2: Oligodeoxycytidylate (oligo (dC))
addition. The pellet containing the plasmid-cDNA:mRNA
was dissolved in 20 ul of 140 mM sodium cacodylate-30 mM
Tris.HCl pH 6.8 buffer containing 1 mM CoCl2, 0.1 mM
dithiothreitol, 0.2 ug of poly (A), 70 uM dCTP, 5 uCi.
32p_dCTP, 3000 ~i/mmole, and 60 U of terminal
deoxynucleotidyl transferase. The reaction was carried
out at 37°C for 5 min. to permit the addition of 10 to 15
residues of dCMP per end and then terminated with 2 ul of_
0.25 M EDTA (pH 8.0) and 1 ul of 10~ SDS. After
extraction with 20 ul of phenol-CHC13, r_he aqueous phase
was mixed with 20 ul of 4 M ammonium acetate, the DNA was
precipitated and reprecipitated with 80 ul of_ ethanol,
and the final pellet was rinsed with ethanol.
Step 3: HindIII endonuclease digestion. The
pellet was dissolved in 30 ul of buffer containing 20 mM
Tris.HCl pH 7.4, 7 mM MgCl2, 50 mM NaCl, anc~ 0.1 mg of_
FiSA per ml and then digested with 10 U of HindIII
endonuclease for 2 hr at 37°C. The reaction was
terminated with 3 ul of 0.25 P1 EDTA (pH 8.0) and 1.5 ul
of 10$ SDS and, after extraction with phenol-CHC13
1 34? 299
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followed by the addition of 30 ul of. 4 P4 ammonium
acetate, the DNA was precipitated with 120 ul of
ethanol. The pellet was rinsed with ethanol and then
dissolved in 10 ul of IO mM Tris.HCl (pH 7.3) and 1 mM
EDTA, and 3 ul of ethanol was added to prevent freezing
during storage at -20°C.
Step 4: Cyclization mediated by the oligo
(dG)-tailed linker DNA. A 9 ul sample of the HindIII
endonuclease-digested oligo (dC)-tailed cDNA:mRNA plasmid
(about 90$ of the sample) was incubated in a mixture (90
ul) containing 10 mM Tris.HCl pH 7.5, 1 mM EDTA, 0.1 M
NaCl, and 1.8 pmol of the oligo (dG)-tailed linker DNA at
65°C for 5 min., shifted to 42°C for 60 min, and then
cooled to 0°C. The mixture (90 ul) was adjusted to a
volume of 900 ul containing 20 mM Tris.HCl pH 7.5, 4 mM
MgCl2, 10 mM (NH4)2S04, 0.1 M KC1, 50 ug of BSA per ml,
and 0.1 mM S-NAD; 6 ug of E. coli DNA lipase were added
and the solution was then incubated overnight at 12°C.
Step 5: Replacement of RNA strand by DNA. To
replace the RNA strand of. the insert, the ligation
mixture was adjusted to contain 40 uM of each of the four
deoxynucleoside triphosphates, 0.15 mM R-NAD, 4 ug of
additional E. coli DNA lipase, 16 U of E. coli DNA
polymerase I ( PolI, ) and 9U of E. coli RNase H. This
mixture (960 ul) was incubated successively at 12°C and
at room temperature for 1 hr each to promote optimal
repair synthesis and nick translation by Poll.
Step 6: Transformation of E. coli.
Transformation was carried out using minor modifications
of_ the procedure described by Cohen et al. (Proc. Nat.
Acad. Sci. U.S.A., 69: 2110-2114 [1972]). E. coli K-12
strain MC1061 (Casadahan, M. and Cohen, S., J. Mol. Biol.
138: 179-207 [1980]) was grown to 0.5 absorbancy unit at
600 nm at 37°C in 300 ml of L-broth. The cells were
collected by centrifugation and suspended in 30 ml of 10
mN4 Pipes (pH 7), 60 mM CaCl2, 15°s glycerol, and
1 341 299
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centrifuged at 0°C for 5 min. The cells were resuspended
in 24 ml of the above buFfer and incubated again at 0°~~
for 5 min.; then 1.0 ml aliduots of the cell suspensions
were mixed with 0.1 ml of the DNA solution (step 5) and
incubated at 0°C for 20 min. Next the cells were kept at
42°C for 2 min. and thereafter at room temperature for 10
min.; then 1 liter of L-broth was added, and the culture
was incubated at 37°C for 60 min. Ampicillin was added
to a concentration of 50 ug/ml. The culture was shaken
for an additional 10 hrs. at 37°C. Dilutions of_ this
culture were spread on L-broth agar containing 50 ug/ml
ampicillin. After incubation at 37°C f_or 12 to 24 hr,
individual colonies were picked with sterile tooth-
picks. In all, approximately 1 x 105 independent cDNA
clones were generated.
D. Screening the pcD Library.
104 single clones were picked at random from
the T-cell cDNA library and propagated individually in
wells of microtiter dishes containing 200 ul L-broth with
ampicillin at 50 ug/ml and dimethyl sulfoxide at 7~. To
focus only on the novel MCGF activity, 53 IL-3 cDNA
clones and one GM-CSF cDNA clone were ident i f ied by
hybridization with the appropriate 32P-labelled cDNA
probes constructed from the clones disclosed by Lee et
al., Proc. Natl. Acad. Sci., Vol. 82, pgs. 4360-4364
(1985), and by Yokota et al., Proc. Natl. Acad. Sci.,
Vol. 81, pgs. 1070-1074 (1984), and were eliminated.
This procedure was carried out as follows: Each plate of
96 cultures was replicated onto nitrocellulose filters
for hybridization screening. Hybridizations were
performed in 6XSSPE (1XSSPE - 180 mM NaCl; 10 mM sodium
phosphate, pH 7.4; 1 mm EDTA), 0.1~ SDS, 100 uq/ml _F.
coli tRNA, 50~ formamide, for 16 hrs. at 42°C.
Hyhridizinc~ clones were identified by autoradiography of
the washed filter. These clones were removed by
1 341 299
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sterilizing the microtiter wells containing these clones
with ethanol prior to the preparation of_ clone pods.
Pools containing up to 48 cDNA clones were prepared from
the microtiter cultures. Two hundred such pools were
grown up in 1 liter cultures of L-broth containing 100
ug/ml ampicillin. Plasmid DNA was isolated from each
culture and purified by twice banding through CsCl
gradients. The DNA representing each pool was .
transfected into COS7 monkey cells as follows. (COS7
cells are described by Gluzman in Cell, Vol. 23, pgs.
175-180 (1981), and are available from the ATCC under
accession number CRL 1651.)
One day prior to transfection, approximately
106 COS 7 monkey cells were seeded onto individual 100 mm
plates in DME containing 10~ fetal calf serum and 2 mM
glutamine. To perform the transfection, the medium was
aspirated from each plate and replaced with 4 ml of DME
containing 50 mM Tris.HCl pH 7.4, 400 ug/ml DEAF-Dextran
and 50 ug of the plasmid DNAs to be tested. The plates
were incubated for four hours at 37°C, then the DNA-
containing medium was removed, and the plates were washed
twice with 5 ml of serum-free DME. DME containing 150 uM
Chloroduine was added back to the plates which were then
incubated for an additional 3 hrs at 37°C. The plates
were washed once with DME, and then DME containing 4~
fetal calf serum, 2 mM glutamine, penicillin and
streptomycin was added. The cells were then incubated
f_or 72 hrs at 37°C. The growth medium was collected and
evaluated in the various bioassays.
An initial set of plasmid pools was screened
primarily by using proliferation assays for TCGF and MCGF
activities with the HT-2 cell line (described more fully
below) and MC/9 cell line, respectively. Among the first
110 pools assayed on these two cell. lines, eir~ht produced
significant activity in the HT-2 TCGF assay. Several of
these pools had weak but siqnif_icant MCGF activity, hut
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because the MCGF activities were generally weaker and
more variable, we did not rely on this assay For
identifying positive pools.
Approximately half of the COS supernatants from
the random pool transfections were also assayed for Ia-
inducing activity on mouse 8 cells. Among the pools
tested, each pool shown to be active for TCGF activity
was found also to have Ia-inducing activity. Thus, there
was a perfect correlation between the TCGF activity and
the Ia-inducing activity.
One pool, 2A, which was reproducibly the most
active in all assays, was subdivided into 48 smaller sub-
pools. Two subpools were positive for both MCGF and TCGF
activities. The single clone, 2A-E3, common to both
subpools was then grown individually and its plasmid DNA
was transfected into COS 7 cells as described above. The
resulting COS supernatant was then assayed for the
presence of various activities, including MCGF, TCGF, Ia-
inducing, and IgE- and IgG-enhancing activities.
A 366 base-pair-long PstI fragment isolated
from clone 2A-E3 (Figure lA) and labelled with 32P was
used as a probe to screen pools which had been positive
for biological activity as well as other untested
pools. The screening was performed by hybridization to
filters replicated with the microtiter cultures as
described above. Nine hybridizing clones were isolated
and their DNA analyzed by restriction mapping. All pools
which exhibited biological activity contained at least
one hybridizing clone which shared a common restriction
cleavage map with clone 2A-E3. The frequency of
hybridizing clones among the 104 which were picked
suggests a frequency of approximately 0.2~ in the total
library. Of the hybridizing clones which were tested,
approximately 90$ expressed a functional protein.
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E. Biological Activities of Culture Supernatants of COS
7 Monkey Cells Transfected with pcD-2A-E3.
Supernatant from COS 7 cells transfected with
pcD-2A-E3 was tested for TCGF activity on the murine
helper T cell line HT-2, described by Watson in J. Exp.
Med., Vol. 150, pg. 1510 (1979). Proliferation of the
HT-2~cells, as determined by the colorimetric assay of
Mosmann (cited above), was used as a measure of TCGF
activity (degree of proliferation being correlated to
optical density (OD) from 570 to 630 nm). Figure 3A
illustrates the relative TCGF activities at various
dilutions of (i) supernatant from COS 7 cells transfected
with pcD-2A-E3 (curve 1), (ii) supernatant from
Cl.Ly1+2-/9 cultures (curve 2), (iii) supernatant from
COS 7 cells transfected with a pcD plasmid carrying IL-2
cDNA (curve 3), and (iv) supernatant from COS 7 cells
transfected with a pcD plasmid containing no cDNA insert
(i.e. a "mock" transfection) (curve 4) .
Similarly, supernatants from pcD-2A-E3
transfected COS 7 cells were tested for MCGF activity on
MC/9 cells, again using the colorimetric assay"of Mosmann
to measure MC/9 proliferation. Figure 3B illustrates
relative MCGF activity of (i) supernatant from COS 7
cells transfected with pcD-2A-E3 (curve 1), (ii)
supernatant from COS 7 cells transfected with a pcD
plasmid carrying IL-3 cDNA (curve 2), (ii.i) supernatant
from Cl.Lyl+2-/9 cells (curve 3), and (iv) supernatant
from mock transfected COS 7 cells (curve 4).
Figure 3C illustrates the results of an Ia
induction assay conducted on (i) supernatant of COS 7
cells transfected with pcD-2-E3 (curve 1), (ii)
supernatants of Cl.Lyl+2-/9 cells (curve 2), and (iii)
supernatants of mock transfected COS 7 cells (curve 3).
The Ia-induction assay was carried out as described by
Roehm et al. (cited above) . Several DBA/2 mice (2-3
months old) were sacrificed and the spleens obtained
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surgically. The erythrocytes were lysed by hypotonic
shock using 0.87 ammonium chloride. Then the T-cella
were lysed by using cytotoxic monoclonal antibodies
directed against T-cell-specific surface markers (Thy-1,
Lyt-1 and Lyt-2) followed by incubation in rabbit
complement. The dead cells were then removed using
ficoll-hypaque density gradients. Adherent cells had
been removed previously by adherence to plastic petri
dishes at 37°C. At this time the cells were washed,
counted and scored for viability. Approximately one
million cells were incubated in 0.5 ml of tissue culture
medium (RPMI 1640 or Minimal essential medium-MEM/Earle's
salts) (Gibco) supplemented with 10~ fetal calf serum, 2-
mercaptoethanol and various antibiotics (penicillin,
streptomycin and gentamicin). In experiments where the
positive control consisted of supernatants from T-cells
induced with the T-cell mitogen Concanavalin A, 10 mg/ml
(final concentration) of alpha-methyl-mannoside was added
to neutralize the mitogen. After 24 hours' incubation,
the cells were harvested and prepared for staining with
anti-I-Ad or anti-I-Abd monoclonal antibodies. These
antibodies were used as first-stage antibodies conjugated
to either the hapten N.I.P. or biotin. T'he staining was
then completed by incubating the cells with
fluoresceinated second-stage reagents (either anti-NIP
antibodies or avidin). The intensity of fluorescence
staining was then determined using either a fluorescence-
activated cell sorter (Becton-Dickinson, Mountain View,
CA) or a Cytofluorograph (Ortho Diagnostics, Cambridge,
MA). Fluorescence units in Figure 3C are calculated by
multiplying the percentage of positive cells in each
sample by the intensity of fluorescent staining.
Figure 3D graphically illustrates the degrees
by which IgE and IgGl production are induced in T-cell-
depleted mouse spleen cells by (i) COS 7 medium alone
(bar 1), (ii) 20$ supernatant from mock transfected COS 7
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cells (bar 2), (iii) 10~ supernatant from Cl.Ly1+2-/9
cells plus 20~ supernatant from mock transfected COS 7
cells (bar 3), and (iv) 20~ supernatant from pcD-2H-E3-
transfected COS 7 cells (bar 4). Levels of IgE and IgGl
were determined by the isotype-specific ELISa described
above.
Murine IL-4 was found to enhance the MCGF
activity of IL-3 in MC/9 cells: Smith and Rennick, Proc.
Natl. Hcad. Sci., Vol. 83, pgs. 1857-1861 (1986). Murine
IL-4 was also found to enhance GM-GSF-stimulated
proliferation of the IL-3-dependent cell line, NFS-60,
described by Holmes et al., in Proc. Natl. Acad. Sci.,
Vol. 82, pgs. 6687-6691 (1985).
F. Structure of pcD-2A-E3 and Nucleotide Sequence of
Its cDNA Insert.
The structure of pcD-2A-E3 is illustrated
diagramatically in Figure 2A, and an expanded restriction
map of~its insert is illustrated in Figure 2B. The
insert was sequenced using both the Maxam and Gilbert
approach (Methods in Enzymolog~, Vol. 65, pgs. 499-560
(1980)) and the Sanger approach (Proc. Natl. Acad. Sci.,
Vol. 74, pgs. 5463-5467 (1977)). The sequence is
illustrated in Figure lA, along with the deduced amino
acid sequence for the longest open reading frame in-phase
with the first ATG start codon. The sin gle long open
reading frame in the mouse 2a-E3 cDNA clone consists of
140 amino acid residues. Because this lymphokine is a
secreted protein, a hydrophobic leader sequence would be
expected to precede the sequence for the mature secreted
form of the protein. Analysis of the hydrophobicity of
the polypeptide and comparison with a proposed consensus
sequence for the processing of signal peptides (Penman
et al., J. Mol. Biol., Vol. 167, pgs. 391-409 (1983))
suggest that cleavage of the precursor polypeptide would
occur following the serine residue at amino acid position
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-67-
20 in Figure lA. Grabstein et al., J. Exp. Med_., Vol.
163, pqs. 1405-1414 (19Fi6), has confirmed that the ~d-
terminal sequence of secreted murine IL-4 begins at the
position 21 His of Figure lA.
Example II. Preparation of Human IL-4 Via a Murine
cDNA Probe to a Human Helper T Cell cDNA
Library and Transient Expression in COS 7
Monkey Cells and Mouse L Cells.
cDNA clones coding for IL-4 were isolated from
cDNA libraries constructed from an induced human helper T
cell, 2F1, and induced human peripheral blood lymphocytes
(PBLs) by way of a murine cDNA probe. Other human cell
lines known to produce BCGF activity include variants of
the CEM line, available from the ATCC under accession
numbers CCL 119, CRL 8436, and TIB 195, and described by
Foley et al., in Cancer, Vol. 18, pgs. 522-529 (1965),
and by Ligler, in Lymphokine Research, Vol. 3, pgs. 183-
191 (1984).
A human helper T-cell clone, 2F1, and human
PBLs were grown in Iscove's medium supplemented with 3~
fetal calf serum. The 2F1 cells were activated with ConA
(10 ug/ml) and PBLs were stimulated with 1 ug/ml PNA for
12 hours, after which ConA at 5 ug/ml was added. The
cells were harvested 4 hr (2F1) or 10 hr (PBLs) after
addition of ConA.
mRNA extraction and cDNA library construction
were carried out as described in Example I. A PstI
fragment was isolated from the mouse pcD-2A-E3 cDNA
clone, labeled by nick translation (1 x 108 cpm/ug) and
used to probe nitrocellulose filters containing plasmid
DNA preparations from ten pools, each representing
approximately 1 x 103 clones of 2F1 cDNA lihrary. Low
stringency hybridization conditions (overnight at 42°C)
were used: 6xSSPE (lxSSPE=180 mM NaCl/l~mM sodium
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phosphate, ~H 7.4/1 mM EDTA) (Maniatis, T. et al.,
Molecular Cloning: A Laboratory Manual (Cold Spring
Harbor Laboratory, N.Y., 1982)), 20$ (vol/vol) formamide,
0.1~ sodium dodecyl sulfate, yeast carrier tRNA at 100
ul. The filters were washed with 2 x SSPE, 0.1~ sodium
dodecyl sulfate at 37°C.
A single clone (pcD=46) was identified in one
of the ten pools. Additional clones were obtained by
screening the PBL cDNA libraries with a probe constructed
from the NheI-EcoRI fragment of ncD-46 (illustrated in
the restriction map of Figure 2D). Analysis by
restriction enzymes indicated that the PBL clones were
identical in structure to pcD-46.
It was discovered that a guanidine-rich region
in the 5', or upstream, direction from the coding region
insert of pcD-46 inhibited expression of the IL-4
polypeptide. Consequently, the insert of pcD-46 was
recloned to remove the guanidine-rich region. The
resulting clone is designated pcD-125. It was also
discovered that expression was improved by transfecting
mouse L cells with pcD-45.
The vector pcD-125 was formed as follows:
pcD-46 was cleaved with Sau3A to isolate a fragment
containing the 5' 162 nucleotides of the cDNA insert
(eliminating the GC segment) and then the fragment was
inserted into the BglII site of p101. The plasmid p101
was derived from pcD-mouse IL-3 (see, Yokota, T. et al.,
[1984] cited above) and is deleted for the sequence from
the PstI site at the S' end of the cDNA to a BqlII site
within the mouse IL-3 eDNA. A BglII site is included at
the junction of_ the deleted sequence. 'the Sau3A fragment
is fused to the SV40 promoter as in pcD-46, except for
the GC segment. The remainder of_ the human cDNA was then
reconstructed with a HindIII/NheI fragment from pcD-46
which carries the 3' end of the cDNA, tile SV40 poly-A
site and all of the pBR322 sequences of pcD-46.
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Supernatants of the pcD-46- and pcD-125-
transfected COS 7 and L cells were assayed for BCGF and.
TCGF activity. TCGF was assayed with a human helper T
cell line JL-EBV transformed with Epstein-Barr virus, and
phytohemagglutinin-stimulated human peripheral blood
lymphocytes.
The human helper T-cell clone JL-EBV was
stimulated with irradiated (4500R) cells of a human EBV-
transformed B-cell line, and subsequently maintained in
RPMI 1640 medium containing 10$ human AB serum, 50
micromolar 2-mercaptoethanol (2ME) and recombinant human
IL-2. Human PBLs were stimulated with PHA (20
microgram/ml) and maintained in RPMI 1640 containing 10~
fetal calf serum, 50 micromolar 2ME and recombinant human
IL-2. Five to ten days after stimulation, JL-EBV cells
or PHA blasts were used as targets in a two-day TCGF
assay using the Mosmann colorimetric method (described
above), or in a three-day TCGF assay using [3HJ thymidine
incorporation.
Figure 4A illustrates the TCGF activities
measured by JL-EBV cells (colorimetric assay) of (i)
supernatant from COS 7 cells transfected with pcD
plasmids expressing human IL-2 (curve A); (ii)
supernatant from L cells transfected with pcD-125 (curve
B); (iii) supernatant from COS 7 cells transfected with
pcD-125 (curve C), (iv) supernatant from COS 7 cells
transfected with pcD-46 (curve D), and (v) supernatant
from mock transfected COS 7 cells (curve E). Figure 4B
illustrates the TCGF activities measured by PHA-
stimulated PBLs (colorimetric assay) of (i) supernatant
from COS 7 cells transfected with pcD plasmids expressing
human IL-2 (curve A), (ii) supernatant from COS 7 cells
transfected with pcD-125 (curve B), and (iii) supernatant
from mock transfected COS 7 cells (curve C). Figure 4C
illustrates the TCGF activities measured by PHA-
stimulated PBLs (tritiated thymidine incorporation assay)
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of (i) supernatant from COS 7 cells transfected with pcD-
125 (curve A), (ii) supernatant from COS 7 cells
transfected with pcD plasmids expressing human IL-2
(curve B), and (iii) supernatant from mock transfected
COS 7 cells (curve C).
BCGF activity of various dilutions of pcD-125
transfection supernatants were compared with the BCGF
activity of a BCGF ("commercial BCGF") described by
Maizel et al., Proc. Natl. Acad. Sci., Vol. 79, pgs.
5998-6002 (1982), and commer-cially available from
Cytokine Technology International (Buffalo, NY). Table
II illustrates the BCGF activities of various dilutions
of COS 7 transfection supernatants on anti-IgM antibody
preactivated B cells. B cells were prepared as described
in the assay section above.
Table II. Effect of the IL-4 cDNA transf ection super-_
natants on anti-IaM-preactivated B cells
(vol/vol) ~H-~fh ymidineIncorporation m)
(cp
of v~k_
supernatants Mock- Clone transfection Clone 125
added transfection 125 + 10$ BOGF + 10~ BCGF
0 278 278 1835 1835
0.2 189 144 1362 2303
1 323 1313 1699 3784
5 408 4314 1518 7921
15 397 4289 1093 3487
Table III illustrates the BCGF activities of various
dilutions of COS 7 transfection supernatants on SAC-
preactivated B cells (prepared as described above).
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Table III. Activity of the IL-4 cDNA transfection
supernatants on SAC-preactivated B cells
(vol/vol) 'H~hymidine Incorporation (c )
of Mock-
supernatants NYxk- Clone transfection Clone 125
added transfection 125 + 10~ BOGF' + 10~ BCGF
0 2237 2237 12,992 12,992
0.2 1789 2682 13,126 5,655
1 740 2374 13,714 6,765
5 1285 2826 5,848 10,023
15 1560 4701 10,128 10,924
Although the human IL-4 of the invention and commercial
BCGF both display BCGF activity, Mehta et al., in J.
Immunol., Vol. 135, pgs. 3298-3302 (1985), demonstrated
that TCGF activity can be biochemically separated from
the BCGF activity of commercial BCGF, indicating that the
activities are caused by separate molecules. Thus, human
IL-4 and commercial BCGF are different molecules because
TCGF activity is inseparable from BCGF activity in human
IL-4, using standard biochemical fractionation
techniques.
Supernatants from COS-7 cells transfected with
plasmids bearing the human IL-4 cDNA induce the
proliferation of normal human T cells and the human T-
cell clone JL-EBV, and activity that is similar to mouse
IL-4. However, the maximum extent of proliferation of
human T cells induced in response to human IL-4 is about
half of that induced by human IL-2, The proliferation-
inducing activity of IL-4 could not be inhibited by
monoclonal antibodies against IL-2 br against the IL-2
receptor when tested. These results suggest that IL-4
acts directly on T cells and not by way of induction of
IL-2 and that its activity is not mediated by the IL-2
receptor. The COS-human IL-4 supernatants also stimulate
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the proliferation of human B cells preactivated with
optimal concentrations of anti-IgM antibodies coupled to
beads and have additive proliferative capacity with
commercial BCGF at saturation levels of the BCGF assay.
This suggests that human IL-4 and commercial BCGF operate
on B cells by different routes, e.g, possibly by
different sets of receptors. The supernatants did not
significantly induce proliferation of B cells
preactivated with SAC, whereas commercial BCGF purified
from supernatants of PBL cultures stimulated with PHA
strongly induced the proliferation of SAC-preactivated
human B cells. These results further indicate that the
human IL-4 cDNA encodes a BCGF activity distinct from
that present in the commercial BCGF.
Supernatant of pcD-125-transfected COS 7 cells
was also tested for its ability to induce Fc-epsilon
receptors on tonsilar B cells. Human tonsil cells were
dispersed into a single cell suspension using standard
techniques. The B cell population was enriched using the
protocol described above, and the enriched cells were
stimulated with anti-IgM antibody for 24 'hours in culture
medium at 37°C. Fc-epsilon receptor-bearing cells were
assayed by a Becton Dickinson FRCS IV cell sorter using a
fluorescently labeled monoclonal anti'oody specific for
the receptor by means of the technique disclosed by
Bonnefoy et al., in J. Immunol. Meth., Vol. 88, pgs. 25-
32 (1986). Figures 5A-SD are histagrams illustrating
cell frequency (ordinate) versus fluorescent intensity
(abscissa). Fluorescence intensity is proportional to
the number of Fc-epsilon receptors present on a cell. In
all the Figures the cells have been stimulated with anti-
IgM. Figures 5A through 5D correspond to exposures to
media consisting of 0~, 0.1~, l~, and 10$ supernatant
from pcD-125-transfected COS 7 cells.
The DNA sequence of the cDNA insert of clone
no. 46 was determined and is shown in Figure 1B. The
1 X41 299
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cDNA insert is 615 by long, excluding the poly(a) tail.
There is a single open reading frame, with the first aTG
codon located at 64 nucleotides from the 5' end followed
by 153 codons ending with the termination codon TAG at
nucleotide positions 523-525. The N-terminal segment of
the predicted polypeptide is hydrophobic, as would be
expected for a secreted protein.
A comparison between the coding regions of a
human 'and a mouse cDNA of the present invention revealed
that the regions of .the human cDNA coding sequence in.
pcD-46 covered by amino acid positions 1-90 and 12.9-149
share approximately 50$ homology with the corresponding
regions of the mouse cDNA (2A-E3) coding sequence. These
regions, and 5' and 3' untranslated regions, share about
70% homology between the two cDNA sequences from the
different species, whereas the region covered by amino
acids 91-128 of the human protein shares very limited
homology with the corresponding mouse region. In all,
six of the seven cysteine residues in the human protein
are conserved in the related mouse protein. Some amino
acid sequence homology exists between a native form of a
human polypeptide of the present invention and mouse IL-
3. Amino acid residues 7-16 and 120-127 are 50~ and 55$
homologous, respectively, to residues 16-27 and 41-49 of
the mouse IL-3 precursor polypeptide (Yokota, T. et al.,
Proc. Natl. Acad. Sci. U.S.A. 81: 1070-1074 [1984]).
As described more fully below, izuman IL-4
purified from pcD-125-transfected COS 7 supernatants was
found to be the 129 amino acid polypeptide having the
sequence illustrated by Figure 1C.
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Example III. Enhanced Expression of_ Human IL-4 in COS 7
Monkey cells by Using an Epstein-Barr
Virus (EBV)-Derived Vector Containing an
RSV-LTR Promoter.
A 10-20 fold enhancement of human IL-4
expression was obtained by recloning the XhoI fragment
of pcD-125 into an EBV-derived vector containing a Rous
sarcoma virus long-terminal repeat (RSV-LTR) promoter.
The EBV-derived vector and the RSV-LTR promoter are
described by Gorman et al., Proc. Nat. Acad. Sci., Vol.
79, pgs. 6777-6781 (1982); and Yates et al., Nature,
Vol. 313, pqs. 812-815 (1985).
A HindIII/XhoI fragment containing the RSV-LTR
promoter was isolated from a pcD plasmid previously
constructed from the RSV-LTR containing AccI/HindIII
fragment described by Gorman et al. (cited above) and a
commercially available pcD vector (e.g. Pharmacia). The
above HindIII/Xhol fragment and a HindIII/XhoI fragment
from a pLl plasmid (Pharmacia) containing an SV40 oriqin-
of-replication (ori) are spliced into plasmid pcDVl
(available from Pharmacia), the orientation of the SV40
o n region not being critical. Between an AatII site and
an NdeI site, the resulting pcD vector contains in
sequence (from the AatII site) an SV40 o n region, an
RSV-LTR promoter, and the SV40 poly A region. After the
XhoI fragment of pcD-125 is isolated anc9 inserted into
the XhoI site of the newly constructed pcD vector, the
unique AatII and NdeI sites on the vector are converted
into SalI sites using standard techniques. Briefly, the
pcD vector is digested with AatII and N<9eI, the IL-4
containing fragment is isolated, and the isolated
fragment is treated with T4 DNA polymerase in the
presence of appropriate concentrations of the nucleoside
triphosphates. The 5'~ 3' DNA polymerase activity of
T4 DNA polymerase fills in the 5' protruding ends of the
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restriction cuts, and the 3'--~ 5' exonuclease activity
of T4 DNA polymerase digests the 3' protruding ends of_
the restriction cuts to leave a blunt-ended fragment, to
which kinased SalI linkers (New England Biolabs) are
ligated using T4 DNA ligase.
The above SalI fragment (illustrated in Figure
11) is inserted in the EBV-derived vector p201 described
by Yates et al. (cited above) at the location of a unique
ClaI site, which had been converted to a SalI site using
standard techniques. Briefly, p201 (illustrated_in
Figure 11) is digested with ClaI and treated with DNA
polymerase I (Klenow fragment) and appropriate
concentrations of nucleoside triphosphates. This
procedure fills in the protruding ends of the ClaI cut to
leave a blunt-ended fragment. Next, the blunt ends are
ligated to a kinased SalI linker. The resulting EBV-
derived vector containing the RSV-LTR promoter and human
IL-4 cDNA insert is referred to herein as pEBV-178.
pEBV-178 was transfected into COS 7 cells using
standard techniques and the culture supernatants were
assayed for TCGF activity as a measure of IL-4
expression.
Example IV. Expression of Native Human IL-4 and Mutein
ISO (Ala-Glu-Phe) in E, coli
Two vectors containing human IL-4 cDNA inserts
were constructed for expression of human IL-4 in _E.
coli: a pIN-III secretion vector which contains the
signal peptide sequence of the om~A protein ("pIN-III-
ompA2"), and a pUCl2 plasmid containing a trpP promoter
and an adjacent ribosome binding site (RBS) region
("TRPC11").
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A. pIN-III-ompA2
Two vectors were constructed using the pIN-III-
ompA2 plasmid, which is described by Ghrayeb et al., in
EMBO Journal, Vol. 3, pgs. 2437-2442 (1984), and Masui et
al., in Biotechnology, Vol. 2, pgs. 81-85 (1984).
The first vector, designated pIN-III-ompA2(1),
was constructed by ligating, in series, the EcoRI/BamHI-
digested pIN-III-ompA2 plasmid, a synthetic linker, and
the BamHI/EcoRV fragment of pcD-125. The synthetic
linker used in this construction resulted in the
secretion of a biologically active IL-4 polypeptide
having the three extra N-terminal amino acids Ala-Glu-
Phe-; i.e. mutein ILO(Ala-Glu-Phe) was secreted. The
synthetic linker consisted of the following sequences of
nucleotides:
AA TTC CAC AAG TGC GAT
G GTG TTC ACG CTA
EcoRI/BamHI-digested pIN-III-ompA2 and the
BamHI/EcoRV fragment of pcD-125 were mixed in a standard
ligation solution (e. g. Maniatis et al., cited above)
containing 0.1 micromolar of the synthetic linker. _E.
coli strain Ab1899 was infected by the pIN-III-ompA2(1)
plasmid and transformants were selected by colony
hybridization using a 32P-labeled IL-4 cDNA probe. Human
IL-4 extracts for assaying were obtained as follows.
After sonication, the bacterial cultures were
centrifuged, and the supernatant removed from the
pellet. The pellet was treated with 1~ SDS, 2rnM
dithiothreitol, and 6M guanidine. The material was
recentrifuged, the supernatant discarded, and the pellet
treated at 45°C with 3$ SDS and 2mM dithiothreitol. The
material was again centrifuged, and the supernatant
assayed by SDS-PAGE.
1341 X99
pIN-III-ompA(2) was constructed so that the
native human IL-4 would be expressed. The three amino
acid addition in the pIN-III-ompA(1) construction was
eliminated by site-specific mutagenesis of the om A
signal peptide sequence of pIN-III-ompA2. The site-
specific mutagenesis was carried out as disclosed by
Zoller and Smith (cited above). Briefly, the XbaI/BamHI
fragment of pIN-III-ompA2 containing the coding sequence
for the o~A signal peptide (see Fig. 1 in Ghrayeb et
al., cited above) was purified, mixed with purified
XbaI/BamHI-digested replicating form (RF) of M13mp19,
ligated, transfected into E. coli K-12 JM101, and
plated. A clear plaque in the presence of IPTG and X-gal
was selected and propagated, and single stranded DNAs
were prepared, e.g. according to the procedures disclosed
by Messing in Methods in Enzymology, Vol. 101 (Academic
Press, New York, 1983) . Separately, the following
oligonucleotide primer (23-mer) containing the indicated
base substitutions (boxed) was synthesized and
phosphorylated:
5' - GGAATTCAGAAGCT TG C G~ GCTAC - 3'.
This sequence introduces a second HindIII site in the
signal peptide coding region of the mutated pIN-III-
ompA2. The oligonucleotide primer was annealed to the
M13mp19 RF containing the XbaI/BamHI fragment of pIN-III-
ompA2, and treated with DNA polymerase in the presence of
appropriate concentrations of nucleoside triphosphates.
The resulting RFs were used to transfect JM101 E. coli,
and mutant-containing plaques were screened by a labeled
oligonucleotide probe. The sequence of the selected RF
was confirmed by dideoxy sequencing using a universal M13
primer. The selected RF was propagated, isolated, and
digested with XbaI and BamHI, and the purified XbaI/BamHI
fragment was inserted into an XbaI/BamHI-digested pIN-
1 X41 299
_,8_
III-ompA2. To form pIN-III-ompA2(2), the mutant pIN-III-
ompA2 was propagated, purified, digested with HindIII and
BamHI, and mixed with the BamHI/EcoRV fragment of pcD-125
in a standard liqation solution containing 0.1 micromolar
of the following synthetic linker:
A GCT CAC AAG TGC GAT
GTG TTC ACG C'.TA
E. coli strain Ab1899 was infected by the pIN-III-
ompA2(2) plasmid and transformants were selected by
colony hybridization using a 32P-labeled IL-4 cDNA
probe. IL-4 extracts, prepared as described above,
exhibited TCGF activity comparable to supernatants of
pcD-125 COS7 cells.
B. TRPC11
The TRPC11 vector was constructed by ligating a
synthetic consensus RBS fragment to ClaI linkers (ATGCAT)
and by cloning the resulting fragments into ClaI-
restricted pMTllhc (which had been previously modified to
contain the ClaI site). pMTllhc is a small (2.3
kilobase) high copy, AMPR, TETS derivative of_ pBR322 that
bears the EcoRI-HindIII polylinker region of the nVX
plasmid (described by Maniatis et al., cited above). It
was modified to contain the ClaI site by restricting
pMTllhc with EcoRI and BamHI, f_illinq in the resulting
sticky ends and lipating with ClaI linker (CATCGATG),
thereby restoring the EcoRI and BamHI sites and replacing
the SmaI site with a ClaI site.
One transformant from the TRPC11 construction
had a tandem RBS seauence flanked by ClaI sites. One of
the ClaI sites and part of the second copy of_ the RBS
sequence were removed by digesting this plasmid with
PstI, treating with Ba131 nuclease, restricting with
EcoRI and treating with T4 DNA polymerase in the presence
1341299
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of all four deoxynucleotide triphosphates. The resulting
30-40 by fragments were recovered via PAGE and cloned
into SmaI-restricted pUCl2. A 248 by E. coli tr~P-
bearing EcoRI fragment derived from pKC101 (described by
Nichols et al. in Methods in Enzymology, Vol. 101, pg.
155 (Academic Press, N.Y. 1983)) was then cloned into the
EcoRI site to complete the TRPC11 construction, which is
illustrated in Figure 12.
TRPC11 was employed as a vector for human IL-4
cDNA by first digesting it with ClaI and BamHI, purifying
it, and then mixing it with the EcoRV/BamHI fragment of
pcD-125 in a standard ligation solution containing 0.1
micromolar of the following synthetic linker:
TCG ATG CAC AAG TGC GAT
AC GTG TTC ACG CTA
The insert-containing vector was selected as described
above and propagated in E. coli K-12 strain JM101. IL-4
was extracted as follows. JM101 cells were sonicated in
their culture medium and centrifuged. The pellet was
r
resuspended in 4M guanidine and 2mM dithiothreitol, and
again centrifuged. The supernatant was tested for
biological activity and found to exhibit TCGF activity
comparable to that of supernatants of pcD-125-transfected
COS7 cells.
Example V. Preparation of Bovine IL-4 cDNAS Via Mouse
and Human IL-4 cDNA Probes to a Bovine
Helper T Cell cDNA Library and Transient
Expression in COS 7 Monkey Cells.
cDNA clones coding for IL-4 are isolated from
cDNA libraries constructed from induced bovine peripheral
blood lymphocytes (PBLs) by way of combined mouse and
human cDNA probes. Alternative sources of bovine cDNAs
1 X41 Z99
-so-
include several bovine cell lines maintained in the
ATCC's NBL animal line collection. Procedures are
substantially identical to those described in Example
II. Cells are harvested about 10 hours after induction
by ConA. mRNA extraction and cDNA library construction
are carried out as in Example II.
The mouse and human cDNA probes can be used
together as a mixture or sequentially to detect bovine
IL-4 cDNAs. As in Example II, the PstI fragment is
isolated from the mouse pcD-2A-E3 cDNA clone. Likewise
the PstI fragment is isolated from the human pcD-125 cDNA
clone. Several other fragments are also available to
construct probes from. Either together or separately the
isolated PstI fragments are labeled by nick translation
(about 1 x 108 cpm/microgram) and are used to probe
nitrocellulose filters containing plasmid DNA
preparations from 10 pools, each representing about 1000
clones of the induced PBL cDNA library. Filter
hybridization is carried out as in Example II. Positive
scoring clones are identified and propagated.
Example VI. Expression of Native Human IL-4 and
Muteins D1-4 and ISO(Gly-Asn-Phe-Val-His-
Gly) in Saccharomyces cerevisiae
Native human IL-4 cDNA and two mutants thereof
were cloned into the plasmid pMF-alpha8 and expressed in
the yeast Saccharomyces cerevisiae. The construction and
application of pMF -alpha8 for expressing non-yeast
proteins is described fully in Miyajima et al., Gene,
Vol. 37, pgs. 155-161 (1985); and Miyajima et al., EMBO
Journal, Vol. 5, pqs. 1193-1197 (1986). prsF-alpha8 is
deposited with the American Type Culture Collection
(Rockville, MD) under accession number 40140, and a map
of the plasmid is given in Figure 13A (designations in
1 341 29 9
-81-
the figure are defined fully in Miyajima et al., Gene,
cited above).
A. Human IL-4 Mutein Dl-4.
Plasmid pcD-125 was isolated and digested with
EcoRV and BamHI. The EcoRV/BamHI fragment containing
the human IL-4 cDNA was isolated, treated with DNA
polymerase I (Klenow fragment) to fill in the BamHI cut,
and kinased (i.e. phosphorylated). pMF-alpha8 was
digested with StuI and combined with the kinased
EcoRV/BamHI fragment of pcD-125 in a standard ligation
solution to form plasmid phIL-4-2. phIL-4-2 was used to
transform S. cerevisiae 20B-12 (MATalpha tr~l-289
pe~4-3), which was obtained from the Yeast Genetic Stock
Center, University of California, Berkeley. Yeast cells
were grown in synthetic medium containing 0.67$ Yeast
Nitrogen Base without amino acids, 2$ glucose, and 0.5$
Casamino acids (Difco). The yeast cells were transformed
with the plasmids by the lithium acetate method of Ito et
al., J. Bacteriol., Vol. 153, pqs. 163-168 (1983), and
transformants were selected in synthetic medium lacking
tryptophan. Supernatant of a transformant culture was
tested for TCGF activity. Figure 13B (curve D)
illustrates the TCGF activity of several dilutions of the
supernatant from phIL-4-2-transformed yeast cells in
comparison with other factors (Curve A ~-- human IL-2;
Curve B -- supernatant from pcD-125-transfected COS 7
cells; and Curve C -- supernatants from phIL-4-1-
transformed yeast cells). Curve E illustrates the TCGF
activity of supernatant from yeast that had been
transformed with pMF-alpha8 lacking the IL-4 cDNA insert,
i.e. the "mock" transformant.
B. Human IL-4 Mutein ISO(Gly-Asn-Phe-Val-His-Gly).
The pMF-alpha8 insert for expression of mutein
ILO(Gly-Asn-Phe-Val-His-Gly) was prepared exactly as for
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mutein ~1 4, except that the NaeI/BamHI fragment from
pcD-125 was used. The resulting plasmir3 was designated
phIL-4-1. Several dilutions of supernatant from phIL-4-
1-transformed yeast cells were tested for TCGF
activity. The results are illustrated by Curve C of
Figure 138. The supernatants were also tested for BCGF
activity on both anti-IgM and SAC-acticrated B
lymphocytes. The assays were performed as described
above, and the results are given in Table IV.
Table IV. BCGF Activity of Supernatants of phIL-4-3
Transformed Yeast Cells
$ (vol/vol) [3Hj-Thymidine Incorporation (cFm)
of
supernatants SAC-activated Anti-IgM Head Activated
added 8 Lymptyocytes B Lymphocytes
.0 3633 t 1239 641 t 69
0.09 7610 t 310 13221 f 472
O.I9 9235 t 181 --- - -----
0.39 10639 t 786 16681 t 310
0.78 10372 t 572 18090 t 1248
1.56 9905 t 328 17631 f 1216
3.12 11354 t 836 18766 t 1179
6.25 10481 t 541 19810 t 1349
12.5 9641 t 30 18136 t 1126
25. 8253 f 857 14750 t 1125
C. Expression of Native Human IL-4 in Yeast.
cDNA coding for native human IL-4 was cloned
into pMF-alpha8 by first inserting bases upstream of the
N-terminal His colon to form a KpnI restriction site.
After cleavage by KpnI and treatment by DNA polymeraseI,
the hlunt-ended IL-4 cDNA was inserted into the StuI site
of pMF-alpha8. The KpnI site was formed by use of
standard site-specif.ic.mutaqenesis. Briefly, pcD-125 was
digested with BamHI, and the f_raqment containing the
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-83-
entire human IL-4 cD~IA was isolated and inserted into the
BamHI site of M13mp8. Single-stranded M13mp8 containing
the insert was isolated and hybridized to the following
synthetic oligonucleotide which served as a primer:
5' - TCCACGGA GGTAC CACAAGTG - 3'
The inserted nucleotides are boxed. The plasmid
containing the mutated IL-4 cDNA was identified by an
oligonucleotide probe, propagated, isolated, and treated
with KpnI and BamHI. The Kpnl/BamHI fragment was
isolated, treated with DNA polymerase I (Klenow fragment)
to generate blunt ends, kinased, and ligated with StuI-
digested pMF-alpha8. Yeast was transformed by the
resulting pMF-alpha8 plasmids, designated phIL-4-3, as
described above, and supernatants were tested for TCGF
activity. The supernatants displayed TCGF activity
comparable to that observed for supernatants of phIL-4-1-
transformed yeast.
Example VII. Construction and Expression of a Synthetic
Human IL-4 Gene in E. coli
A synthetic human IL-4 gene is constructed
which substantially comprises bacterial-preferred codons,
includes a series of unique restriction endonuclease
sites (referred to herein as "unique restriction sites"),
and permits rapid and convenient expression of a wide
variety of human IL-4 muteins. The nucleotide sequence
of the synthetic gene is illustrated in Figure 6A.
Techniques for constructing and expressing the synthetic
gene of this example are standard in the art of molecular
biology, e.g. especially Sproat and Gait-, ~lucleic Acids
Research, Vol. 13, pgs. 2959-2977 (1985); and rFerretti et
al., Proc. Natl. acad. Sci., Vol. 83, pgs. 599-603
(1986); but also Mullenbach et al., J. Biol. Chem., Vol.
1 X41 X99
-84-
261, pgs. 719-722 (1986); ~~Iells et al., Gene, Vol. 34,
pgs. 315-323 (1985); and Estell 'et al., Science, Vol.
233, pgs. 659-663 (1986). Briefly, the synthetic human
IL-4 gene is assembled from a plurality of chemically
synthesized double-stranded DNA fragments. Base
sequences of the synthetic gene are selected so that the
assembled synthetic gene contains a series of unique
restriction sites.
The series of unique restriction sites defines
a series of segments which can be readily excised and
replaced with segments having altered base sequences.
The synthetic fragments are inserted either directly or
after ligation with other fragments into a suitable
vector, such as a pUC plasmid, or the like. The above-
mentioned segments roughly correspond to the "cassettes"
of Wells et al. (cited above). The synthetic fragments
are synthesized using standard techniques, e.g. Gait,
Oliqonucleotide Synthesis: A Practical Approach (IRL
Press, Oxford, UK, 1984). Preferably an automated
synthesizer is employed, such as an Applied Biosystems,
Inc. (Foster City, CA) model 380A, pUC plasmids and like
vectors are commercially available, e.g. Pharmacia-PL, or
Boehringer-Mannheim. Cloning and expression can be
carried out in standard bacterial systems, for example E.
coli K-12 strain JM101, JM103, or the like, described by
Viera and Messing, in Gene, Vol. 19, pgs. 259-268 (1982).
Restriction endonuclease digestions and ligase
reactions are performed using standard protocols, e.g.
Maniatis et al., Molecular Cloning; A Laboratory Manual
(Cold Spring Harbor Laboratory, New York, 1982).
The alkaline method (Maniatis et al., cited
above) is used for small-scale plasmid preparations. For
large-scale preparations a modification of the alkaline
method is used in which an equal volume of isopropanol is
used to precipitate nucleic acids from the cleared
lysate. Precipitation with cold 2.5 M ammonium acetate
1341299
-85-
is used to remove RNA prior to cesium chloride
equilibrium density centrifugation and detection with
ethidium bromide.
For filter hybridizations Whatman 540 filter
circles are used to lift colonies which are then lysed
and fixed by successive treatments with 0.5M NaOH, 1.5M
NaCl; 1M Tris.HCl pH8.0, 1.5M NaCl (2 min each); and
heatin4 at 80°C (30 min). Hybridizations are in 6xSSPE,
20$ formamide, 0.1$ sodium dodecylsulphate (SDS), 100
ug/ml E. coli tRNA, 100 ug/ml Coomassie Brilliant Blue G-
250 (Biorad) at 42°C for 6 hrs,using 32P-labelled
(kinased) synthetic DNAs. (20xSSPE is prepared by
dissolving 174 g of NaCl, 27.6 q of NaH2P04'H20, and 7.4
q of EDTA in 800 ml of H20, ad j ust i ng pH to 7 . 4 wi th
NaOH, adjusting volume to 1 liter, and sterilizing by
autoclaving.)
Filters are Washed twice (15 min, room
temperature) with lxSSPE, 0.1% SDS. After
autoradiography (Fuji~RX film), positive colonies are
located by aligning the reqrown colonies with the hlue-
stained colonies on the filters.
DNA is sequenced by either the chemical
degradation method of Maxam and Gilbert, Methods in
Enzymoloqy, Vol. 65, pq. 499 (1980), or the dideoxy
method of Sanger et al. Proc. Natl. Acad. Sci., Vol. 74,
pg. 5463 (1977). Templates for the dideoxy reactions are
either single-stranded DNAs of relevant regions recloned
into Ml3mp vectors, e.q. Messing et al. Nucleic Acids
Res., Vol. 9, pg. 309 (1981), or double-stranded DNA
prepared by the minialkaline method and denatured with
0.2M NaOH (5 min, room temperature) and precipitated from
0.2M NaOH, 1.43M ammonium acetate by the addition of_ 2
volumes of ethanol. Dideoxy reactions are done at 42°C.
DNA is synthesized by phosphoram-idite chemistry
using Applied Biosystems 380A synthesizers. Synthesis,
deprotection, cleavage and purification (7M urea PAGE,
_~Y'G~e~yo a r
1341299
-86-
elution, DEAF-cellulose chromatography) are done as
descrihed in the 380A synthesizer manual. Complementary
strands of synthetic DNAs to be cloned ( 400ng each ) are
mixed and phosphorylated with polynucleotide kinase in a
reaction volume of 50 ul. This DNA is ligated with 1 ug
of vector DNA digested with appropriate restriction
enzymes, and liQations are done in a yo:Lume of 50 ul at
room temperature for 4 to 12 hours. Conditions for
phosphorylation, restriction enzyme digestions,
polymerase reactions, and ligation. have been described
(Maniatis et al., cited above). Colonies are scored for
lacZ+ (when desired) by plating on L agar supplemented
with ampicillin, isopropyl-1-thio-beta-D-galactoside
(IPTG) (0.4 mM) and 5-bromo -4-chloro-3-:indolyl-beta-D-
qalactopyranoside (x-gal) (40 ug/ml).
The TAC-RBS vector was constructed by a
sequence of steps, starting with filling-in with DNA
polymerase the single BamHI site of the tacP-bearing
plasmid pDR540 (Pharmacia). The product was then ligated
to unphosphorylated synthetic olic~onucleotides
(Pharmacia) which form a double-stranded fragment
encoding a consensus ribosome binding site (RBS,
GTAAGGAGGTTTAAC). After ligation, the mixture was
phosphorylated and reliqated with the SstI linker
ATGAGCTCAT. The resulting complex was then cleaved with
SstI and EcoRI, and the 173 by fragment isolated via
polyacrylamide gel electrophoresis (PAGE:) and cloned into
EcoRI/SstI-restricted pUCl8 (Pharmacia) (as described
below). The sequence of the RBS-ATG-polylinker regions
of the final construction (called TAC-RBS) is shown in
Figure 8.
The synthetic human IL-4 gene is assembled into
a pUCl8 plasmid in six steps. At each step inserts free
of deletions and/or inserts can he detected after cloning
by maintaining the lacZ(a) gene of pUCl~i in frame with
the ATG -start codon inserted in step 1. Clones
1 349 29g
_$7_
containing deletion and/or insertion changes can be
filtered out by scoring for blue colonies on L-ampicillin
plates containing x-gal and IPTG. Alternatively, at each
step sequences of inserts can be readily confirmed using
a universal sequencing primer on small-scale plasmid DNA
preparations, e.g. available from Boehringer Mannheim.
In step 1 the~TAC-RBS vector is digested with
SstI, treated with T4 DNA polymerase (whose 3'
exonuclease activity digests the 3' protruding strands of
the SstI cuts to form blunt-end fragments), and after
deactivation of T4 DNA polymerase, treated with EcoRI to
form a 173 base pair (bp) fragment containing the TAC-RBS
region and having a blunt end at the ATG start codon and
the EcoRI cut at the opposite end. Finally, the 173 by
TAC-RBS fragment is isolated.
In step 2 the isolated TAC-RBS fragment of step
1 is mixed with EcoRI/SstI-digested plasmid pUClB and
synthetic fragment lA/B, which as shown in Figure 7A has
a blunt end at its upstream terminus and a staggered
(sticky) end corresponding to an SstI cut at its
downstream terminus. The fragments are ligated to form
the pUCl8 of step 2.
In step 3 synthetic fragments 2a/B and 3A/B
(illustrated in Figure 7B and 7C) are mixed with
SstI/BamHI-digested pUClB of step 2 (after amplification
and purification) and ligated to form pUCl8 of step 3.
Note that the downstream terminus of fragment 2A/B
contains extra bases which form the BamHI staggered
end. These extra bases are cleaved in step 4. Also
fragments 2A/B and 3A/B have complementary 9-residue
single-stranded ends which anneal upon admixture, leaving
the upstream SstI cut of 2A/B and the downstream BamHI
cut of 3A/B to ligate to the pUCl8.
In step 4 MluI/XbaI-digested pUCl8 of step 3
(after amplification and purification) is repurified,
1341299
-8$_
mixed with synthetic fragment 4A/B (Figure 7D), and
ligated to form pUCl8 of step 4.
In step 5 XbaI/SalI-digested pUCl8 of step 4
(after amplification and purification) is mixed with
synthetic fragment of 5A/B (Figure 7E) and ligated to
form the pUCl8 of step 5.
In step 6 SalI/HindIII-digested pUCl8 of step 5
(after amplification and purification) is mixed with
synthetic fragment oA/B (Figure 7F) and ligated to form
the final construction.
Figure 6B is a cleavage map of the unique
restriction sites present in the pUCl8 construction just
described. When the disclosed synthetic human IL-4 gene
is used as an insert of pUClB, each pair of unique
restriction sites defines a segment which can be readily
excised and replaced with altered synthetic segments.
The set of unique restriction sites includes EcoRI, HpaI,
SacI (SstI), EcoRV, PstI, MluI, BclI, Xbal, NaeI, SalI,
XhoI, and HindIII.
The pUCl8 containing the synthetic IL-4 gene is
inserted in E. coli K-12 strain JM101. After culturing,
protein is extracted from the JM101 cells and dilutions
of the extracts are tested for biological activity.
Example VIII. Construction and Expression of Human IL-4
Mutein I1e52 in E. coli.
Leu at position 52 (relative to the N-ter.rninu.s
of the native human IL-4) is changed to Ile to form human
IL-4 mutein I1e52. The pUCI8 plasmid of Example VII
containing the synthetic human IL-4 gene of Figure 6A is
digested with PstI and MluI and purified. The above
purified pUClB is mixed with the synthetic double-
stranded fragment illustrated below and ligated. The
altered part of the base sequence is boxed. The
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_89_
resulting pUCl8 is transfected into E. coli K-12 strain
JM101, or the like, and expressed.
GA GCT GCT ACC GTT ATC CGT
ACG TCT CGA CGA TGG CAA TAG
GCA
CAG TZ'C TAC TCT CAC ' GAA
CAC AAA
GTC AAG ATG AGA GTG~ GTG CTT
TTT
GAC A
CTG TGC GC
PstI/MluI Replacement Fragment For_
Generating Auman IL '4 Mutein I1e52
After culturing, protein is extracted from the
JM101 cells using standard techniques, and dilutions of
the extracts are tested for biological activity.
Example IX. Construction and Expression of Human IL-4
Mutein (I1e52, Asplll).
The modified pUClB plasmid of Example VIII
(containing the I1e52 coding sequence) is digested with
SalI and XhoI, and the large fragment is isolated. The
isolated fragment is mixed with the synthetic double-
stranded fragment illustrated below in a standard
ligation solution. The altered part of the sequence is
boxed. The resulting plasmid is transfected into E. coli
K-12 strain JM101, or the like, and expressed.
TCG ACT CTG GAA GAC TTC C
GA GAC CTT CTG AAG GAG CT
1 349 299
-90-
SalI/XhoI Replacement Fragment
After culturing, protein is extracted from the
JM101 cells using standard techniques, and dilutions of
the extracts are tested for biological activity.
Example X. Sequence of Human IL-4 Purified from
Transfection Supernatants
Human IL-4 was purified from culture
supernatants of cells transiently transfected with
vectors containing human IL-4 cDNA. The sequence of the
secreted native human IL-4 was determined from the
purified material.
A. Biological Assay for Purification.
TCGF activity was used to assay human IL-4
during the separation procedures. The assay was
substantially the same as that described in Example II.
Briefly, blood from a healthy donor was drawn into a
heparinized tube and layered onto Ficoll.-Hypaque; e.g., 5
ml of blood per 3 ml Ficoll-Hypaque in a 15 ml centrifuge
tube. After centrifugation at 3000 x q for 20 minutes,
cells at the interface were aspirated and diluted in a
growth medium consisting of RPMI 1640 cantaininq 10~
fetal calf serum, 50 micromolar 2-mercaptoet~hanol, 20
microqram/ml phytohemaqqlutinin (PHA), and recomhinant
human IL-2. After 5-10 days of. incubation at 37°C, the
PHA-stimulated peripheral blood lymphocytes (PRLs) were
washed and used in 2-day colorimetric assays: Mossmann,
J. Immunol. Methods, Vol. 65, pqs. 55-63 (1983). Serial
two-fold dilutions of the IL-4 standard (supernatants
from either pcD-125- or pEHT-178-transfected COS 7 cells)
or the fraction to be tested were performed in 96-well
trays utilizing the growth medium described above to
yield a final volume of 50 microliters/well. 50
microliters of the PHA-stimulated PBLs at about 4-8 x 106
1341299
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cells/ml were added to each well and the trays were
incubated at 37°C for 2 days. Cell growth was then
measured according to Mosmann (cited above).
Units of human IL-4 TCGF activity are defined
with respect to supernatants of either pcD-125-
transfected COS 7 cells (Example II) or pEBV-178-
transfected COS 7 cells (Example III).
For purification, units are based on the
activity of pcD-125-transfection supernatants, which are
produced as follows. About 1 x 106 COS 7 cells are
seeded onto 100 mm tissue culture plates containing
Dulbecco's Modified Eagle's medium (DME), 10$ fetal calf
serum, and 4 mM L-glutamine. About 24 hours after
seeding, the medium is aspirated from the plates and the
cells are washed twice with serum-free buff ered (50 mM
Tris) DME. To each plate is added 4 ml serum-free
buffered DME (with 4 mM L-glutamine), 80 microliters
DEAE-dextran, and 5 micrograms of pcD-125 DNA. The cells
are incubated in this mixture for 4 hours at 30°C, after
which the mixture is aspirated off and the cells are
washed once with serum-free buffered DME. After washing,
5 ml of DME with 4 mM L-glutamine, 100 micromolar
Chloroquine, and 2~ fetal calf serum is added to each
plate, and the cells are incubated for 3 'hours and then
twice washed with serum-free buffered DME. Next, 5 ml.
DME with 4 mM L-glutamine and 4$ fetal calf serum is
added and the cells are incubated at 37°C for 24 hours.
Afterwards the cells are washed 1-3 times with DME or
PBS, 5 ml serum-free DME (with 4 mM L-glutamine) is
added, and the cells are incubated at 37°C until culture
supernatants are harvested 5 days later.
One unit, as used herein, is the amount of
factor which in one well (0.1 rnl) stimulates 50~ maximal
proliferation of 2 x 104 PHA-stimulated PBLs over a 48
hour period.
1341299
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B. Purification
Purification was accomplished by a sequential
application of cation-exchange chromatography, gel
filtration and reverse-phase high-pressure liquid
chromatography. All operations were performed at 4°C.
Af to r remova l of the COS 7 cel l s by
centrifugation, the supernatant was concentrated about 10
fold by ultrafiltration and stored at -80°C until further
processed. IL-4 titers were determined by assaying for
the ability of the protein to stimulate proliferation of
phytohemagqlutinin-induced human peripheral blood
lymphocytes, i.e. by TCGF activity using the standard
assay descrihed above.
Concentrated COS 7 supernatant, having TCGF
activity of about 104-106 units/ml and a protein content
of about 15-20 mg/ml, is dialyzed against 2 changes of 50
mM sodium HEPES, pH 7.0 over a 24 hour period (each
change being approximately 10-15 times the volume of one
concentrate). The dialysate was applied to a column (1 x
2.5 cm) of S-Sepharose (flow rate: 0.2 ml/min) pre-
eQUilibrated with 50 mM sodium HEPES, pH 7Ø The column
was washed with 15 column volumes of equilibrating buffer
followed by elution with 20 column volumes of a linear
sodium chloride gradient extending from 0 to 0.5 M sodium
chloride in 50 mM sodium HEPES, pH 7Ø The gradient was
terminated by elution with 5 column volumes of uniform
concentration: 50 mM sodium HEPES, 0.5 M NaCl, pH 7Ø
1.5 ml and 1.8 ml fractions were collected from
respective batches. IL-4 titers were found fir both
chromatoqraphies to elute between 300 mM and 500 mM
sodium chloride.
Six fractions from each S-Sepharose column
containing IL-4 titers were separately combined f_or total
volumes of 9.0 and 10.8 ml. Both volumes were
concentrated to 1.9 ml by ultrafiltration using an Amicon
YM5 membrane (molecular weight cut-off: 5000). The
W''~ G7~e/~'~a Y
1 341 2 9 9
-93-
recovery of protein from this step was about 80~. The
concentrated IL-4 solution was applied to a Sephadex 6-
100 column (1.1 x 58 cm) pre-equilibrated in 50 mri HEPES,
0.4 M NaCl, pH 7.0, and the column was eluted with the
same buffer at 0.15 ml/min. A total of 50 fractions (1.0
ml/fraction) were collected and analyzed for IL-4
titers. A peak in biological activity was observed at an
apparent molecular weight of 22,000 daltons. The
Sephadex G-100 was calibrated for apparent molecular
determination with bovine serum albumin (65,000 daltons),
carbonic anhydrase (30,000 daltons) and cytochrome C
(11,700 daltons).
A fraction from the Sephadex G-100 column
containing IL-4 activity was concentrated 3-4 fold in
vacuo and was injected onto a Vydac C-4 guard column (4.6
x 20 mm). A linear gradient of 0 to 72% (v/v)
acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA) was
produced in 15 minutes at a column temperature of 35° and
a flow rate of 1.0 ml/min. Three peaks resulted that
were detected at 214 nm with retention times of 7, 8.2
and 8.7 min. (peaks 1, 2, and 3 of Figure 10,
respectively). A 40 microliter aliquot of peak 2 (8.2
min. elution time) cvas lyophilized and redissolved in
minimal essential medium containing 10$ fetal calf
serum. This solution showed a positive TCGF response. A
300 microliter aliquot of peak 2 was evaporated to
dryness and redissolved in 200 ul of 0.1$ (w/v) sodium
dodecyl sulfate (SDS). A 2 ul aliquot was diluted in 200
ul of 1$ (v/v) TFA and rechromatoc~raphed. The HPLC of
this sample demonstrated a single peak at 215 nm. Peak 2
material indicated an activity of about 7 x 108 units/mg.
C. Amino Acid Sequence Analysis
Amino Acid sequence determination was performed
by automated gas-phase Edman degradation (Hewick, R.M.,
Hunkapillar, M.W., Hood, L.E. and Dryer, W.J. (1981) J.
i ad~~~U,-lC
1 ~4' 299
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Biol. Chem. 256: 7990) employing an Applied Biosystems
microsequenator. A 90 microliter aliduot of the peak 2
HPLC fraction, dissolved in 0.1$ SDS as described above,
was applied to the glass fiber filter cartridge in the
presence of Polybrene. Amino acid sequence information
was obtained up to the 35th residue. The N-terminal
sequence was determined as follows
His - Lys - Asp - Ile Thr - Leu - Gln - Glu
- - -
Ile - Ile - Lys - Thr - Leu - Asn - - Leu - Thr
Ser -
Glu - Gln Lys - Thr Leu - Thr - Glu - Leu
- - - -
- Val - Thr - Asp - Ile - Phe - - Ala
Ala
wherein the blanks indicate the lack of an identifiable
amino acid.
Blanks in the the amino-terminus at positions 3
and 23 were consistent with the presence of cysteine,
which cannot be detected in this system. The blank at
position 28, corresponding to a threonine in the cDNA-
predicted sequence, may have been due either to the
variability of phenylthiohydantoin-threonine detection or
to the presence of O-linked glycosylation or
esterification.
A 100 microliter aliquot of the HPLC fraction
on which the amino-terminal sequence was performed was
evaporated to dryness and redissolved in 70~ formic
acid. A 50-fold molar excess of cyanoqen bromide was
added and the solution was allowed to stand at room
temperature for 2.5 hours. The cleaved protein was
sequenced on the Applied Biosystems gas-phase sequenator,
as described above. Two sequences were identifiable:
( 1 ) Arg - Glu - Lys - Tyr - Ser - Lys
(2) His - Lys - - Asp - Ile - Thr
~ X41 X99
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Sequence (1) is identical to the sequence predicted from
the cDNA to have been released f_ollowinq cyanogen bromide
cleavage of the methionine residue at position 120. The
last two residues of the C-terminus may have been present
but may not have been detectable owing to insufficient
sample. Sequence (2) is identical to the amino-terminal
'sequence obtained for the native IL-4 protein, as
described above. The relative amounts of amino-terminal
and carboxyl-terminal phenylthiohydantoin-amino acids
released suggested equimolar amounts of both sequences in
the sample. This result supports the conclusion that the
protein sample that was sequenced contained predominantly
a single polypeptide chain with amino and carboxyl
termini predicted from the cDNA sequence of human IL-4.
Example XI. Construction and Expression of Human IL-4
Mutein (Ilesz, s~l, IS94(Ala))
The modified pUCl8 plasmid of Example VIII
(containing the I1e52 coding sequence) is digested with
MluI and BclI, and the large fragment is isolated. The
isolated fragment is mixed with the synthetic douhle-
stranded fragment illustrated below in a standard
liqation solution. The resulting plasmid is transf_ected
into E. coli K-12 JM101, or the like, and propagated.
CG CGT TGT CTC GGC GCC ACT
A ACA GAG CCG CGG TGA
GCG CAG TTC CAC CGT CAC AAA GAG CT
CGC ~ GTC AAG GTG GCA GTG TTT GTC GAC TAG
Deletion
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MluI/BclI Replacement rr~ragment
The modified plasmid is isolated and digested
with XbaI and NaeI, and the large fragment is isolated.
The isolated fragment is mixed with the synthetic double-
stranded fragment illustrated below in a standard
ligation solution. The added codon is boxed. The
resulting plasmid is transfected into E. coli K-12 strain
JM101, or the like, and expressed.
CTA GAC CGT AAC CTG TGG GGC
TG GCA TTG GAC ACC CCG
CTG GCC GCC
GAC CGG CGG
XbaI/NaeI Replacement Fragment
After culturing, protein is extracted from the
JM101 cells using standard techniques, and dilutions of
the extracts are tested for biological activity.
Example XII. Induction of DR Antigens on Cells from a
Patient Suffering from Bare Lymphocyte
Syndrome.
Bare lymphocyte syndrome is characterized by
the lack of expression of class I and/or class II HLA
antigens on cell surfaces, and is frequently associated
with severe immunodeficiency, e.g. Touraine, Lancet, pgs.
319-321 (February 7, 1981); Touraine and Bethel, Human
Immunology, Vol. 2, pgs. 147-153 (1981); and Sullivan et
al., J. Clin. Invest., Vol. 76, pgs. 75-79 (1985). It
was discovered that human IL-4 was capable of inducing
the expression of the class II DR antigen on the surfaces
1 341 299
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of cells derived from a patient suffering from bare
lymphocyte syndrome.
Peripheral blood lymphocytes (PBLs) were
obtained from a patient suffering from non-expression of
HLA class II antigens. B cells were purified from the
PBLs essentially as described above, and a B cell line
(designated UD31) was established by transformation with
Epstein-Barr virus (EBV). The EBV-transformed cells were
cultured for 48 hours in Yssel's defined medium
(described above) with 2$ fetal calf serum and a 5$ (v/v)
concentration of supernatant from pcD-125-transfected COS
7 cells. The cells were harvested, fixed, stained with
fluorescently-labeled anti-DR monoclonal antibody (e. q.
Becton Dickinson, L243), and analyzed by flow
cytometry. Figure 9 illustrates histograms of cell
fre4uency versus fluorescence intensity for a control
population of the EBV-transformed cells harvested prior
to IL-4 treatment (Curve A), and for the population of_
EBV-transformed cells after IL-4 treatment (Curve B).
Example XIII. MCGF Activity of Cl.Ly1+2-/9 (C1.1)
Supernatants.
Purified IL-3 and supernatant from COS 7 cells
transfected with an IL-3 cDNA clone (recombinant IL-3;
IL-3R) stimulated proliferation of the mast cell line
MC/9 to the same extent (Fig. 14A). However, this level
was a third to a half of that induced by C1.1 T cell
supernatant. The presence of ConA in the T cell super-
natant (2 ug/ml) does not contribute significantly to its
MCGC activity. One explanation for the effect noted
above is that IL-3 does not account for all of the MCGF
activity produced by C1.1 cells. Alternatively, the mast
cell line used for assays could (theorei;ically) be
contaminated with a second factor-dependent population of
cells. This latter possibility is excluded, however,
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because re-cloning the MC/9 cell line yielded subclones,
each of which responds to IL-3 and the r_ell supernatant
in the same way as the original MC/9 clone.
Since C1.1 supernatant also contains lympho-
kines other than IL-3, these were tested for their
ability to promote the growth of MC/9 cells. However,
MC/9 cells were unresponsive'to various concentrations of
recombinant IL-2, IFN and G~1-CSF, as well as to
supernatant containing IL-1 (P388D 1 ce:Lls) (Fig. 14A).
Moreover, supplementation of cultures containing varying
concentrations of recombinant IL-3 with IL-2, GM-CSF,
IFN-y, or IL-1 did not stimulate proliferation of MC/9
cells above the level obtained with IL-3 alone. This
indicates that the increased MCGF activity of the T cell
supernatant is not due to its content of these other
known factors.
Comparative Studies with Other Factor-Dependent Cell
Lines.
Two other mast cell lines, DX-2 and MM3, also
gave higher proliferative responses with C1.1 supernatant
than with IL-3 (representative data for DX-2 shown in
Figure 14B). These cells also chid not respond to IL-2,
IFN-y, GM-CSF or supernatant of P388D-1 cells. Thus, the
enhanced proliferative response of MC/9 cells to the T-
cell supernatant may be typical of mast cells in
general. In contrast, granulocytic-type cells (NFS-60)
were stimulated almost equally by IL-3 and C1.1 super-
natant (Fig. 14C). Although NFS-60 cells were
unresponsive to IL-2, IFN-Y and P388D-1 supernatant,
there was a small but reproducible stimulation by GM-
CSF. The fact that IL-3 and the T cell supernatant
induce comparable levels of proliferation suggests that
the NFS-60 cell line may be relatively insensitive to
additional factors present in the T cell supernatant.
Thus, NFS-60 cells were used to assay IL-3 during
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attempts to separate IL-3 from other MCGF activities by
protein fractionation.
The TCGF activity of Cl.l supernatant was
assessed using the IL-2 dependent T cell line, HT2.
Saturating concentrations of C1.1 supernatant invariably
stimulated 3H-thymidine incorporation wE~ll below the
maximum level achieved with recombinant IL-2 (Fig.
14D). Similar results were obtained with two other cell
lines. The supernatant does not contain an inhibitory
substance since its addition to HT2 cultures containing
IL-2 showed no reduction in 3H-thymidine incorporation
(Fig. 14D). These results suggest C1.1 supernatant
contains a TCGF activity distinct from IL-2. Biochemical
evidence in support of this conclusion is presented
below.
Preliminary Chromatographic Fractionation of. C1.1
Supernatant.
C1.1 supernatant was fractionated by various
chromatographic methods with the immediate goal of
separating IL-3 from other MCGF activities. Prolifera-
tion of NFS-60 cells was used to track IL-3 specifically
against the background of total MCGF activity revealed by
MC/9 proliferation. TCGF activity was assessed by HT2
cell proliferation.
When C1.1 supernatant was fractionated by
cation exchange chromatography at neutral pH,
approximately 98$ of the loaded proteinr 97~ of the IL-3
units (assessed by NFS-60 proliferation) and 1~ of the
TCGF units appeared in the flow-through (Fig. 15).
Elution of bound protein with a gradient of sodium
chloride released TCGF activity at 0.19M NaCl (fraction
60). A small but reproducible peak of TCGF activity also
appeared at 1M NaCl. No further significant TCGF
activity could be eluted at higher NaCl concentrations
(up to 3M). A small amount of IL-3 also eluted in
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approximately the same region as the TCGF peak
(detectable NFS-60 response). Only fractions
corresponding to peak TCGF activity (fractions 59-61) and
the flow-through (fractions 1-20) stimulated high levels
of MC/9 proliferation, comparable to the unfractionated
supernatant (arrows, Fig. 15). Since titration curves
suggested fractions 59-61 contained more of the high
level MCGF activity than did the flow-through, attempts
were made to deplete further the IL-3 activity co-eluting
with the TCGF peak~and again assess MC/9 proliferation
levels. Fractions 59-61 were therefore re-
chromatographed twice more under identical conditions.
After the third column pass, more than 95$ of
the TCGF activity consistently eluted at 0.19 M NaCl.
There was no measurable IL-3 (NFS-60) response) in the
flow-through or in any of the column fractions. However,
still co-eluting with the TCGF peak was an MCGF activity
that now stimulated a plateau level of MC/9 proliferation
that was significantly lower than that obtained with C1.1
supernatant or IL-3 (see below). Since these fractions
were devoid of measurable IL-3 activity (absence of NFS-
60 response), apparently the new MCGF by itself was
incapable of stimulating MC/9 cells to the same degree as
the crude T cell supernatant.
To further attempt separation of MCGF from
TCGF, the 3X fractionated material (fractions 59-61) was
diluted lOX with 0.1$ TFA in H20 to pH 2 and loaded
directly onto a C8 reverse phase column. Fiq. 16A shows
the elution profile of bound protein released with a
gradient of acetonitrile, 0.1~ in TFA. No MCGF or TCGF
activity appeared in the flow-through: both activities
co-eluted at 37$ acetonitrile (fractions 26-29).
However, the saturating MCGF response was now only about
one half that produced by IL-3 alone (Fig. 17A). ~When
all fractions were re-assayed in the presence of
saturating levels of IL-3, only fractions 26-29 showed an
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-lol-
MC/9 proliferation response in excess of IL-3 alone
(arrow, Fig. 16A). In fact, the magnitude of the
response was similar to that of the unfractionated
supernatant itself (Fig. 17A). Further, proliferation
levels equivalent to those of unfractionated supernatant
were also observed with fractions 26-29 were assayed in
the presence of IL-3 containing flow-through or IL-3
purified form WEHI 3. Thus, the combination.of IL-3 and
the unique MCGF/TCGF stimulates levels of MC/9
proliferation characteristic of the original T cell
supernatant.
Altough reverse phase chromatagraphy fails to
resolve the MCGF f rom the TCGF, the TCGF was shown to be
distinct from IL-2 by two criteria. First, when
recombinant IL-2 in COS-7 cell supernatants (IL-2R) and
supernatant from an IL-2 producing (ConA stimulated)
murine T cell line (GF15-1) are both chromatographed on
the same column under identical conditions, the TCGF
activity elutes at 45~ acetonitrile in a sharp peak that
does not overlap with the 37$ acetonitri.le position
r
marking the C1.1 TCGF (Fig. 16B). Second, the saturating
response of HT2 cells to IL-2R is very different from
that which characterizes the Cl.l supernatant (Fig. 16B,
insert). Further, no obvious synergy was detected
between the partially purified TCGF and IL-2 (Fig. 17B).
The chromatofocussing pattern of C1.1 super-
natant is shown in Fiq. 18. The IL-3 activity (NFS-60
response) displayed a very heterogeneous profile,
including activity with pI values higher than 7.1, but
the TCGF activity appeared more homogeneous, with the
major TCGF species (fraction 12) showing an approximate
pI of 6.2. This coincides with the maximum MCGF activity
(MC/9 response) after addition of saturating levels of
IL-3.
13412gg
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SDS PAGE of Cl.l Supernatant and of Partially Purified
Factor.
Figure 19 compares the electrophoretic profiles
of unfractionated C1.1 supernatant (Fiq» 19A) with that
of the ca n on exchanged material, fractions 59-61 (Fig.
19B). Protein was eluted directly from gel slices and
activities were determined by proliferation. Both the
cation exchange fraction and Cl.l supernatant show TCGF
peaks: at 20 Kd and 15 Kd under non-reducing conditions,
and at 21 Kd and 16-Kd under reducing conditions. Since
the cation exchanged material was depleted of IL-3, only
low levels of MC/9 proliferation were induced and this
MCGF activity was still coincident with TCGF peaks.
Addition of saturating amounts of IL-3R to all fractions
raised the MC/9 proliferation response to C1.1
supernatant levels only in the peak MCGF/TCGF fractions
(arrows, Fig. 19B). IL-3 activity of the supernatant was
greatest at the 24 Kd position, but also appears in broad
peaks about the 20 Kd region. Significantly, MC/9
proliferation levels well above that of IL-3 occur at the
20 Kd position of electrophoresed supernatant (arrow,
Fig. 19A). A silver-stained SDS gel of the most highly
purified MCGF/TCGF material (fraction 2~3 of the reverse
phase column) also showed prominent protein bands at 20
Kd and 15 Kd .
Microgram quantities of the 20 kd form of the
factor have been purified to homogeneity by successive
fractionation of ConA-supernatants from activated C1.1 T-
cells by (1) cation exchange chromatography, (2) Heparin
Sepharose chromatography, and (3) c4 reverse phase
chromatography as described previously. The final
material shows only a single band at 20 kd by silver-
stained SDS=PAGE, which corresponds to a single UV
absorbing peak an the c4 elution profile. Biological
characterization of the purified factor confirmed its
ability to stimulate low levels of T-cell and mast cell
1341299
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proliferation and to enhance IL-3-dependent mast cell
proliferation.
As shown in Table V, the factor possesses
several B-cell stimulatory activities ascribed to BSF1
(Roehm, N.W. et al., J. Exp. Med. 169: 679-694 (1984);
Howard, M. et al., Proc. Natl. Acad. Sci. U.S.A. 78: 5788
(1981); Roehm, N.W. et al., Proc. Natl. Acad. Sci. U.S.A.
81:.6149-6153 (1984). First, it induces increased
expression of Ia on resting B-cells as compared to the
control polulation (Table V). Second, the factor, in
conjunction with anti-Ig antibodies, induces significant
[3H]-thymidine incorporation, indicating that it
functions as an accessory factor in the stimulation of B-
cell proliferation.
Table V
B-cell Stimulation Assays
[3]-thymidine incorporation I-A
Source of anti-Ig treated B cells* induction**
Unfractionated
Cl.Ly1+2-/9 33,788 cpm f 5,454 162.3
Supernatant
Purified factor 44,125 cpm t 2,775 157.1
Medium
(negative control) 4,128 cpm t 734 35.5
* results reported as average cpm t SEM of triplicate cultures
supplemented with saturatir~ levels of factor.
** results reported as average relative mean fluorescence of
triplicate cultures supplemented with saturating levels of
factor.
These polypeptides can exhibit several
biological activities, differing in both the apparent
function mediated and the type of cell affected. As
described, these activities include synergy in the
induction of proliferation of B-cells and mast cell
1 341 299
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lines, early activation of resting B-cells, stimulation
of the proliferation of_ T-cells, and selective
enhancement of IgGl and IgE production of mitogen-
activated B-cells. Purification of the polypeptides to
homogeneity has clarified the socpe of these activities.
From the foregoing, it will be appreciated that
the purified proteins of the present invention provide
those skilled in the art with novel therapeutic
capabilities. The ability to produce signi.f_icant
Quantities of these factors will permit improved in vitro
culture of selected mammalian cell lines, as well as an
overall improved understanding of the mammalian immune
response.
cDNA clones pcD-2A-E3, pcD-46 (pcD-2F1-13),
pcD-125, and yeast vector pMF-alpha8 have been deposited
with the American Type Culture Collectio n, Rockville, MD,
USA (ATCC), under accession numbers 53330, 53337, 67029,
and 40140, respectively. These. deposits have been made
under the Budapest Treaty (1977) on the International
Recognition of the Deposit of Micro-organisms for the
purposes of Patent Procedure.
