U.S. patent number 5,317,087 [Application Number 07/630,331] was granted by the patent office on 1994-05-31 for purification of the il-2 receptor.
This patent grant is currently assigned to Immunex Corporation. Invention is credited to Douglas P. Cerretti, Cosman, Steven K. Dower, Alf D. Larsen, Carl J. March, David L. Urdal.
United States Patent |
5,317,087 |
Cerretti , et al. |
May 31, 1994 |
Purification of the IL-2 receptor
Abstract
Interleukin-2 receptor derived from normal and malignant cells
has been purified by use of various techniques including affinity
chromatography in conjunction with a monoclonal antibody directed
to the receptor. The purification process also includes reversed
phased high performance liquid chromatography. By these techniques,
interleukin-2 receptor has been purified to homogeneity. The high
purification of the interleukin-2 receptor has made possible the
sequencing of the amino acid residues at the N-terminal of this
protein molecule. Double-stranded cDNA is prepared from
polyadenylated RNA extracted from cell lines or other sources known
to produce IL-2 receptor. The cDNA is inserted within a plasmid
vector and then the recombinant plasmid employed to transform an
appropriate host. Transformed hosts are identified and grouped into
pools. Plasmid DNA prepared from these pools is hybridized with a
labeled synthetic oligonucleotide probe corresponding to a portion
of the amino acid sequence of the purified IL-2 receptor. The cDNA
clone isolated with the probe is characterized by restriction
enzyme mapping and sequenced by chain-termination method. The
particular DNA clone that actually contains the gene coding for the
functional IL-2 receptor is identified by expressing the clones in
COS-7 monkey kidney cells and assaying for the expressed IL-2
receptor by its ability to bind IL-2 or a monoclonal antibody
directed against the IL-2 receptor.
Inventors: |
Cerretti; Douglas P. (Seattle,
WA), Cosman;; David J. (Seattle, WA), Dower; Steven
K. (Redmond, WA), March; Carl J. (Seattle, WA),
Urdal; David L. (Seattle, WA), Larsen; Alf D. (Seattle,
WA) |
Assignee: |
Immunex Corporation (Seattle,
WA)
|
Family
ID: |
26840873 |
Appl.
No.: |
07/630,331 |
Filed: |
October 22, 1990 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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143277 |
Jan 5, 1988 |
|
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670883 |
Nov 13, 1984 |
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Current U.S.
Class: |
530/350;
435/69.1; 435/69.5; 435/69.52; 530/351; 530/395 |
Current CPC
Class: |
C07K
16/2866 (20130101); C07K 14/7155 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 16/18 (20060101); C07K
16/28 (20060101); C07K 14/715 (20060101); C07K
013/00 (); C07K 015/00 () |
Field of
Search: |
;530/350,351,395,387
;435/69.5,69.52,69.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Matson et al. LC-GC, vol. 4(7) 1986, pp. 624-633. .
Deutscher, Method Enzymology 182, 1990, pp. 779-780. .
A Guide to the Properties and Uses of Detergents in Biology and
Biochemistry, 1987, ed Neugebauer, pp. 15-17. .
Biochemica Information, 1st ed. ed. Keesey, 1987 pp. 181-198. .
Sabe et al, Mol Biol Med 1984 (2), pp. 379-396. .
Nikaido et al. Nature 311, 1984, pp. 631-635. .
High Performance Liquid Chromatography of Proteins and Peptides,
ed. Hearn et al. 1983 (index only). .
Malek et al., PNAS 80, 1983, pp. 5694-5698. .
Rafh et al., I. Exp Med 1981 vol. 154 pp. 1455-1474. .
Leonard et al. Nature vol. 300 1982 p. 267. .
Aviv and Leder, "Purification of Biologically Active Globin
Messenger RNA by Chromatography on Oligothymidylic Acid-Cellulose,"
69 Proc. Nat. Acad. Sci. USA 1408 (1972). .
Smith and Birnstiel, "A Simple Method for DNA Restriction Site
Mapping," 3 Nucl. Acids Res. 2387 (1976). .
Chirgwin et al., "Isolation of Biologically Active Ribonucleic Acid
from Sources Enriched in Ribonuclease," 18 Biochemistry 5294
(1979). .
Nowinski et al., "The Isolation of Hybrid Cell Lines Producing
Monoclonal Antibodies against the p15(E) Protein of Ecotropic
Murine Leukemia Viruses," 97 Virology III (1979). .
Land et al., "5'-Terminal Sequences of Eucaryotic mRNA can be
Cloned with High Efficiency," 9 Nucl. Acids Res. 2551 (1981). .
Robb et al., "T-Cell Growth Factor Receptors. Quantitation,
Specificity and Biological Relevance," 154 J. Exp. Med. 1455
(1981). .
Uchiyama et al., "A Monoclonal Antibody (Anti-Tac) Reactive With
Activated and Functionally Mature Human T-Cells-I. Production of
Anti-Tac Monoclonal Antibody and Distribution of Tac (+) Cells,"
126 J. Immunol. 1393 (1981). .
Uchiyama et al., "A Monoclonal Antibody (anti-Tac) Reactive with
Activated and Functionally Mature Human T Cells-II. Expression of
Tac Antigen on Activated Cytotoxic Killer T Cells, Suppressor
Cells, and on One of Two Types of Helper T Cells," 126 J. Immunol.
1398 (1981). .
Leonard et al., "A Monoclonal Antibody that Appears to Recognize
the Receptor of Human T-Cell Growth Factor; Partial
Characterization of the Receptor," 300 Nature (London) 267 (1982).
.
Third International Lymphokine Workshop: Interleukins, Lymphokines,
and Cyctokines, 70 Cell Immunol. 380-407 (1982). .
Summary of the Third International Lymphokine Workshop, I
Lymphokine Research, (1982). .
"Studies on Transformation of Escherichia coli with Plasmids," 166
J. Mol. Biol. 557 (1983). .
Robb and Greene, "Direct Demonstration of the Identity of the T
Cell Growth Factor Binding Protein and the Tac Antigen," 158 J.
Exp. Med. 1332 (1983). .
Depper et al., "Blockage of the Interleukin-2 Receptor by Anti-Tac
Antibody: Inhibition of Human Lymphocyte Activation," 131 J.
Immunol. 690 (1983). .
Leonard et al., "Characterization of the Human Receptor for T-Cell
Growth Factor," 80 Proc. Natl. Acad. Sci. USA 6957 (1983). .
Smith et al., "Production and Characterization of Monoclonal
Antibodies to Human Interleukin 2: Strategy and Tactics," 131 J.
Immunol. 1808 (1983)..
|
Primary Examiner: Draper; Garnette D.
Attorney, Agent or Firm: Foley & Lardner
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
07/143,277, filed Jan. 5, 1988, which, in turn, is a continuation
of U.S. application Ser. No. 670,883, filed Nov. 13, 1984, both of
which are abandoned.
Claims
The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. A homogeneous interleukin 2 receptor protein, precipitatable by
monoclonal antibody 2A3-A1H, that has a molecular weight by SDS
polyacrylamide gel electrophoresis of about 55,000 to 60,000
daltons and that has a specific activity from approximately 5,000
to approximately 21,000 femtomoles of interleukin 2 receptor per
microgram of protein, wherein said protein comprises an N-terminal
sequence Glu-Leu-Cys-Asp-Asp-Asp-Pro-Pro-Glu-Ile.
2. A homogeneous interleukin 2 receptor protein according to claim
1, obtained by cloning and expression of a DNA sequence that
hybriding under stringent conditions to a synthetic oligonucleotide
probe corresponding to a portion of the amino acid sequence of an
interleukin 2 receptor.
3. A homogeneous interleukin 2 receptor protein according to claim
2, wherein said DNA sequence is expressed in a mammalian cell.
Description
TECHNICAL FIELD
The present invention relates to interleukin-2 receptor
(hereinafter "IL-2 receptor"), and more particularly to: purified
interleukin-2 receptor derived from normal and malignant cells; a
process for producing same; the cloning of IL-2 receptor gene by
use of a synthetic oligonucleotide probe derived from the amino
acid sequence of the purified IL-2 receptor to screen a
complementary deoxyribonucleic acid ("cDNA") library synthesized
from IL-2 receptor messenger ribonucleic acid ("mRNA"); and, the
characterization of the screened IL-2 receptor gene.
BACKGROUND OF THE INVENTION
A large number of normal immune responses require the participation
of T-cells. The proliferation of T-cells to sufficient numbers to
assume an effective role in immune responses is controlled by the
presence of interleukin-2 (hereinafter "IL-2"), Gillis and Smith,
28 Nature 154 (1977). Although the mechanism by which IL-2 controls
the growth of T-cells is not fully understood, it is known that
IL-2 acts on T-cells via a specific, high-affinity, plasma membrane
receptor, i.e., IL-2 receptor. Also, in order to continue to
divide, IL-2 dependent T-cells must express the IL-2 receptor and
the IL-2 must bind to a portion of the IL-2 receptor, Robb et al.,
154 J. Exp. Med. 1455 (1981). A more complete knowledge of the
biochemistry of the IL-2 receptor would foster a better
understanding of the interaction between IL-2 and T-cells. To date,
this has been hampered, at least in part, by the unavailability of
sufficient amounts of IL-2 receptor in purified form.
Leonard et al., 300 Nature (London) 267 (November 1982), reported
employing a murine monoclonal antibody, designated as anti-Tac, to
significantly block the binding of radiolabelled IL-2 to the human
lymphoma T-cell line, HUT-102. This antibody resulted from the
immunization of mice with long term cultures of human T-cells. The
anti-Tac antibody was reported as binding both to a glyco-protein
having a molecular weight of about 47,000-53,000 daltons and also
to proteins having molecular weights of about 113,000 and 180,000
daltons. Leonard et al. hypothesized, but did not establish, that
the cell surface determinant (i.e., the 47,000-53,000 molecular
weight protein) to which the anti-Tac antibody bounded to was the
IL-2 receptor.
Robb and Green, 158 J. Exp. Med. 1332 (1983), reported employing
the anti-Tac antibody in conjunction with mitogen-activated normal
lymphocytes to immunoprecipitate a protein having a molecular
weight of about 52,000-57,000 daltons. They found that this same
protein also bound to IL-2. These researchers opined that this
reactive molecule contained the binding site for IL-2 for normal
lymphocytes.
Leonard et al., 80 Proc. Natl. Acad. Sci. (USA) 6957 (1983)
observed that receptors recognized by anti-Tac antibody on HUT-102
cells and on phytohemaggelutinin-activated normal T-cells appear to
be larger on reducing gels than on nonreducing gels, thus
suggesting the presence of intrachain disulfide bonds. Also, the
HUT-102 cell receptor was reported to exhibit an isoelectric point
of from 5.5 to 6.0. From post-translational studies, Leonard et al.
suggested that the HUT-102 receptor is composed of a peptide
backbone of 33,000 daltons that is initially glycosylated by an
N-linked mechanism to achieve a 35,000-37,000 daltons doublet and
then glycosylated by an O-linked mechanism to increase the weight
of the molecule by about 13,000-15,000 daltons. Although the
researchers stated that their studies "suggested" that the HUT-102
cell receptor recognized by the anti-Tac antibody is the human
receptor for IL-2, they admitted that actual proof would require
purifying the receptor, which prior to the making of the present
invention had not been accomplished.
Recombinant DNA techniques have been developed for economically
producing a desired protein once the gene coding for protein has
been isolated and identified. A discussion of such recombinant DNA
techniques for protein production is set forth in the editorial and
supporting papers in Vol. 196 of Science (April, 1977). However, to
take advantage of the recombinant DNA techniques discussed in these
references, the gene coding for the IL-2 receptor must first be
isolated.
SUMMARY OF THE INVENTION
The present invention relates to the production of IL-2 receptor
derived from malignant and normal T-cells, to the purification of
the IL-2 receptor to homogeneity and to the determination of the
amino acid sequence of the amino terminal portion of the IL-2
receptor molecule. The IL-2 receptor of the present invention is
purified by a combination of affinity chromatography and reversed
phased high performance liquid chromatography. The affinity
chromatography procedure employs a highly specific monoclonal
antibody that recognizes an epitope on the receptor molecule.
Once purified to homogeneity, the amino acid sequence of the amino
terminal portion of the receptor molecule can be ascertained by use
of a protein sequencer. This information is used to construct a
hybridization probe to isolate the IL-2 receptor from a cDNA
library constructed from mRNA receptors isolated from cells known
to express IL-2. To this end, total RNA is extracted from cell
lines or other sources known to produce relatively high levels of
IL-2 receptor molecules. Polyadenylated mRNA is isolated from the
total RNA extract. A cDNA library is constructed by reverse
transcription of the polyadenylated mRNA with reverse
transcriptase. The DNA is rendered double-stranded with DNA
polymerase I and inserted into a cloning vector, and the
recombinant vector is used to transform a host.
Transformed hosts are identified and grouped into pools. Plasmid
DNA prepared from these pools is hybridized with a labeled
synthetic oligonucleotide probe corresponding to a portion of the
amino acid sequence of the IL-2 receptor. The pool(s) of clones
that give a positive signal to the probe are identified, replated
as single colonies, and hybridized with the synthetic
oligonucleotide probe to identify the particular host colony
containing the IL-2 receptor gene. Plasmid DNA is prepared from
this host colony and characterized by restriction enzyme mapping.
The IL-2 receptor gene is sequenced to establish its entire
nucleotide and amino acid composition. In addition, the IL-2
receptor gene is cloned in a mammalian cell system to express
mature IL-2 receptor and then a binding assay is conducted to
confirm that the expressed protein product is the IL-2
receptor.
BRIEF DESCRIPTION OF THE DRAWINGS
The details of typical embodiments of the present invention will be
described in connection with the accompanying drawings, in
which:
FIG. 1 illustrates partial restriction maps of the IL-2 Rec N4
("N4") and IL-2 Rec N1 ("N1") clones in side-by-side comparative
relationship to each other;
FIG. 2 illustrates the nucleotide sequence and the corresponding
amino acid sequence of the IL-2 receptor gene as contained in the
N4 nucleotide fragment, with the nucleotides being numbered from
the position of the initiator methionine codon and the amino acids
being numbered from the mature NH.sub.2 -terminus of the protein,
i.e., the Glu residue, as marked with a star;
FIG. 3A illustrates the strategy employed to clone the coding
regions of the N4 and N1 fragments in plasmid vectors used to
transfect mammalian cells to determine whether one or both cDNA
clones would encode a functional IL-2 receptor; and
FIGS. 3B and C illustrate the ability of the transfected mammalian
cells to bind to IL-2 and to a monoclonal anti-IL-2 receptor
antibody.
DESCRIPTION OF THE INVENTION
Preparation of IL-2 Receptor Extracts From Malignant and Normal
Cells
Malignant cells are cultured in vitro in a suitable culture medium
supplemented with serum and various additives. After an optimum
culture period, the cells are harvested and IL-2 receptor
containing extracts formed from the cells. The malignant cell lines
which may be employed as a source of IL-2 receptors include
T-lymphoma or T-leukemia cell lines. These cell lines are produced
by either a spontaneous occurrence, via viral transformation or via
transformation by chemical carcinogen or irradiation. The present
invention has been carried out in conjunction with a naturally
occurring lymphoma cell line, designated as HUT-102. The cell line
is available from a wide variety of sources and has been used
extensively by researchers. See, for instance, Leonard et al., 80
Proc. Natl. Acad. Sci (USA), 6957 (1983) and Leonard et al., 300
Nature (London), 267 (November 1982).
The present invention also includes producing IL-2 receptor
molecules from normal cells. For instance, human peripheral blood
mononuclear cells are separated from human blood by Ficoll-Hypaque
centrifugation, such as described by Boyum, 18 Scand. J. Clin. Lab.
Invest. Suppl. 77 (1966). Adherent cells are removed by plastic
adherence and then nonadherent cells are cultured in vitro in serum
containing medium in the presence of an activating agent, such as a
T-cell mitogen. After a suitable period of time, the cells are
harvested by centrifugation. Examples of T-cell mitogens that may
be used as activating agents, include phytohemagglutinin ("PHA"),
concanavalin A ("Con A") or pokeweed mitogen ("PKM").
The numbers of IL-2 receptors expressed by stimulation of the
peripheral blood leukocytes with a plant mitogen varies with time.
Optimum levels of IL-2 receptor expression are reached at
approximately 72 hours after mitogen stimulation.
The culture medium used to expand the IL-2 receptor bearing
malignant and normal cells may consist of commercially available
medium, such as Roswell Park Memorial Institute ("RPMI") medium.
Dulbecco's Modified Eagle Medium ("DMEM") and Click's Medium.
Additives, which may be individually or in combination added to the
culture medium, include serum, such as fetal calf serum ("FCS") or
normal human serum. Additional additives include glutamine and
various antibiotics, such as penicillin and streptomycin.
The process of culturing the malignant and normal cells to induce
receptor formation may be carried out in various environmental
conditions. Preferably, however, the cultures are maintained in the
temperature range of approximately 35.degree.-38.degree. C. in a
humidified atmosphere of approximately 5-10% CO.sub.2 in air. Also,
the pH of the culture should be kept in slightly alkaline
condition, in the range of approximately pH 7.0-7.4.
IL-2 receptor containing extracts are prepared from the cultivated
normal and malignant cells by harvesting the cells by
centrifugation. The cells are then washed with a buffered saline
solution and resuspended in the buffered saline solution together
with a detergent and phenylmethylsulfonylfluoride ("PMSF"). After a
period of time the detergent extract is centrifuged to remove
nuclei and insoluble debris and then is stored frozen until
used.
Preparation of Monoclonal Antibody Against IL-2 Receptor
The present invention also concerns the production of a monoclonal
antibody having a high affinity to an epitope on the IL-2 receptor
molecule. The antibody is used as a bound ligand in the affinity
chromatography procedures during purification of the IL-2 receptor.
The antibody is also employed in a radioimmune precipitation assay
and in soluble receptor assays to monitor the IL-2 receptor protein
during purification steps, as more fully discussed below.
A preferred procedure for generating the monoclonal antibody
against the IL-2 receptor is generally disclosed in U.S. Pat. No.
4,411,993, incorporated herein by reference. In the procedure,
BALB/c mice are injected with PHA activated human peripheral blood
leukocytes ("PHA/PBL") several times at weekly intervals. Prior to
the first injection, the PHA/PBL is emulsified in complete Freund's
adjuvant and prior to the remainder of the injections the PHA/PBL
is emulsified in incomplete Freund's adjuvant.
During the course of immunization, serum samples from the mice are
tested by an enzyme linked immunoabsorbant assay ("ELISA"), as is
well known in the art, for the presence of antibody reaction with
the immunization cells. Once an antibody titer is detected, the
animals are given an intravenous injection of PHA/PBL suspended in
saline. Several days later the animals are sacrificed and their
spleens harvested. Single cell suspensions from the splenocytes are
cultured in tissue culture medium supplemented with various
additives to expand the number of antibody producing cells. The
antibody producing cells are isolated from the culture and purified
by standard techniques for subsequent fusion with myeloma cells to
produce hybrid cells that express anti-IL-2 receptor antibody. The
fusion process is detailed in U.S. Pat. No. 4,411,933 and in
Nowinski et al., 93 Virology 111 (1979), incorporated herein by
reference.
After fusion, the hybrid cells are resuspended in a tissue culture
medium supplemented with various additives and selected suppressing
agents to preclude the growth of unfused myeloma cells, double
myeloma cells, unfused spleen cells and double spleen cell hybrids,
thereby allowing the anti-IL-2 receptor antibody producing cells to
grow. Such growth inhibitors or suppressants may include
hypoxanthine, aminopterin and thymidine (hereinafter collectively
referred to as "HAT").
After several days of culture, the hybridoma cells, which are
generated, are screened by ELISA assay for anti-IL-2 receptor
antibody responses. These hybrid cells are tested for production of
antibody capable of inhibiting both mitogen and antigen induced
proliferation of human peripheral blood leukocytes. The hybrid
cells which give positive ELISA results are gradually weaned to
HAT-free medium and then cultured in vitro in large volumes for
bulk production of antibody. Alternatively, the cells may be
expanded in vivo by injecting the hybridoma cells in the peritoneal
cavities of mice and thereafter collecting the intraperitoneal
ascites which contain high concentrations of the antibody. The
antibodies contained in the ascites fluid can be isolated and
concentrated by established techniques, such as by ammonium sulfate
precipitation followed by gel column chromatography. If required,
the antibody can be further purified by ion exchange chromatography
and/or affinity chromatography. By the above process, a particular
hybridoma, designated as 2A3, was found to produce antibody that
significantly inhibited both mitogen and antigen induced
proliferation of human peripheral blood leukocytes.
The present invention also includes identifying potent cell line
sources of anti-IL-2 receptor antibody by cloning cell lines known
to produce this antibody, for instance, the 2A3 cell line. The
cloning is accomplished by the limiting dilution procedure, as is
well known in the art and as is detailed in U.S. Pat. No.
4,411,993. By this procedure, one particular subclone, designated
as 2A3-A1H was found to produce antibody that substantially
entirely inhibited both mitogen and antigen induced proliferation
of human peripheral blood leukocytes. The 2A3-A1H antibody has been
characterized as of the .gamma..sub.1 K isotype with an unusually
high affinity to the human IL-2 receptor.
A control antibody preferably is employed to confirm the processes
of the present invention utilizing anti-IL-2 receptor antibody and
as a reagent in the purification of the receptor. The control
antibody should be of the same isotype as the anti-IL-2 receptor
antibody. Applicants have identified the antibody secreted by the
mouse myeloma cell line MOPC-21 as a suitable control antibody for
the 2A3-A1H antibody. The MOPC-21 cell line is widely available
from numerous private and commercial sources.
Soluble IL-2 Receptor Assays
Assays employing the 2A3-A1H monoclonal antibody are used in
conjunction with the present invention to monitor the quantitative
amount of IL-2 receptor present in the initial cell lysates and
during purification procedures. These assays hinge on the discovery
by applicants that the 2A3-A1H antibody has an extremely high
affinity for the IL-2 receptor, the affinity constant being in
excess of 5.times.10.sup.9 M.sup.-1 and that the 2A3-A1H antibody
can be radioiodinated to high specific activity and still retain
its capacity to bind to the IL-2 receptor.
One such preferred assay involves ascertaining the extent to which
samples of cell lysate or column chromatography fractions
containing IL-2 receptors are capable of inhibiting the binding of
radiolabelled IL-2 antibody to glutaraldehyde fixed, intact
receptor bearing cells. This assay relies on the observation by
applicants that IL-2 receptor is stable to glutaraldehyde fixation,
i.e., the receptor cannot be extracted from such cells with
nonionic detergents, such as Triton X-100, and the presence of
detergent does not affect the binding of radiolabelled 2A3-A1H
antibody to the fixed cells. Preincubation of a subsaturating dose
of iodinated 2A3-A1H antibody with detergent solutions containing
the IL-2 receptor inhibits the subsequent binding of the 2A3-A1H
antibody to the glutaraldehyde fixed cells. This assay will
hereinafter be referred to as the "soluble inhibition assay."
For use in the soluble inhibition assay, the 2A3-A1H antibody is
radiolabeled with iodine 125 (".sup.125 I") by a chloramine-T
method, as is well known in the art and as described by Segal and
Hurwitz, 118 J. Immunol. 1338 (1977). The standard labeling
conditions employed are: 50 micrograms ("ug") 2A3-A1H IgG; 4
nanomoles ("nM") of chloramine-T (Sigma Chemical Company, St.
Louis, Mo.); and, 2.5 microcurins ("mCi") of .sup.125 I sodium
iodide (New England Nuclear, Boston, Mass.), in a final volume of
60 microliters ("ul"). This protocol has resulted in preparations
of .sup.125 I-2A3-A1H, which routinely have specific activities in
the range of 2 to 5.times.10.sup.15 counts per minute/millimole
("cpm/mMol") (1.3-3.3.times.10.sup.7 cpm/ug). Also, 2A3-A1H
antibodies labeled in this way were found to be more than 95
percent bindable to IL-2 receptor bearing cells and had apparent
affinity constants in excess of 5.times.10.sup.9 M.sup.-1.
In the soluble inhibition assay, 50 ul of .sup.125 I-2A3-A1H
[(2.times.10.sup.-10 M in RPMI-1640 medium containing 2% bovine
serum albumin ("BSA"), 20 mM HEPES buffer (pH 2.7) and 0.2% sodium
azide ("NaN.sub.3 ") (collectively "binding medium")] is mixed with
50 ul of cell lysate or column fraction diluted in phosphate
buffered saline ("PBS") containing 1% (w/v) Triton X-100 detergent
(Sigma Chemical Company, St. Louis, Mo.). This mixture is incubated
for one hour at room temperature in round bottom 96 well plates
(Linbro, Hamden, Conn.). At the end of the incubation period,
10.sup.7 glutaraldehyde fixed, PHA activated human T-cells in 50 ul
of binding medium are added to detect uncomplexed .sup.125
I-2A3-A1H. Incubation is continued for one hour at room
temperature. Duplicate 60 ul aliquots of the mixture are then
transferred to precooled 400 ul polyethylene centrifuge tubes
containing 200 ul of a phthalate oil mixture and the cell bound
antibody is separated from unbound antibody by centrifugation. The
details of the well-known phthalate oil separation method are set
forth in Segal and Hurwitz, supra. The percent of specific
inhibition caused by the lysate or column fraction is calculated by
using 50 ul of PBS-2% Triton X-100 instead of a test sample for the
positive control. Also, 15 ul of PBS-2% Triton X-100 containing
10.sup.-8 M unlabeled 2A3-A1H is used as a negative control.
The nitrocellulose dot assay ("dot assay") is used as a second
soluble IL-2 receptor assay to quantify the amount of IL-2 receptor
molecules present in a sample of cell lysate or column fraction.
Briefly, in the dot assay, solutions are made of a log.sub.2
dilution series of potential IL-2 receptor containing samples and
PBS containing 1% Triton X-100. Samples of 5 ul of these solutions
are then applied to a piece of dry nitrocellulose (Schleicher and
Schuell, Keene, N.H.). The nitrocellulose is then blocked by
overnight incubation in 10 ml of 0.5 M TRIS, (pH 7.5), 0.15M NaCl,
3% BSA (hereinafter TBS-3% BSA). After the blocking step, the
nitrocellulose is incubated for one hour at room temperature in 10
ml of TBS-3% BSA containing 0.05 ug/ml .sup.125 I-2A3-A1H and 0.6
ug/ml unlabeled MOPC-21. The nitrocellulose is then washed three
times in TRIS buffered saline and twice in TRIS buffered saline
containing 1% (w/v) Nonidet P-40 detergent (Gallard Schlesinger
Chemical Manufacturing Corp., Carle Place N.Y.), 1% (w/v) sodium
deoxycholate, and 0.1% (w/v) sodium lauryl sulfate. Each of these
washes lasts 30 minutes at room temperature. After the final wash,
the nitrocellulose sheet is blotted dry, covered with a clear
plastic sheet and then exposed at -70.degree. C. to Kodak X-omat
AR.RTM. film.
Radioimmune Precipitation Assay
The specificity of the IL-2 receptor antibody is ascertained with a
radioimmune precipitation assay involving forming precipitations
between samples of radiolabeled IL-2 receptor molecules and an
antibody to the receptor and then employing polyacrylamide gel
electrophoresis and either fluorography or autoradiography to
visualize the receptor proteins that were precipitated. In this
assay technique, the IL-2 receptor molecules are labeled either by
surface iodination metabolically before extraction.
A surface podination of the IL-2 receptor on cell membranes after
extraction is performed by the .sup.125 I-IODO-GEN method (Pierce
Cl. Co., Rockford, Ill.). The details of this radiolabeling
technique are well known in the art and described by Urdal et al.,
1 Cancer Metastasis Reviews 65 (1982); and, Markwell et al., 17
Biochemistry (Wash.) 4807 (1978). The use of .sup.35 S methionine
to label the receptor molecules metabolically also is well known in
the art and is described by, for instance, Robb and Greene,
supra.
After labeling with .sup.125 I or .sup.35 S methionine, the cells
are washed with PBS and then extracted with PBS containing 1%
Triton X-100 and 2 mM PMSF. Affinity supports for the radioimmune
precipitation assay are prepared by coupling purified antibodies
(2A3-A1H and MOPC-21) to Affi-gel-10. Briefly, one volume of moist
Affi-gel-10 is added to one volume of antibody (3-5 mg/ml) in
borate buffered saline ("BBS") and then the mixture incubated
overnight at 4.degree. C. Thereafter, 100 ul of 1M glycine
ethylester is added per ml of gel to couple any of the unreacted
groups on the Affi-gel-10. Applicants have found that routinely
from 3 to 4 mg of antibody are coupled per ml of the gel under
these conditions. Before use, each gel is washed extensively with
PBS. Each gel is also washed with a buffer solution composed of
PBS-1% Triton X-100 and 0.5 M TRIS, pH 7.5, containing 0.5M NaCl,
1% (w/v) NP 40 detergent, 1% (w/v) sodium deoxycholate, and 0.1%
sodium dodecyl sulfate ("SDS") (collectively "RIPA buffer").
The radioimmune precipitations are performed by mixing 50 ul of
radiolabeled cell extract with 75 ul of PBS-1% percent Triton X-100
containing 20% (v/v) of affinity gel having antibody coupled
thereto. The mixture is incubated over night at 4.degree. C. and
then the gel washed four times with RIPA buffer and twice with 0.1M
TRIS, pH 8.0, containing 0.5M NaCl, 5 mM, ethylene diamine tetra
acetate ("EDTA"), and 0.5% NP-40 detergent. After the final wash,
the resulting gel pellets are suspended in 40 ul of SDS
polyacrylamide gel sample buffer (0.06M TRIS, pH 6.8, 2% SDS, 10%
glycerol, 5% 2-mercaptoethanol and boiled for three minutes to
break apart the bonds between the antibody and the IL-2 receptor
molecules. A 30 ul sample of the supernate is then analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE) (8-12% polyacrylamide
gel for .sup.125 I labeled receptor; 12% polyacrylamide gel for
.sup.35 S methionine labeled receptor) according to the stacking
gel procedure of Laemmli, 227 Nature (London) 680 (1970).
In the lysate analysis the receptor proteins employing the .sup.35
S methionine gels are visualized by fluorography. To this end, the
.sup.35 S methionine gels were impregnated with Enhance (New
England Nuclear, Boston, Mass.) prior to drying and fluorography.
The receptor proteins immunoprecipitated with the .sup.125 I gels
are visualized by autoradiography. To this end, the .sup.125 I gels
are stained with Coomassie blue prior to drying and
autioradiography. Both the .sup.35 S methionine and .sup.125 I gels
are exposed to Kodak X-omat AR.RTM. film at -70.degree. C. for 24
to 72 hours.
Gel Electrophoresis of Chromatography Column Fractions
Fractions eluted from the affinity chromatography and reversed
phase HPLC columns employed in the purification processes of the
present invention are assayed by gel electrophoresis. 50 ul
aliquots are removed from the eluate fractions. The aliquots are
dried under vacuum after addition of 2 ul of 10% SDS (w/v) to each
aliquot. The dried residue is dissolved in 40 ul of SDS
polyacrylamide gel sample buffer and then boiled for 3 minutes. The
solution is applied to an 8% polyacrylamide gel and electrophoresis
is then carried out by the stacking gel procedure of Laemmli,
supra. The resulting gel samples are silver stained by the method
described by Oakley et al., 105 Anal. Biochem. 361 (1980).
Purification of IL-2 Receptor
Cell extracts from the malignant and normal cells produced by the
above procedures are initially concentrated by affinity
chromatography techniques employing the same affinity supports used
in the radioimmune precipitation assay described above. The
procedure employed involves applying cell extracts first to an
MOPC-21 column and then to a second column prepared with a mixture
of MOPC-21 antibody and 2A3-A1H antibody so that in the second
column from 3 to 4 mg of total IgG is coupled to each ml of gel,
but only 10 to 30% of the antibody is composed of 2A3-A1H. This
technique is used to counteract the extremely high affinity between
the 2A3-A1H antibody and the IL-2 receptor.
In the purification procedure, the cell extracts, as prepared
above, are first applied to the MOPC-21 column that has been
preequilabrated with a buffer containing a detergent, thereby to
remove proteins in the cell extract that might nonspecifically bind
to mouse immunoglobulin. The flow through from the MOPC-21 column
is then applied to the 2A3-A1H column. Elution from this column is
carried out with a guanidine-HCL detergent solution. The recovered
fractions are then dialyzed against decreasing concentrations of
the eluting agent to optimize the recovery of biological
activity.
Fractions are collected and assayed by gel electrophoresis and
silver staining, as described above. Applicants have found that by
use of the affinity chromatography procedure, IL-2 receptor from
malignant cells which constitutively produce the receptor is
purified approximately 1600 times from initial cell lysate. A
somewhat lower purification level is typically attained for IL-2
receptor from activated normal cells.
The pooled active fractions from the above affinity chromatography
procedure is employed as a starting material for the HPLC
procedures. The HPLC technique used in the present invention
preferably employs a reversed phase, tetra methyl bonded silica
column having a pore size sufficiently large to be optimumly
utilized with the proteineaceous IL-2 receptor, i.e., a pore size
of at least 300 .ANG..
Suitable reversed phased HPLC columns for use in the practice of
the present invention are articles of commerce. A preferred column
for this purpose is the Vydac C-4 reversed phase column
commercially available from Separations Group, Hesperia, Calif.
This column consists of tetra methyl silane groups covalently
bonded by means of a siloxane (silicon-oxygen-silicon) bond to the
surface of the 300 .ANG. pore diameter silica gel which has been
classified to a mean particle size of 5 microns. Alternative HPLC
columns which may be employed in the present invention include
those constructed with octylsilane (Vydac C-8) or octyldecylsilane
(Vydac C-18) resins covalently bonded to silica gel.
The elution of the proteins from the HPLC column is carried out in
a manner well known in the art. A suitable elution procedure for
removing the bonded receptor molecule proteins from the tetra
methyl column involves the use of a linear gradient of
acetonitrile. A preferred gradient for this purpose is 0 to 95
percent (v/v) acetonitrile gradient in 0.1% (v/v) trifluoroacetic
acid (TFA), pH 2.1.
The eluted protein can be conveniently monitored with detection
systems that are well known in the art. The relative protein
concentration in the fractions eluted from the HPLC columns can be
determined by measuring absorbance of the eluded material in an
automated ultraviolet light spectrophotometer, at 214 nanometers
light wave length. The suitable automated ultraviolet light
absorbance detection apparatus is available from Waters Associates,
Millford, Mass. Final identification of the IL-2 receptor is
dependent on the detection of the receptor by use of the soluble
receptor assay and by use of gel electrophoresis as described
above.
By use of the above-described soluble receptor assay techniques,
applicants have found that the specific activity of the IL-2
receptor after HPLC purification is very high, i.e., approximately
21,000 fmole IL-2 receptor/ug protein for IL-2 receptor derived
from malignant cells. This is approximately a 16,700 fold level of
purification over the specific activity of the IL-2 receptor in the
starting cell lysate. The specific activity of the IL-2 receptor
from normal T-cells was about 1/3 of the specific activity from
malignant cells. By polyacrylamide gel electrophoresis and silver
staining, applicants ascertained that the molecular weight of the
IL-2 receptor from normal cells is approximately 60,000 daltons, as
opposed to 55,000 daltons for receptor molecules found
constitutively on the malignant cells.
Amino Acid Sequencing
The ability to prepare homogeneous IL-2 receptor has permitted
applicants to determine the amino acid sequence of the amino
terminal portion of this molecule. This information may be employed
to assist in the cloning of the IL-2 receptor gene and the
production of large quantities of pure IL-2 receptor for clinical
trials and ultimately for widespread medical use. Moreover, the
availability of homogeneous IL-2 receptor will no doubt lead to a
more complete understanding of the biology of IL-2. While the prior
art has said to have partially "characterized" the IL-2 receptor,
applicants are not aware of any instances in which this protein has
been truly purified to homogeneity to the extent that the receptor
can be analyzed for amino acid composition and sequence.
Samples of homogeneous IL-2 receptor, as prepared above, can be
analyzed for amino acid composition and sequence, for instance with
an automated sequencer, such as with an Applied Biosystems model
470A protein sequencer. Ideally, several sequencing runs are made
to confirm the accuracy of the sequence. Through this technique,
applicants have found that the first 15 residues of the amino
terminal portion of the IL-2 receptor molecule are composed of the
following sequence:
Glu-Leu-Cys-Asp-Asp-Asp-Pro-Pro-Glu-Ile-Pro-His-Ala-Thr-Phe.
Sources of IL-2 Receptor Producing Cells
Preferably, a cDNA library, from which the gene coding for the IL-2
receptor will be sought, is constructed from cells known to produce
high levels of IL-2 receptor. As noted above, these sources may
include malignant cell lines that have previously been identified
as high level IL-2 receptor producers, such as the human lymphoma
T-cell line designated as HUT-102, and human peripheral blood
mononuclear cells.
Preparation of RNA from IL-2 Receptor Bearing Cells
Total RNA from the IL-2 receptor bearing cells is extracted by
standard methods, such as disclosed by Chirgwin et al., 18
Biochemistry 5294 (1979) and Maniatis et al., Molecular Cloning, a
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1982).
As is well known, when extracting RNA from cells, it is important
to minimize ribonuclease ("RNase") activity during the initial
stages of extraction. One manner in which this is accomplished is
to denature the cellular protein, including the RNase, at a rate
that exceeds the rate of RNA hydrolysis by RNase. In the procedures
of Chirgwin et al., supra, and Maniatis et al., supra at 196, this
is carried out by use of guanidinium thiocyanate, together with a
reducing agent, such as 2-mercaptoethanol (to break up the protein
disulfide bonds). The RNA is isolated from the protein by standard
techniques, such as phenol/chloroform extraction, ethanol
precipitation or sedimentation through cesium chloride.
Although several techniques have been developed to separate the
polyadenylated mRNA from the extracted protein, one preferred
method is to chromatograph the polyadenylated mRNA on oligo
(dT)-cellulose in the well known manner described by, for instance,
Edmonds et al., 68 Proc. Natl. Acad. Sci. (USA) 1336 (1971); Aviv
and Leder, 69 Proc. Natl. Acad. Sci. (USA) 1408 (1972); and
Maniatis et al., supra at 197. The oligo (dT)-cellulose column is
prepared with a loading buffer and then the mRNA applied to the
column. Thereafter, the column is initially washed with a buffer
solution to remove the unpolyadenylated mRNA and then the
polyadenylated mRNA is eluted from the column with a buffered, low
ionic strength, eluent. The integrity of the polyadenylated mRNA is
verified by gel electrophoresis.
Preparation of cDNA from mRNA
A library of double-stranded cDNA corresponding to the mRNA is
prepared by known techniques employing the enzyme reverse
transcriptase. One such procedure which may be employed in
conjunction with the present invention is detailed by Maniatis et
al., supra at 230. Briefly, the polyadenylated mRNA is reverse
transcribed by using oligo-dT that has been hybridized to the
polyadenylated tail of the mRNA, as a primer for a first cDNA
strand. This results in a "hairpin" loop at the 3' end of the
initial cDNA strand that serves as an integral primer for the
second DNA strand. Next, the second cDNA strand is synthesized
using the enzyme DNA polymerase I and the hairpin loop is cleaved
by S1 nuclease to produce double-stranded cDNA molecules. The
double-stranded cDNA is fractionated by any convenient means to
remove the shorter strands thereby avoiding the needless cloning of
small cDNA fractions.
It is to be understood that in accordance with the present
invention, alternative well known procedures may be employed to
prepare double-stranded cDNA from mRNA. One such alternative
technique is disclosed by Land et al., 9 Nucl. Acids Res. 2251
(1981). In the Land et al. protocol, the hairpin loop is not used
as a primer for the second cDNA strand. Rather, the 3' end of the
first cDNA strand is tailed with dCMP residues using terminal
deoxynucleotidyl transferase ("TdT"). This produces a 3' tail of
poly-C residues. Then the synthesis of the second strand is primed
by oligo-dG hybridized to the 3' tail. This technique is said to
help avoid losing portions of the 5' tail of the second cDNA strand
which might occur if the hairpin is cleaved with S1 nuclease, as in
the Maniatis et al. protocol.
Cloning of cDNA
Next, the double-stranded cDNA is inserted within a cloning vector
which is used to transform compatible prokaryotic or eukaryotic
host cells for replication of the vector. Thereafter, the
transformants are identified and plasmid DNA prepared
therefrom.
To carry out the present invention, various cloning vectors may be
utilized to clone the cDNA. Although the preference is for a
plasmid, the vector may be a bacteriophage or a cosmid. If cloning
occurs in mammalian cells, viruses also can be used as vectors.
If a plasmid is employed, it may be obtained from a natural source
or artificially synthesized. The particular plasmid chosen should
be compatible with the contemplated transformation host, whether a
bacteria such as Escherichia coli ("E. coli"), yeast, or other
unicellular microorganisms. The plasmid should have the proper
origin of replication for the particular host cell to be employed.
Also, the plasmid should have a phenotypic property that will
enable the transformed host cells to be readily identified and
separated from cells that do not undergo transformation. Such
phenotypic characteristics can include genes providing resistance
to growth inhibiting substances, such as an antibiotic. Plasmids
are widely available that encode genes resistant to various
antibiotics, such as tetracycline, streptomycin, sulfa drugs,
penicillin, and ampicillin.
If E. coli is employed as the host cell, many possible cloning
plasmids are commercially available which may be used in
conjunction with the present invention. A preferred plasmid for
performing the present invention is pBR322. This plasmid is widely
commercially available and has been fully sequenced, as set forth
in Sutcliffe, 43 Cold Spring Harbor Symp. Quant. Biol. 77 (1979). A
significant advantage of this plasmid is that it has 11 known
unique restriction sites at which the plasmid may be cleaved by a
specific enzyme, including the Pst I site in the ampicillin
resistant gene. This feature is particularly useful for cloning by
the homopolymer tailing method.
If a bacteriophage is used instead of a plasmid, such phages should
have substantially the same characteristics noted above for
selection of plasmids. This includes the existence of a phenotypic
marker and ligatable termini for attachment of foreign genes.
The double-stranded cDNA prepared from mRNA, having blunt ends, may
be inserted within a plasmid cloning vector by various methods that
are well-known in the art. One such technique involves attaching
linkers to the ends of the cDNA strands. The linkers are composed
of approximately 8 to 10 base pair oligonucleotides that are
synthesized chemically and added to the double-stranded cDNA by
using DNA ligase. The linkers are then cleaved with a restriction
enzyme to generate cohesive termini for insertion within a plasmid
cleaved with the same restriction enzyme.
An alternative procedure, and of preference in the present
invention, is to insert the double-stranded cDNA into a plasmid
vector by homopolymeric tailing. In this technique, complementary
homopolymer tracks are added to the strands of the cDNA and to the
plasmid DNA. The vector and double-stranded cDNA are then joined
together by hydrogen bonding between complementary homopolymeric
tails to form open, circular hybrid molecules capable of
transforming host cells, such as E. coli.
In one procedure for homopolymeric tailing, approximately 50 to 150
dA nucleotide residues are added to the 3' ends of linearized
plasmid DNA. A similar number of dT nucleotide residues are added
to the 3' ends of the double-stranded cDNA and then the cDNA and
plasmid joined together.
In an alternative tailing method, dG tails are added to the 3' ends
of the cloning vector that has been cleaved with an appropriate
restriction enzyme. For instance, if the pBR322 plasmid is
employed, the restriction enzyme Pst I may be used to digest the
plasmid at the ampicillin resistant gene. Complementary dC tails
are added to the 3' ends of the double-stranded cDNA prior to
insertion of the cDNA segment in the plasmid with an appropriate
annealing buffer.
The recombinant DNA plasmids, as prepared above, are used to
transform host cells. Although the host may be any appropriate
prokaryotic or eukaryotic cell, preferably, it is a well-defined
bacteria, such as E. coli or a yeast strain. Such hosts are readily
transformed and capable of rapid growth in culture. However, in
place of E. coli, other unicellular microorganisms may be employed,
for instance, fungi and algae. In addition, various forms of
bacteria, such as salmonella or pneumococcus may be substituted for
E. coli. Whatever host is chosen, it should not contain a
restriction enzyme that would cleave the recombinant plasmid.
If E. coli is employed as a host, a preferable strain is MM294.
Protocols for transformation of this particular host by a plasmid
vector are well known, for instance, see Maniatis et al., supra at
255; and, Hanahan, 166 J. Mol. Biol. 557 (1983). Other strains of
E. coli which also could serve as suitable hosts include RR1, DH1
(ATCC No. 33849) and C600. These strains and the MM294 strain are
widely commercially available.
During transformation, only a small portion of the host cells are
actually transformed, due to limited plasmid uptake by the cells.
The cells that have been transformed can be identified by placing
the cell culture on agar plates containing suitable growth medium
and a phenotypic identifier, such as an antibiotic. Only those
cells that have the proper resistance gene (e.g., to the
antibiotic) will survive. If the recombinant pBR322 plasmid is used
to transform E. coli strain MM294, transformed cells can be
identified by using tetracycline as the phenotypic identifier.
Preparation of a Synthetic Oligonucleotide Screening Probe
A radiolabeled synthetic oligonucleotide corresponding to a portion
of the amino acid sequence of the IL-2 receptor, as determined
above, is used as a probe to screen the cDNA library. The
hybridization of the synthetic oligonucleotide probe with plasmid
cDNA prepared from the library clones is subsequently identified by
autoradiography.
The amino terminal portion of the IL-2 receptor molecule has been
identified and partially sequenced, above. A portion of this amino
acid sequence, composed of the residues: Cys-Asp-Asp-Asp-Pro-Pro,
is employed as the basis for the synthetic oligonucleotide probe.
This particular portion of the amino acid sequence of the IL-2
receptor has the advantage of being short enough to be easily
chemically synthesized, while also being long enough to be useful
as a direct probe for the IL-2 receptor gene. Also, this sequence
corresponds to a particular codon composition that is relatively
free of ambiguity.
Applicants have developed two synthetic oligonucleotides from the
above amino acid sequence for use as probes to screen plasmid DNA
thought to contain the IL-2 receptor genes. The probes are composed
of the following two sequences each having 17 bases: 5'
G-G-.sub.C.sup.T -G-G-G-T-C-G-T-C-G-T-C-A-C-A 3'. The particular
compositions of these probes were arrived at after conducting
initial primer extension analysis which enabled applicants to
eliminate other possible oligonucleotide sequences corresponding to
the above-identified amino acid sequence. The compositions of the
probes are the same except for the third nucleotide from the 5'
end, which in one oligonucleotide is composed of thymidine and in
the other is composed of cytosine. Also, the last nucleotide of Pro
residue was not employed thereby to reduce the ambiguity of the
oligonucleotide probes.
Although the described oligonucleotide sequences are the preferred
composition of the synthetic probes of the present invention, it is
to be understood that probes of other compositions corresponding to
additional partial amino acid sequences of the IL-2 receptor
molecule can be employed without departing from the spirit or scope
of the present invention.
The synthetic oligonucleotide probes may be chemically synthesized
by well-known techniques, such as by phosphodiester or triester
methods. Methods for triester synthesis are set forth in Sood et
al., 4 Nucl. Acid Res. 2557 (1977); and, Hirose et al., 28 Tet.
Lett. 2449 (1978). After synthesis, the oligonucleotide probe is
labeled with T4 polynucleotide kinase and .sup.32 P-ATP, for
instance by the protocol set forth in Maniatis et al., supra at
122. Advantageously, the oligonucleotide probes can be synthesized
with OH 5' termini thereby avoiding the phosphatase procedure
typically required.
Screening of cDNA Library
In the screening procedure of the present invention, the
transformed bacteria cultures are pooled into groups. After the
replicated plasmids have been extracted from the transformants, DNA
is prepared by cleaving the plasmids at the Pvu II and Hind III
restriction sites, both being unique sites on the hybrid plasmid.
The resulting DNA segments are fractionated by electrophoresis on
agarose gels and then directly analyzed by Southern blotting, for
instance as described in 98 J. Mol. Biol. 503 (1975). The DNA that
binds to the nitrocellulose filter in the Southern blotting
procedure is hybridized with the labeled oligonucleotide probes.
The specific DNA fragments that hybridize to the probe are
identified by autoradiography.
The particular pool(s) of clones that give a signal following
autoradiography are plated out and used in direct bacterial colony
hybridization on a nitrocellulose filter with the same
above-identified oligonucleotide probes. After completion of the
hybridization, the nitrocellulose filter is monitored by
autoradiography to identify positive colonies. In the present
invention, applicants discovered two such positive colonies.
Plasmid DNA, designated as IL-2 Rec N4 (hereinafter "N4") and IL-2
Rec N1 (hereinafter "N1") were prepared from the two particular
positive colonies identified.
Characterization of Screened cDNA
The plasmid DNA prepared above is initially characterized by
restriction enzyme mapping. Various strategies for restriction
enzyme mapping are discussed by Maniatis et al., supra at 374. One
preferred technique involves the partial digestion of end-labeled
fragments of linear DNA. This technique, developed by Smith and
Birnstiel, 3 Nucl. Acids Res. 2387 (1976), is now well known in the
art. Partial restriction enzyme maps of the N4 cDNA clone in the
region of the IL-2 receptro gene and of the N1 cDNA clone are shown
in FIG. 1. A distance scale for 100 nucleotide base pairs ("bp") is
also shown. The Pst I sites shown in brackets are those generated
by the cloning procedures.
The mapped plasmid cDNAs are initially partially sequenced to
determine whether they are homologous to the amino acid sequence of
the IL-2 receptor. Although applicants have ascertained that both
cDNA clones illustrated have nucleotide sequences corresponding to
the known N-terminus amino acid sequence of the IL-2 receptor, as
discussed below, only the pN4 cDNA clone contains the gene coding
for IL-2 receptor. The N-terminus of the mature IL-2 receptor
protein is located at the Sst I site of the N4 clone.
Thereafter, the cloned cDNA's are sequenced using chain-termination
method. This method of nucleotide sequencing was originated by
Sanger et al., 70 Proc. Natl. Acad. Sci. (USA) 5463 (1977). See
U.S. Pat. No. 4,322,499. Methods for chain-termination sequence
determination are set forth in the Amersham Handbook entitled, M13
Cloning and Sequencing, Blenheim Cresent, London (1983)
(hereinafter "Amersham Handbook"); Messing, 2 Recombinant DNA
Technical Bulletin, NIH Publication No. 79-99, 2, 43-48 (1979);
Norrander et al., 26 Gene 101 (1983); Cerretti et al., 11 Nucl.
Acids Res. 2599 (1983); and, Biggin et al., 80 Proc. Natl. Acad.
Sci. (USA) 3963 (1983). M13 filamentous phage are employed as
vectors to clone the DNA sequences of interest. These phage vectors
provide single-stranded DNA templates which are readily sequenced
by chain-termination method, which involves priming a
single-stranded template molecule with a short primer strand having
a free 3' hydroxyl group and then using DNA polymerase to copy the
template strand in a chain extension reaction using all four
deoxyribonucleotide triphosphates, i.e., dATP, dCTP, dGTP, and dTTP
(collectively referred to as "dNTPs"), with one of them being
radiolabeled. In the synthesis reaction, a nucleotide specific
chain terminator lacking a 3'-hydroxyl terminus, for instance, a
2', 3' dideoxynucleotide triphosphate ("ddNTP"), is used to produce
a series of different length chain extensions. The terminator has a
normal 5' terminus so that it can be incorporated into a growing
DNA chain, but lacks a 3' hydroxyl terminus. Once the terminator
has been integrated into the DNA chain, no further deoxynucleotide
triphosphates can be added so that growth of the chain stops. Four
separate synthesizing reactions are carried out, each having a
ddNTP of one of the four nucleotide dNTPTs, i.e., dATP, dCPT, dGTP
and dTTP. One of the normal dNTPs is radiolabeled so that the
synthesized strands after having been sorted by size on a
polyacrylamide gel, can be autoradiographed. The chain extensions
from the four reactions are placed side by side in separate gel
lanes so that the pattern of the fragments from the autoradiography
corresponds to the DNA sequence of the cloned DNA.
The DNA and corresponding amino acid sequences of the N4 and N1
clones from the 5' ends to the Xba I restriction site, as
determined by the above techniques, are illustrated in FIG. 2. As
detailed below, the gene coding for IL-2 receptor is contained in
the N4 clone and not in the N1 clone. In FIG. 2, the nucleotide
sequence shown is from the N4 clone except for the sequences
upstream from the arrow, which are derived from the N1 clone. The
arrow marks the 5' end of the insert in the N4 clone. The
nucleotides are numbered from the position of the initiator
methionine codon to the TAG termination codon. The amino acids are
numbered beginning from the mature NH.sub.2 -terminus of the IL-2
receptor protein, i.e., the Glu residue, marked with a star, and
extending to the Ile residue (251) located adjacent the termination
codon TAG. The IL-2 receptor gene, extending from the initiator
methionine codon to the TAG termination codon, is shown as a box
portion in FIG. 1. Correspondingly, the coding region of the N1
clone is shown as a box portion. The restriction enzyme cleaving
sites identified in FIG. 1 are also indicated in FIG. 2.
The base sequence of the N1 clone differs from the N4 clone, in
that the N4 clone contains a 216 base pair insert sequence not
present in the N1 clone, extending from nucleotides 370 to 585
(underlined in dots in FIG. 2). This 216 base pair insert is shown
in FIG. 1 as the unshaded box portion of the N4 clone. The two
clones also differ at nucleotides 148, 183, 322 and 327. In
addition, as shown in FIG. 2, three of these sequence differentials
would cause amino acid changes. In terms of similarities, both
clones contain the sequence of the oligonucleotide probe employed
above, with a single base pair mismatch, and both encode the amino
acid sequence determined above for the NH.sub.2 -terminus of the
IL-2 receptor. Both also encode a stretch of 15 amino acids
immediately preceding the NH.sub.2 -terminus sequence, which starts
with a methionine residue and has many of the characteristics of a
hydrophobic signal peptide expected from membrane or secreted
proteins.
In preparation for the sequencing procedures, the cDNA clones shown
in FIG. 1 are digested with various restriction endonucleases in
various combinations and then the resulting DNA fragments cloned
into M13 phage vectors to form single stranded DNA templates. A
universal primer is used to sequence the sense and nonsense
strands. Rather than relying on the sequencing results obtained
from sequencing the entire length of the fragments with a single
chain termination procedure, in the longer fragments additional
synthetically produced primers are used to initiate the chain
termination procedure from intermediate locations along the lengths
of the fragments. By this process, both strands of the cDNA clones
shown in FIG. 1 are sequenced in overlapping fashion, thereby
serving to redundantly confirm the sequences.
It is to be understood that rather than employing the
chain-termination technique outlined above, other knonw methods may
be utilized to sequence the IL-2 receptor gene without departing
from the spirit or scope of the present invention. For instance,
the chemical degradation method of Maxam and Gilbert as set forth
in 74 Proc. Nat'l Acad. Sci. (USA) 560 (1977) can be used.
Expression Of Functional IL-2 Receptor From cDNA Clones
To determine whether the cDNA coding regions of the N1 or N4 clones
could encode a functional IL-2 receptor, the clones are expressed
in mammalian cells. Hybrid cDNA fragments containing the coding
regions of the N4 and N1 clones are inserted into a plasmid vector
derived in part from simian virus 40 ("SV40"). The genome of this
virus consists of a single, small, covalently closed circular DNA
molecule whose entire nucleotide sequence has been determined,
Fiers et al., 237 Nature, (London) 113-120 (1978), and Reddy et
al., 200 Science 494-502 (1978). The two constructed vectors,
designated as pMLSV-N1/N4-S and pMLSV-N1/N4-X, having the coding
regions of the N4 and N1 clones, respectively, are illustrated in
FIG. 3A.
The above-delineated vectors are transfected into mammalian cells.
After subsequent incubation, the cells are harvested and assayed
for expression of mature IL-2 receptor by their ability to bind to
labeled IL-2 or the labeled 2A3-A1H monoclonal antibody directed
against the IL-2 receptor. Labeled 2A3-A1H monoclonal antibody may
be prepared as described above. IL-2 may be prepared by established
methods, such as set forth in U.S. Pat. No. 4,401,756, and in Urdal
et al., 296 J. Chromatog. 171 (1984) and then radiolabeled, for
instance by use of a radioiodination reagent such as
Enzymobead.RTM. (BioRad Laboratories, Richmond, Calif.). As shown
in sections B and C of FIG. 3, the mammalian cells transfected with
the pN1/N4-S vector specifically bound to both IL-2 and the 2A3-A1H
monoclonal antibody. However, neither pN1/N4-X or mock-transfected
cells (prepared as a control) specifically bound to IL-2 or the 2A3
monoclonal antibody. Since the pN1/N4-S vector contained the coding
region of the N4 clone, this indicated that this clone contains the
gene coding for the functional IL-2 receptor protein, whereas the
N1 clone does not.
The processes and products of the present invention are further
illustrated by the following examples.
EXAMPLE 1
Preparation of IL-2 Receptor Containing Extracts From Malignant
Cell Line
Hut-102 cells in a concentration of 2.times.10.sup.5 cells per ml
were cultured in 100-500 ml volumes in various plastic culture
flasks or bottles (Falcon Plastics, Oxnard, Calif.) in RPMI-1640
medium. The medium was supplemented with 10% FCS, 2 mM glutamine,
100 U/ml penicillin and 100 ug/ml streptomycin. Since the HUT-102
cells have been reported to produce human T-cell leukemia virus
(HTLV-1), work with this cell line was performed in a P-3 isolation
facility.
The cells were cultured for 3-5 days in a humidified atmosphere of
5% CO.sub.2 in air. After this period of time, viable cells were
harvested by centrifugation and washed three times in PBS.
Thereafter, the cell pellet was suspended in a volume that is three
times the volume of the cell pellet in a solution composed of PBS
containing 1% (w/v) Triton X-100 detergent and 2 mM PMSF. This
mixture was kept on ice and periodically vortexed for 30 minutes.
The extract was then centrifuged at 20,000.times.g for 20 minutes
to remove nuclei and insoluable debris. The cell extract, as thus
prepared, was then stored at -70.degree. C. until used.
EXAMPLE 2
Preparation of IL-2 Receptor Containing Extracts From Lectin
Activated Normal Cells
Human peripheral blood mononuclear cells were prepared by
Ficoll-Hypaque density gradient centrifugation as described by
Boyum, supra. The resulting cells were incubated separately in
100-mm plastic petri dishes in 8% FCS at a concentration of
2-5.times.10.sup.6 per ml. The adherent cells were recovered with a
rubber policeman after removing nonadherent cells with three media
washes. The E.sup.- adherent cells together with the E.sup.+
nonadherent cells in a ratio of 1:25 were placed in bulk culture in
75-cm.sup.2 flasks at a concentration of about 1-2.times.10.sup.6
cells/ml in RPMI-1640 medium supplemented with 10% FCS, 100 U/ml
penicillin and 100 ug/ml streptomycin. Activation was accomplished
with 1% (v/v) PHA (Difco Laboratories, Detroit, Mich.). The
cultures were incubated at 37.degree. C. in an humified atmosphere
of 5% CO.sub.2 in air. Aliquots containing approximately
1-2.times.10.sup.7 cells were removed at various times for analysis
of cell surface IL-2 receptors.
Cells were harvested by centrifugation approximately 72 hours after
mitogen stimulation, and washed three times with PBS. The resulting
cell pellet was suspended in a volume three times the volume of the
pellet in a solution composed of PBS containing 1% (w/v) Triton
X-100 detergent and 2 mM PMSF. The resulting mixture was kept on
ice with periodic vortexing for 30 minutes. Thereafter, the extract
was centrifuged at 20,000.times.g for 20 minutes to remove nuclei
and insoluable debris. The resulting cell extracts were stored at
-70.degree. C. centrigrade until used.
EXAMPLE 3
Production of Monoclonal Antibody To IL-2 Receptor
Female BALB/c (Jackson Laboratories, Bar Harbor, Me.) of ages of
from 8-12 weeks were immunized intradermally in the hind legs with
10.sup.7 PHA/PBL. Prior to immunization, the PHA/PBL cells were
prepared as an emulsion by mixing these cells with 0.4 ml of
complete Freund's adjuvant (Difco Laboratories). After the initial
immunization, the mice were rechallenged weekly for four weeks with
10.sup.7 PHA/PBA in incomplete Freund's adjuvant.
Periodically, serum from the mice was collected and tested
individually for binding to PHA/PBL by ELISA, in a manner well
known in the art. The animals found to have the highest response
were given an additional intravenous injection of 10.sup.7 PHA/PBL
in PBS. Four days later, the animals were sacrificed by cervical
dislocation. The spleens of the animals were harvested and single
cell suspensions prepared therefrom. The spleen cells were cultured
in medium.
Fusion was achieved by mixing approximately 20.times.10.sup.6
spleen cells with approximately 10.times.10.sup.6 NS-1 murine
myeloma cells in a 50 ml conical centrifuge tube. The cell mixture
was pelleted by centrifugation for 5 minutes at 200.times.g, and
then the supernate removed by aspiration. The centrifuge tube with
its intact cell pellet was transferred into a 37.degree. C. water
bath. Then polyethylene glycol 15 w (Eastman, Inc.) (50% (w/v) in
RPMI-1640 was added to the cell pellet in dropwise manner at a
ratio of 1 ml of PEG/1.6.times.10.sup.8 spleen cells. Thereafter,
one volume of RPMI-1640 and 10 volumes RPMI 1640 containing 15% FCS
and 1 mM pyruvate were slowly added during gentle stirring. Then,
the cell suspension was centrifuged at 200.times. g for 5 minutes
and the supernate discarded to complete the fusion process.
The hybrid cells were selected by resuspending the resulting cell
pellet in Click's medium containing 15% FCS and 100 mM sodium
pyrvate. The unfused myeloma driver cells (NS-1), double NS-1
hybrids, unfused spleen cells and double spleen cell hybrids were
prevented from proliferation by the addition to the medium of
approximately 13.6 mg/L of hypoxanthane, 0.176 mg/L aminopterin and
3.88 mg/L of thymidine. The suspension was then divided into 200 ul
aliquots in flat-bottom microliter plates (No. 3596, Costar Inc.,
Cambridge, Mass.). The cultures were maintained at approximately
37.degree. in a humified atmosphere of 5% CO.sub.2 in air.
After 10 days of culture, a 100 ul aliquot of supernate was removed
from each viable culture and tested in an ELISA assay for binding
to PHA/PBL (IL-2 receptor positive) or PBL (IL-2 receptor
negative). Hybrids which demonstrate significant binding to PHA/PBL
and little or no binding to PBL were transferred to 1 ml cultures
and gradually weaned to HAT-free media. These hybrids were
subcloned by limiting dilution cultures. Through this process,
applicants have identified one particular hybrid clone, designated
as 2A3-A1H, which significantly inhibits both mitogen and antigen
induced proliferation of human PBL. Samples of this cell line are
on deposit with the American Type Culture Collection ("ATCC"),
Rockville, Md., under accession No. HB 8555. The 2A3-A1H monoclonal
antibody has been characterized as of the .gamma..sub.1 K isotype
that exhibits a very high affinity to the human IL-2 receptor. This
antibody inhibits the binding of IL-2 to its receptor and is
antagonistic of IL-2 action.
EXAMPLE 4
In Vivo Production of Hybridoma Cells Producing Monoclonal
Anti-IL-2 Receptor Antibodies
Anti-IL-2 receptor antibody was produced in high concentration in
vivo by intraperitoneal injection of BALB/c mice with approximately
1-10.times.10.sup.6 hybridoma cells. One week prior to hybridoma
cell injection, recipient BALB/c mice were given approximately 1.0
ml of pristane intraperitoneally as an ascites inducing irritant.
From 8 to 14 days after hybridoma injection, intraperitoneal
ascites were collected and each volume of fluid is mixed with 0.9
volume of 45% saturated ammonium sulfate and stirred overnight. The
precipitate was separated by centrifugation and redissolved in
phosphate buffer (0.05M), pH 6.8. Residual ammonium sulfate was
removed by dialysis against the same buffer.
The protein solution was then passed over a 5 ml bed volume DE-52
column (Whatman, Clifton, N.J.) and the fronting peak of protein
was pooled. The pooled fractions were dialyzed against 0.02M sodium
borate, 0.1M sodium NaCl, pH 8.5, ("BBS") and then applied to a
2.6.times.90 cm ACA-34 (LKB, Bromma, Sweden) gel filtration column
previously equilibrated in the same buffer. The fractions
corresponding to IgG were collected and pooled. Yields typically
were in the range of 3 mg IgG/ml of ascites.
EXAMPLE 5
Purification of IL-2 Receptor By Affinity Chromotography
Cell extracts from normal and malignant cells produced by the
procedures of Examples 2 and 3 were concentrated by affinity
chromatography technique employing an initial gel column having
control antibody for removing protein that might nonspecifically
bind to mouse IgG and a second column having 2A3-A1H antibody bound
thereto. The control antibody used in the initial column was
secreted by the myeloma cell line MOPC-21. This antibody is of the
same isotype as the 2A3-A1H antibody and is readily available.
To prepare the columns, purified 2A3-A1H and MOPC-21 antibodies
were coupled to Affi-gel-10 (BioRad, Richmond, Calif.) according to
the manufacturer's instructions. Equal volumes of moist Affi-gel-10
and antibody (3-5 mg/ml) in PBS were mixed together and incubated
overnight at 4.degree. C. Thereafter, unreacted sites on the
Affi-gel-10 were blocked by addition of 100 ul of 1M glycine ethyl
ester per ml of gel. Applicants found that the antibody-coupled gel
routinely contained from 3 to 4 mg of antibody per ml of gel.
Because the 2A3-A1H antibody exhibits such an extremely high
affinity for the IL-2 receptor, the receptor yield from the
chromatography columns was improved by employing columns prepared
with a mixture of MOPC-21 and 2A3-A1H antibody. A total of 3 to 4
mg IgG was still coupled per ml of gel, but only 10-30% of the IgG
is composed of 2A3-A1H. The column having both MOPC-21 and 2A3-A1H
antibody bound thereto will be referred to as the "2A3-A1H"
column.
Prior to use, each gel was washed extensively with PBS and RIPA
buffer. The MOPC-21 and 2A3-A1H gel columns were poured in 3 ml
syringes that have their open ends closed with a cork and tubing,
thereby to enable the columns to be run in either direction. The
cell extracts, as prepared in Examples 1 and 2 above, were first
applied to the MOPC-21 column at a flow rate of 0.1 ml/min at
4.degree. C. to remove proteins the nonspecifically bind to the
mouse IgG. This absorption was repeated once more and then the
flow-through from the MOPC-21 column is twice applied to the
2A3-A1H column.
The 2A3-A1H column was then washed with 10 column volumes of PBS-1%
Triton X-100, 10 column volumes of RIPA buffer and lastly, 10
column volumes of PBS-1% Triton X-100. Thereafter, the receptor was
eluted from the column with 6M guanidine hydrochloride ("GuHCl")
and 0.5% Triton X-100. Eluate fractions in 1.2 ml volume were
collected and each fraction was dialyzed against 3M GuHCl in 0.5%
Triton X-100 for four hours. This was followed by dialysis against
1.5M GuHCl in 0.5% Triton X-100. Final dialysis was performed
against PBS containing 0.5% Triton X-100. Aliquots at each stage of
the purification were saved for analysis of: biological activity by
the above-described soluble receptor assays; protein concentration
by fluorescamine assay with bovine serum albumin as a standard, as
is well known in the art; and, protein heterogeneity by
polyacrylamide gel electrophoresis with the protein being detected
by silver staining, as also described above. From these assays, the
IL-2 receptor from the HUT-102 cells was found to have a specific
activity of approximately 2,000 fm receptor/ug protein. The
specific activity from the PHA-PBL cells was somewhat less.
EXAMPLE 6
Reversed Phase High Performance Liquid Chromatography
The active fractions obtained in Example 5 were pooled for use as
the starting material for the HPLC process. These fractions were
pumped directly onto a 3.9 mm times 15 cm Vydac C-4 column, which
had been previously equilibrated with 0.1 percent TFA in water, at
a flow rate of about 1 ml/min with a Waters M-45 A solvent pump
(Waters Associates, Millford, Me.). The loaded column was initially
washed with 0.1% TFA to remove nonbound components until the
absorbence at 214 nanometers as detected with a Waters Model 441
absorbence detector drops to base line. Elution of bound proteins
was accomplished with a linear gradiant of 0-95% acetonitrile in
0.1 percent TFA (v/v) at a rate of 1% per minute. The IL-2 receptor
protein was found to elute off the column in the 50 to 55%
acetonitrile fractions.
One minute fractions were collected (1 ml) and 50 ul aliquots were
removed from each fraction for analysis by polyacrylamide gel
electrophoresis followed by silver staining. This technique
resulted in the separation of a single band of protein at a
molecular weight of 55,000 daltons for the HUT-102 receptor
molecule. The PHA-PBL receptor molecule, which eluted at the same
position on the HPLC as the HUT-102 receptor molecule, exhibited a
single band of protein having a molecular weight of 600,000
daltons.
Aliquots in 50 ul volumes were also removed from the minute
fractions for biological assay. The aliquots were dried under
vacuum in the presence of 50 ug BSA. The dried residue was
dissolved in PBS-2% Triton X-100 for analysis by the soluble
receptor assay techniques discussed above. This assay indicated
that the IL-2 from HUT-102 receptor had been purified from 1.26
fmole receptor/ug in protein the cell lysate starting material to
approximately 21,000 fmole receptor/ug protein after the HPLC
purification step. This equates to an increase in purification of
the IL-2 receptor of about 16,670 times. The specific activity of
the PHA-BPL receptor after the HPLC purification step was found to
be approximately 5,000 fmole receptor/ug protein. It is clear from
the single protein bands which resulted from the polyacrylamide gel
electrophoresis and silver staining of the fractions collected
after HPLC, and also from the specific activities of the fractions
analyzed by the soluble receptor assays, essential homogeneity of
the IL-2 receptor molecule was achieved.
EXAMPLE 7
Protein Sequencing
Purified IL-2 receptor from Example 6 was dried under vacuum to a
final volume of approximately 100 ul and then subjected to
automated amino terminal Edman degration using an Applied
Biosystems Model 470A protein sequencer. Fractions from the
sequencing cycles were evaporated to dryness and then resuspended
in acetonitrile/H.sub.2 O (50:50) before injection into an HPLC
column for residue identification.
By the above process, the amino-terminal amino acid sequence for
the IL-2 receptor from both the HUT-102 and PHA-PBL cells were
found to be the same. The first 15 residues of the N-terminal
portion of the IL-2 receptor molecule was determined to be composed
of the following sequence:
Glu-Leu-Cys-Asp-Asp-Asp-Pro-Pro-Glu-Ile-Pro-His-Ala-Thr-Phe. This
amino acid sequence was compared with known protein sequences
contained in the National Biomedical Research Foundation protein
data base "SEARCH" (January, 1984), and was not significantly
homologous to any protein sequence contained in this data base.
EXAMPLE 8
Preparation of Polyadenylated mRNA
Hut-102 cells at a concentration of approximately 2.times.10.sup.5
cells/ml were cultured in 100-500 ml volumes in RPMI-1640 medium
supplemented with 10% FCS (v/v), 2 mM glutamine, 100 U/ml
penicillin and 100 ug/ml streptomycin. The cells were cultured for
3-5 days in a humidified atmosphere of 5% CO.sub.2 in air. After
this period of time, viable cells were harvested by
centrifugation.
Total RNA was extracted from the Hut-102 cells by the method as
described by Chirgwin et al., supra. In this procedure guanidinium
thiocyanate was used to denature the cellular protein including the
RNase at a rate that exceeds the rate of RNA hydrolysis by RNase.
The mRNA was removed from the cellular protein by
ultracentrifugation through a dense cushion of cesium chloride.
Thereafter, polyadenylated mRNA was separated from the extracted
protein on an oligo (dT)-cellulose chromatography column using the
method disclosed by Maniatis et al., supra at 197. Briefly, the
column was prepared with application buffer composed of 20 mM
Tris-Cl (pH 7.6), 0.5M NaCl, 1 mM ethylene diamine tetraacetate
("EDTA") and 0.1% sodium dodecyl sulfate ("SDS"). The pellet was
dissolved in water and application buffer and then loaded onto the
column. The nonadsorbed material was removed by initial washings
with application buffer followed by additional washings with
application buffer containing 0.1M NaCl. The retained
polyadenylated mRNA was removed with buffers of reduced ionic
strength composed of 10 mM Tris-C;l (pH 7.5), 1 mM EDTA and 0.05%
SDS. The eluted polyadenylated mRNA was precipitated at -20.degree.
C. with 1/10 volume sodium acetate (3M, pH 5.2) and 2.2 volumes of
ethanol. After elution of the polyadenylated mRNA from the oligo
(dT)-cellulose column, the integrity of the polyadenylated mRNA was
confirmed by electrophoresis through agarose gels as detailed in
Maniatis et al., supra at 199.
EXAMPLE 9
Construction of cDNA Library
A library of double-stranded cDNA corresponding to the mRNA was
prepared from the purified mRNA in Example 8 by employing the
procedure detailed by Maniatis et al., supra at 229. Oligo-dT was
hybridized to the polyadenylated tail of the mRNA to serve as the
primer for the reverse transcription of the first cDNA strand. The
enzyne avian myeloblastosis virus ("AMV") reverse transcriptase was
employed to synthesize the first DNA strand by using the mRNA as a
template. This procedure resulted in a hairpin loop being formed at
the 3' end of the initial cDNA strand. The hairpin loop served as
an integral primer for the second cDNA strand. After the mRNA
strand was degraded with NaOH, the second cDNA strand was
synthesized with DNA polymerase I. The hairpin was then removed
with nuclease S1 to produce doublestranded cDNA molecules.
The double-stranded cDNA was fractionated into size classes by
Sephacryl S-400 column chromatography and monitored by alkaline
agarose electrophoresis using end-labeled fragments of pBR322 DNA
as molecular-weight markers. Strands having a length of less than
500 bp were culled out to avoid needless cloning of these
undersized cDNA fractions.
The double-stranded cDNA fractions, as prepared above, were
inserted into the Pst I site of the pBR322 plasmid. The
double-stranded cDNA was tailed with poly (dC) at its 3' ends. The
plasmid pBR322 was digested with Pst I endonuclease and then tailed
with poly (dG) at its 3' ends. The tailed plasmid DNA and the
tailed cDNA were annealed in annealing buffer (0.1M NaCl, 10 mM
Tris-Cl (pH 7.8) and 10 mM ETDA) to form novel recombinant
plasmids.
The recombinant plasmids were transformed into E. coli strain MM294
by using the procedure of Hanahan, supra in which the E. coli cells
were prepared by growth in elevated levels of Mg.sup.2+. The
transformation hosts were plated and then transformants are
identified by use of tetracycline as a phenotypic identifier. By
use of this technique, applicants obtained approximately
2.times.10.sup.6 independent transformants.
EXAMPLE 10
Preparation of Synthetic Oligonucleotide Screening Probes
Synthetic oligonucleotides were employed as a probe in screening
the cDNA library prepared as set forth above in Example 9. The
probes were composed of the following compositions: 5'
G-G-.sub.C.sup.T -G-G-G-T-C-G-T-C-G-T-C-A-C-A 3'. These
oligonucleotide probes were chemically synthesized by triester
method using the well known techniques of Sood et al., supra and
Hirose et al., supra.
After chemical synthesis had been completed, the 5' ends of the
oligonucleotide probes were labeled with .sup.32 P. To facilitate
labeling, the 5' ends of the oligonucleotide were synthesized with
OH termini, thereby eliminating the phosphatese treatment which
typically must be employed when labeling DNA fragments. The
labeling protocol included adding 1 ul of the synthetic
oligonucleotides to 16 ul of .sup.32 P-ATP (7000 Ci/mM), 1
microliter ("ul") (10 U) of T4 polynucleotide kinase and 2 ul of
10.times.kinase buffer I. The 10.times.kinase buffer I was composed
of 0.5M Tris-Cl (pH 7.6), 0.1M MgCl.sub.2, 50 mM dithiothreitol, 1
mM spermidine and 1 mM ETDA. The reaction was carried out at
37.degree. C. for 30 minutes, and thereafter the synthesized
oligonucleotides were extracted with phenol/chloroform. The labeled
probes were separated from unlabeled oligonucleotides by
chromatography on or centrifugation through Sephadex G-50
columns.
EXAMPLE 11
Screening of cDNA Library
To facilitate initial screening of the cDNA library prepared in
Example 9 above, the transformed bacteria cultures were grouped
into pools each having approximately 5,000 different clones.
Plasmid DNA was removed from samples of the host bacteria by the
well known alkaline lysis method, for instance as described by
Ish-Horowicz and Burke, 9 Nucl. Acids Res., 2989 (1981).
The isolated plasmids were separated into two fragments. This was
accomplished by initially digesting the plasmids to completion with
Pvu II and Hind III. The plasmids were redissolved in 20 ul of
1.times.Hind III buffer (7 mM Tris, (pH 7.4), 7 mM magnesium
chloride, 60 mM NaCl) and then 1 ul of Pvu II and 1 ul of Hind III
restriction endonucleases were added. This mixture was incubated at
37.degree. C. for two hours.
Next, the plasmid digests were fractionated by electrophoresis
through 0.8% agarose gel wtih markers of appropriate size. The
agarose gel was blotted onto nitrocellulose filter using the well
known method described by Southern, supra. After the transfer
process, the filter was air dried and baked for two hours at
approximately 80.degree. C. under a vacuum to bind the DNA
fragments to the nitrocellulose.
The bound DNA was next hybridized with the labeled oligonucleotide
probes. Briefly, the baked nitrocellulose was presoaked in
6.times.saline sodium citrate ("SSC") (20 X SSC is composed of
175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H.sub.2
O, with pH adjusted to 7.0 with 10N NaOH) and then incubated at
50.degree. C. for 2-4 hours in prehybridization buffer composed of
6.times.SSC, 0.5% NP40 detergent, 0.1% sarcosyl, 5.times.Denhardt's
solution (0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% BSA) and
100 ug/ml denatured salmon sperm DNA (Sigma Type III, sodium salt).
The filter was then incubated overnight at 50.degree. C. with the
.sup.32 P-labeled oligonucleotide probe (10.sup.6 cpm/ul) (from
Example 10) in hybridizing solution as above. After overnight
hybridization, the filter was washed extensively with 6.times.SSC
at room temperature and then for 5 minutes at 50.degree. C. with
6.times.SSC. After air drying, the filter was subjected to
autoradiography at -70.degree. C.
From the autoradiography, applicants found several pools of
transformants generating hybridizing bands. The appropriate pools
of the transformants were plated out and then used in direct
bacterial colony hybridization on nitrocellulose paper with the
labeled oligonucleotide probe under the same hybridizing conditions
as above. By this process, two positive colonies were
identified.
EXAMPLE 12
Restriction Enzyme Mapping of Screened cDNA
Plasmids, designated as N4 and N1, were prepared from the
identified positive colony by the procedures set forth in Example
9. Samples of the N4 and N1 plasmids transformed into E. coli
strain MM294 are on deposit with the ATCC, under Accession Nos.
39752 and 39751, respectively. Thereafter, the N4 and N1 plasmids
were analyzed by restriction enzyme mapping using the standard
method developed by Smith and Birnstiel, supra, involving partial
digestion of end-labeled fragments of the linearized DNA. The DNA
fragments were labeled at their 5' termini with .sup.32
P-phosphoryl groups using polynucleotide kinase and .sup.32 P-ATP.
The labeled DNA strands were then cleaved asymmetrically with a
suitable restriction enzyme to provide two fragments, each labeled
at only one of its ends. These labeled fragments were isolated by
gel electrophoresis. Each of the two fragments was partially
digested by appropriate restriction enzymes. Although a large
spectrum of digestion fragments were produced, the labeled
fragments formed a simple overlapping series each having a common
labeled terminus. These fragments were fractionated by gel
electrophoresis and then examined by autoradiography. The locations
of the fragments on the gel correspond directly to the order of the
restriction sites along the plasmid DNA.
By this procedure, applicants partially mapped the restriction
sites, as shown in FIG. 1, of the N4 and N1 plasmid cDNAs in the
region of the IL-2 receptor gene.
EXAMPLE 13
Sequencing of Screened cDNA
The DNA fragments shown in FIG. 1 were initially partially
sequenced by the dideoxy chain termination method. From the
sequencing results, applicants confirmed that the N4 DNA fragment
shown in FIG. 1 contains the gene coding for the IL-2 receptor,
with the N-terminus of the mature IL-2 receptor protein being
located at the Sst I site of the DNA fragment shown in FIG. 1.
Thereafter, the portions of the N4 and N1 clones from the 5'
terminals to the Xba I restriction sites were sequenced by the
chain termination protocol essentially as described in the Amersham
Handbook, supra, with the variations set forth below. The N4 and N1
clones were digested with Pst I, Sst I and Xba I restriction
endonucleases in various combinations and then the resulting DNA
fragments were cloned into strains mp18 and mp19 of the M13
single-stranded filamentous phage vector (Amersham, Arlington
Heights, Ill.). The mp18 and mp19 phage vectors, as set forth in
Norrander et al. supra, contain the following unique cloning sites:
Hind III; Sph I; Pst I; Sal I; Acc I; Hinc II; Xba I; BamHI; Xma I;
Sma I; Kpn I; Sst I; and, EcoRI. The composition of the mp18 and
mp19 vectors are identical, with the exception that the order of
the above-identified restriction sites are reversed in the mp19
vector so that both strands of a DNA segment may be conveniently
sequenced with the two vectors. The mp18 and mp19 vectors, with
fragments of the N4 and N1 clones inserted therein, were used to
transform E. coli JM103 and JM105 of the strain K12 (Bethesda
Research Laboratories, Bethesda, Md.) to produce relicate
single-stranded DNA templates containing single-stranded inserts of
the sense and antisense strands.
The synthetic universal primer: 5'-CCCAGTCACGACGTT-3' (P-L
Biochemicals, Milwaukie, Wis.), was annealed to the single-strand
DNA templates and used to prime DNA synthesis as described above at
page 23. Thereafter, the extension fragments were size-separated by
gel electrophoresis and autoradiographed from which the nucleotide
sequences of the fragments were deduced.
An additional primer was employed to prime synthesis from an
intermediate location along the sense strands of the N4 and N1
clones. A primer having the composition: 5'-GTGACACCTCAACCTGA-3',
corresponds to nucleotides 262 through 278 (FIG. 2). The
composition of this primer strand was established from the
sequencing information previously obtained by the sequencing of the
N4 and N1 clones from their 5' termini with the universal primer.
An additional synthetic primer of the composition:
5'-TGTGACGAGGCAGGAAG-3' (corresponding to nucleotides 613 through
629 in FIG. 2) was used in sequencing the antisense strands between
the Xba I and Sst I sites of the N4 and N1 clones. By the above
"walk down" method, the strands of the N4 and N1 clones were
sequenced from their 5' terminals to their Xba I sites in an
overlapping, redundant manner thereby confirming the nucleotide
sequence of these clones. It is to be understood that other
synthetic primers could have been employed to initiate chain
extensions from other locations along the N4 and N1 clones, without
departing from the scope of the present invention.
Deoxyadenosine 5' (alpha-[.sup.35 S] thio) triphosphate
(hereinafter "dATP [alpha-.sup.35 S]") was used as the radioactive
label in the dideoxy sequencing reactions. Also, rather than using
the gel set forth at page 36 of the Amersham Handbook, a 6%
polyacrylamide gel was employed (6% polyacrylmide gel, 0.4 mm
thick, containing 7M, urea 100 mM Tris borate (pH 8.1), and 2 mM
EDTA).
As noted above, the nucleotide sequences of the N4 and N1 clones
from their 5' terminals to the Xba I sites are illustrated in FIG.
2. This segment of DNA was found to include the coding regions of
the clones. The nucleotides are numbered from the position of the
initiator methionine codon. The corresponding amino acids, as
determined by the nucleotide sequence and by protein sequence
analysis, are set forth above the appropriate codons. The amino
acid composition of the IL-2 receptor gene extends from the mature
NH.sub.2 -terminus of the IL-2 receptor molecule, i.e., the Glu
residue, as marked with a star in FIG. 2 (from which the numbering
of the amino acid residues begins), to the Ile residue (No. 251)
immediately preceding the termination codon TAG. Various
restriction enzyme cleaving sites are also indicated in FIG. 2. The
portions of the coding regions of the N4 and N1 clones in FIG. 2
are illustrated as boxed regions in FIG. 1, with the solid box
portions indicating substantially corresponding portions of the
clones and the open box portion depicting the 216 base pair
sequence only present in the N4 clone.
EXAMPLE 14
Expression of Mature IL-2 Receptor In Mammalian Cells
The coding regions of the N4 and N1 clones were inserted into a
plasmid vector for transfection of mammalian cells to ascertain
whether either coding region encodes a functional IL-2 receptor.
The transfected cells were assayed for expression of IL-2 receptor
by their ability to bind either labeled IL-2 or a labeled
monoclonal antibody directed against the IL-2 receptor, i.e.,
2A3-A1H monoclonal antibody. Hybrid cDNAs containing the coding
regions of the N4 and N1 clones (illustrated in FIG. 3A),
designated as pN1/N4-S and pN1/N4-X, respectively, were inserted
into the pMLSV phage vector, shown as a circle, to produce the
plasmids pMLSV-N1/N4S and pMLSV-N1/N4X, respectively.
The pMLSV vector was derived principally from SV40 whose genome
consists of a single, small covalently closed DNA molecule that has
been entirely sequenced, Fiers et al., supra, and Reddy et al.,
supra. The pMLSV vector is composed of four parts, including the
stippled box portion shown in FIG. 3A which contains the control
region of the SV40 plasmid (including the origin of DNA
replication, enhancer elements and early and late promoters) (SV40
coordinates 5107-208). This vector portion was originally derived
from the psV2-dhfr vector as a Hind III-Pvu II fragment, Subramani
et al., 1 Mol. Cell Biol. 854-864 (1981) and Lebowitz and Weissman,
87 Current Topics in Microbiology and Immunology 43 (1979). For use
in the pMLSV plasmid, the Pvu II site was converted into a BamHI
site and the Hind III site converted to Xba I site.
Downstream from the early promoter, the pMLSV vector includes a
synthetic polylinker of the composition: ##STR1## This polylinker
has Xba I cohesive termini and contains the following restriction
sites: Hind III; Kpn I; Pvu II; Pst I; Bgl II; Xho I; EcoRI; Cla I;
and, Xba I. The hatched box portion of the plasmid contains the
SV40 small t antigen donor and acceptor splice junctions (SV40
coordinates 4035-4656) and the SV40 polyadenylation signal (SV40
coordinates 2469-2706), originally derived from the pSV2-dhfr
plasmid as a Bgl II-BamHI fragment, Subramani, supra. The Bgl II
site was converted to a Xba I site for correspondence with the
adjacent terminal of the synthetic polymer.
The long thin line portion of the pMLSV plasmid is derived from the
plasmid pML2d, a derivative of plasmid pBR322, that lacks sequences
inhibitory to DNA replication in mammalian cells, Sarver et al., 79
Proc. Natl. Acad. Sci. (USA) 7147-7151 (1982); and, Luskey and
Botchan, 293 Nature 79-81 (1981).
Because it is known that the presence of dG-dC tails at the 5' end
of a cDNA insert can inhibit its expression in mammalian cells (for
instance, see Riedel et al., 3 EMBO Journal 1477 (1984)), hybrid
cDNAs were constructed by combining portions of the N4 and N1 cDNA
clones with the sequences derived from the N4 clone shown as open
boxes and the sequences derived from the N1 clone shown as solid
boxes in FIG. 3A. As illustrated, the pN1/N4S hybrid fragment
includes the portion of the N4 clone from the BamHI site to the 5'
Sst I site to which is attached the 5' Pst I-Sst I fragment from
the N1 clone, and thus contains the coding region of the N4 clone.
The pN1/N4X hybrid cDNA contains a 5' Pst I-Xba I fragment from the
N1 clone and a Xba I-BamHI fragment from the N4 clone, and thus
contains the coding region of the N1 clone. It will be appreciated
that both of the hybrid cDNAs take advantage of the "natural" Pst I
site in the 5' prime noncoding region of the N1 clone that lacks
tails. The pN1/N4-S and pN1/N4-X hybrid cDNAs having Pst I and
BamHI cohesive ends were inserted into the Pst I and Bgl II sites
of the pMLSV plasmid by standard techniques, for instance, as
detailed in Maniatis et al., supra, to form plasmid vectors
pMLSV-N1/N4-S and pMLSV-N1/N4-X, respectively. The pMLSV-N1/N4-S
plasmid vector has been deposited with the ATCC under Accession No.
39890.
The plasmids as prepared above were transfected into COS-7 monkey
kidney cells (ATCC, Rockville, Md.) by standard techniques, for
instance, by essentially using the procedures described by Lauthman
and Magnusson, 11 Nucl. Acid Res. 1295 (1983). Monolayers of COS-7
cells (10.sup.6 cells per 10 cm plate) were washed twice with
Tris-buffered saline ("TBS") and exposed to 10 ug of hybrid
pMLSV-pN1/N4-S or pMLSV-pN1/N4-X DNA per plate in 1 ml TBS
containing 500 ug/ml DEAE-Dextran (molecular weight
5.times.10.sup.5 ; Sigma Chemical Company, St. Louis, Mo.) for 30
minutes at room temperature. The cells were washed once more with
TBS and fed with growth medium (Dubecco's Modified Eagle's Medium
with 10% (v/v) fetal bovine serum) containing 100 uM chloroquine
(St. Louis, Mo.). After incubation for five hours at 37.degree. C.,
the medium was replaced by growth medium without chloroquine. The
cells were then incubated at 37.degree. C. for 48 hours, after
which time they were harvested by scraping.
The transfected COS-7 cells were screened for IL-2 receptor
expression by ascertaining the ability of the cells to bind to
.sup.125 I-labeled anti-IL-2 receptor antibody 2A3-A1H (FIG. 3B)
and also to .sup.125 I-labeled IL-2 (FIG. 3C). The 2A3-A1H
monoclonal antibody was prepared and radiolabeled to a specific
activity of 9.8.times.10.sup.14 cpm/mM, as described above.
Purified IL-2 was radiolabeled using the Enzymobead radioiodination
reagent (BioRad Laboratories, Richmond, Calif.) essentially by the
manufacturer's specifications. Fifty ul aliquotes of IL-2
(5.times.10.sup.6 units) in 65% acetonitrile and TFA (pH 2.1) were
combined with 50 ul of 0.2M sodium phosphate (pH 7.2) and then the
acetonitrile evaporated under nitrogen. Fifty ul of Enzymobead
reagent, 10 ul of .sup.125 I (1 mCi) and 10 ug of 2.5%
Beta-D-glucose (BioRad Laboratories, Richmond, Calif.) were added
and then the mixture incubated at 25.degree. C. for 10 minutes.
Twenty ul of 25 mM sodium azide and 10 ul of sodium metabisulfite
(5 mg/ml) were then added, and after 5 minutes of incubation at
25.degree. C., iodinated IL-2 was separated from unbound .sup.125 I
by chromatography on a 2 ml column of Sephadex G-25 equilibrated in
0.05M sodium phosphate (pH 7.2) containing 0.1% v/v BSA and eluted
with this same buffer. Based on an initial biologic specific
activity for IL-2 of 1.times.10.sup.6 units/ug protein, the
radiolabeled preparation had an estimated specific activity of
1.times.10.sup.15 cpm/mM.
The binding assays were performed as described in Dower et al., 132
J. Immunol. 751 (1984). COS-7 cells (1.2.times.10.sup.6) were
incubated with either 5.times.10.sup.-9 M.sup.125 I-2A3-A1H
monoclonal antibody or 1.3.times.10.sup.-8 M.sup.125 -IL-2 in a
final volume of 150 ul of binding medium for 30 minutes at
37.degree. C. Nonspecific binding was measured in the presence of
1000-fold molar excess of unlabeled 2A3-A1H monoclonal antibody or
150-fold molar excess of unlabeled IL-2. Replicate 70 ul aliquots
of the above incubation mixtures were centrifuged through phthalate
oil to separate the .sup.125 I bound to COS-7 cells from the
unbound cells (.sup.125 I labeled IL-2 or 2A3-A1H).
The results of the .sup.125 I binding assay are set forth in panels
B and C of FIG. 3. As shown, only the pMLSV-N1/N4-S transfected
COS-7 cells bound to the labeled IL-2 and labeled 2A3-A1H
monoclonal antibody. Neither the pMLSV-N1/N4-X transfected COS-7
cells or the mock-transfected COS-7 control cells showed any
specific binding of IL-2 or the monoclonal anti-IL-2 receptor
antibody. Since only the pMLSV-N1/N4-S hybrid contains the N4
coding region, the functional IL-2 receptor protein is encoded
thereby.
As will be apparent to those skilled in the art to which the
invention is addressed, the present invention may be carried out by
using cell lines, culture media, culture media additives, culture
conditions, assays, antibodies, purification restriction mapping
and sequencing techniques, and chromatography columns other than
those specifically discussed above without departing from the
spirit or essential characteristic of the invention. The particular
materials and processes described above are therefore to be
considered in all respects as illustrative and not restrictive. The
scope of the present invention is as set forth in the appended
claims rather than being limited to the examples of the methods and
procedures set forth in the foregoing description.
* * * * *