U.S. patent application number 11/784789 was filed with the patent office on 2007-08-16 for biologically active synthetic thyrotropin and cloned gene for producing same.
Invention is credited to Bruce D. Weintraub, Fredric E. Wondisford.
Application Number | 20070190577 11/784789 |
Document ID | / |
Family ID | 23139856 |
Filed Date | 2007-08-16 |
United States Patent
Application |
20070190577 |
Kind Code |
A1 |
Wondisford; Fredric E. ; et
al. |
August 16, 2007 |
Biologically active synthetic thyrotropin and cloned gene for
producing same
Abstract
Substantially pure recombinant TSH has been prepared from a
clone comprising complete nucleotide sequence for the expression of
the TSH. Diagnostic and therapeutic applications of the synthetic
TSH are described.
Inventors: |
Wondisford; Fredric E.;
(Rockville, MD) ; Weintraub; Bruce D.; (Rockville,
MD) |
Correspondence
Address: |
KNOBBE, MARTENS, OLSON & BEAR, LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
23139856 |
Appl. No.: |
11/784789 |
Filed: |
April 10, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11543498 |
Oct 5, 2006 |
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11784789 |
Apr 10, 2007 |
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11376778 |
Mar 15, 2006 |
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11543498 |
Oct 5, 2006 |
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11148604 |
Jun 9, 2005 |
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11376778 |
Mar 15, 2006 |
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10737469 |
Dec 16, 2003 |
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11148604 |
Jun 9, 2005 |
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09892266 |
Jun 27, 2001 |
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10737469 |
Dec 16, 2003 |
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09569141 |
May 11, 2000 |
6284491 |
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09892266 |
Jun 27, 2001 |
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08310923 |
Sep 22, 1994 |
6117991 |
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09569141 |
May 11, 2000 |
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08110639 |
Aug 23, 1993 |
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08310923 |
Sep 22, 1994 |
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07882231 |
May 8, 1992 |
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08110639 |
Aug 23, 1993 |
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07295934 |
Jan 11, 1989 |
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07882231 |
May 8, 1992 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
A61P 43/00 20180101;
G01N 33/57407 20130101; A61P 35/00 20180101; A61K 38/00 20130101;
C07K 16/26 20130101; G01N 33/76 20130101; C07K 14/59 20130101; G01N
2500/00 20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. Substantially pure, biologically active recombinant human
thyrotropin (rTSH).
2. The thyrotropin of claim 1 labeled isotopically with .sup.131I,
.sup.125I, chemiluminiscently or fluorescently.
3. The thyrotropin of claim 1 produced by recombinant genetic
process using constructs with gene elements that enhance
thyrotropin production.
4. A clone comprising complete nucleotide sequence for the
expression of the thyrotropin of claim 1 in a suitable expression
vector.
5. The clone of claim 4 further comprising first untranslated exon
of TSH-.beta..
6. A method for producing TSH, comprising: (a) allowing expression
of TSH by the clone of claim 4 in a suitable expression vector; and
(b) then recovering substantially pure TSH by conventional
purification and isolation methodology.
7. A method for producing TSH, comprising: (a) allowing expression
of TSH by the clone of claim 5 in a suitable expression vector; and
(b) then recovering substantially pure TSH by conventional
purification and isolation methodology.
8. A method for producing TSH, comprising: (a) allowing expression
of TSH by the clone of claim 4, wherein TSH.alpha. is about 3 to 5
times in excess of TSH.beta.; (b) then recovering substantially
pure TSH by conventional purification and isolation
methodology.
9. A method for producing TSH, comprising: (a) allowing expression
of TSH by the clone of claim 5, wherein TSH.alpha. is about 3 to 5
times in excess of TSH.beta.; (b) then recovering substantially
pure TSH by conventional purification and isolation
methodology.
10. A TSH antagonist produced by a mutant of the clone of claim
4.
11. A TSH agonist produced by a mutant of the clone of claim 4.
12. A kit comprising containers separately containing: (a)
universal standard of substantially pure unlabeled rTSH; (b)
substantially pure, labeled rTSH; (c) antibodies against purified
rTSH; and (d) instructional material describing the use of reagents
(a), (b) and (c).
13. Anti-rTSH antibodies without interfering cross-reactivity with
non-TSH hormones.
14. A method for determining the level of TSH in a sample,
comprising reacting an aliquot of a sample in which the amount of
TSH is to be determined with the antibodies of claim 13 and
comparing the level of antibody reactivity with a predetermined
standard antibody-rTSH reactivity curve to determine the amount of
TSH present in said sample.
15. A method of diagnosing the extent of thyroid cancer, comprising
administering rTSH of claim 1 to a patient to maximize .sup.131I
uptake, and then administering a visualizing dose of .sup.131I to
said patient; and then visualizing the cancer by standard
visualizing means.
16. A method for treating thyroid cancer, comprising administering
therapeutic regimen of a combination of rTSH of claim 1 and
.sup.131I or .sup.131I-labeled rTSH to a patient afflicted with
thyroid cancer.
17. A method of blocking TSH activity, comprising inhibiting TSH
activity by competitive amount of the antagonist of claim 10.
18. A method of stimulating TSH activity, comprising inducing TSH
production by the agonist of claim 11.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
11/543,498 filed Oct. 5, 2006, which is a is a continuation of
application Ser. No. 11/376,778 filed Mar. 15, 2006, now abandoned,
which is a continuation of application Ser. No. 11/148,604 filed
Jun. 9, 2005, now abandoned, which is a continuation of application
Ser. No. 10/737,469 filed Dec. 16, 2003, now abandoned, which is a
continuation of application Ser. No. 09/892,266 filed Jun. 27,
2001, now abandoned, which is a continuation of application Ser.
No. 09/569,141 filed May 11, 2000, now U.S. Pat. No. 6,284,491,
which is a continuation of application Ser. No. 08/310,923 filed
Sep. 22, 1994, now U.S. Pat. No. 6,117,991, which is a continuation
of application Ser. No. 08/110,639 filed Aug. 23, 1993, now
abandoned, which is a continuation of application Ser. No.
07/882,231 filed May 8, 1992, now abandoned, which is a
continuation of application Ser. No. 07/295,934 filed Jan. 11,
1989, now abandoned, all of which are hereby expressly incorporated
by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention is related generally to the isolation
and characterization of new genes and proteins. More particularly,
the present invention is related to providing isolated,
substantially pure, biologically active human thyrotropin (hTSH)
synthesized by a cloned gene.
BACKGROUND OF THE INVENTION
[0003] Thyrotropin (TSH) is a pituitary peptide hormone which
regulates important body functions. However, heretofore there was
no stable, reliable and economic means of synthesizing this
important hormone.
SUMMARY OF THE INVENTION
[0004] It is, therefore, an object of the present invention to
provide biologically active, synthetic human thyrotropin in
substantially pure, isolated form.
[0005] It is another object of the present invention to provide a
cloned gene which directs the expression of biologically active
human thyrotropin in a suitable vector.
[0006] It is a further object of the present invention to provide
an assay kit for measuring thyroid-stimulating hormone as well as
other thyrotropin substances such as thyroid-stimulating
immunoglobulins and the like.
[0007] It is a further object of the present invention to provide a
method of diagnosing and treating human thyroid cancer.
[0008] Other objects and advantages will become evident from the
following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] These and other objects, features and many of the attendant
advantages of the invention will be better understood upon a
reading of the following detailed description when considered in
connection with the accompanying drawings wherein:
[0010] FIG. 1 shows schematic construction of the expression
vectors. pSV2.G and pAV2 are pBR322 derived plasmids with the
origin of replication (ori) and ampicillin resistance gene (amp r)
as shown. pSV2.G contains the early promoter of SV40 upstream of
the HindIII cloning site, rabbit .beta.-globin cDNA, and
poly-adenylation site/intron of SV40. pAV2 has the entire
adenovirus-associated virus genome (4.7 kb) with its three
promoters P5, P19, P40, and polyadenylation site. The HindIII
cloning site is downstream of the P40 promoter. Human TSH.beta. and
hCG.alpha. was inserted into the HindIII site of either plasmid,
forming pAV2-hTSH.beta., pAV-hCG.alpha., pSV2.GhTSH.beta., and
pSV2.G-hCG.alpha..
[0011] FIG. 2 shows Northern blot analysis of transfected 293 and
COS cells. Total cellular RNA was separated on a 1%
agarose-formaldehyde gel and transferred to a nylon membrane. Forty
micrograms of total RNA from a control of transfected cell culture
were applied to each lane. Human CG.alpha. and hTSH.beta. were
abbreviated .alpha. and .beta. in construct names and in other
figures. Cells that were not transfected are labeled control. Cells
transfected with a calcium phosphate precipitate lacking DNA are
labeled mock. Lanes 1-4 are total RNAs derived from 293 cells;
lanes 5-9 are RNAs derived from COS cells. The migration position
of an RNA standard in kilobases and hTSH.beta. mRNA from human
pituitary is shown to the left of the autoradiograph. Below the
autoradiograph is a simplified version of FIG. 1 showing the pAV2
and pSV2.G plasmid as a single line, the promoters as blackened
circles, the 2.0 kb hTSH.beta. genomic fragment as a box containing
two exons (blackened regions) and known polyadenylation signal-site
sequences as open arrowheads. Below each construct, pAV2-.beta. and
pSV2.G-.beta., is the predicted RNA initiating at the specified
promoter, and splicing as shown. Solid arrowheads, poly(A) tails.
Predicted size in kilobases (kb) is shown to the right of each mRNA
species.
[0012] FIG. 3 shows the results of gel chromatography. Cell medium
from 293 cells transfected with
pAV2-hCG.alpha./pAV2-hTSH.beta./pVARNA was chromatographed on a
Sephadex G-200 fine column. In addition, standard preparations of
hTSH, hCG.alpha., and hTSH.beta. (described in the text) were
chromatographed on the same column. RIA of human .alpha. and
TSH.beta. and IRMA for hTSH was done on each 1.5-ml fraction.
Elution position of bovine thyroglobulin (void, V.sub.o), BSA
(67,700 (67 k)) and ovalbumin (45,000 (45 k)) is marked, as well as
those of the standard preparations of hTSH, hCG.alpha., and
hTSH.beta..
[0013] FIG. 4 shows the results of human TSH IRMA. A highly
sensitive and specific hTSH IRMA was performed on two pituitary
hTSH standards. World Health Organization 80/558 (WHO STD) and NIH
I-6 (1-6 STD), and the medium from 293 cells after transfection
with pAV2-hCG.alpha., pAV2-hTSH.beta., and pVARNA
(pAV2.alpha./.beta./pVARNA). A logit transformation of assay
binding was plotted vs. arbitrary units of sample volume added to
the assay.
[0014] FIG. 5 shows the results of in vitro bioassay of hTSH in rat
thyroid cells. The human pituitary TSH standards and medium from
transfected 293 cells used in this assay are defined in the legend
to FIG. 4. This in vitro bioassay measures TSH stimulated .sup.125I
uptake into rat thyroid cells (FRTL5). Iodide trapping by pituitary
standards and medium from a transfected culture are normalized to
TSH immunoactivity in an IRMA.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0015] The above and various other objects and advantages of the
present invention are achieved by the cloning of complete
nucleotide sequence which directs the synthesis of biologically
active human thyrotropin in a suitable expression vector and
isolating substantially pure form of the synthesized hormone.
[0016] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned hereunder are incorporated herein by
reference. Unless mentioned otherwise, the techniques employed
herein are standard methodologies well known to one of ordinary
skill in the art.
[0017] The term "substantially pure" as used herein means as pure
as can be obtained by employing standard conventional purification
techniques known in the art.
[0018] The term "biologically active" as used herein means that the
recombinant hormone, even though not identical in physical or
chemical structure or composition as the naturally occurring
hormone, yet is functionally equivalent thereto.
Materials and Methods
Materials
[0019] Restriction and modifying enzymes were obtained from
Bethesda Research Laboratories (Gaithersburg, Md.) and Pharmacia
(Piscataway, N.J.). .sup.32P and .sup.35S compounds were purchased
from both DuPont New England Nuclear (Boston, Mass.) and
Amersham/Searle Corporation (Arlington Heights, Ill.). Gene Screen
and Gene Screen Plus membranes (New England Nuclear) were used in
all DNA and RNA transfer procedures. A transformation competent
strain of Escherichia coli, HB101, was obtained from Bethesda
Research Laboratories and used in all transformations.
Oligonucleotides were purchased from the Midland Certified Reagent
Company (Midland, Tex.). Cloning and propagation of DNA was done in
accordance with NIH guidelines. Sephadex G-200 fine and
concanavalin A-Sepharose were obtained from Pharmacia Fine
Chemicals. .alpha.-Methyl glucoside and .alpha.-methyl mannoside
were purchased from Sigma (St. Louis, Mo.). Human TSH, hCG.alpha.,
and hTSH.beta. were provided by the NIDDK National Hormone and
Pituitary Program (Bethesda, Md.). Protein standards were purchased
from Sigma or Pierce Chemical Co. (Rockford, Ill.).
Genomic Screening
[0020] Independent recombinant phage clones (1.times.10.sup.6) of
an EMBL3 human genomic leukocyte library were screened for the
presence of human TSH.beta. using a radiolabeled mouse TSH.beta.
cDNA obtained from W. Chin, Brigham and Women's Hospital, Harvard
Medical School, Boston, Mass., and two separate clones were
identified. A 34 base oligonucleotide, with the same sequence as
the first 34 bases of the 5'-untranslated sequence of bovine
TSH.beta. cDNA, was 5'-end labeled with [.tau.-.sup.32P]ATP to a
specific activity of 5-8.times.10.sup.6 cpm/picomol using
polynucleotide kinase; mouse TSH.beta. cDNA was [.alpha.-.sup.32P]
dCTP labeled with a random primer to a specific activity of
1-5.times.10.sup.9 cpm/.mu.g. Both were used to probe Southern
blots of restriction digests of one clone.
Subcloning and Sequencing
[0021] Genomic fragments were subcloned into pUC18 and mp13 to
facilitate restriction mapping and sequencing using the dideoxy
chain termination method of Sanger (Sanger et al. 1977 Proc Natl
Acad Sci USA 74:5463-5467).
Expression Vectors
[0022] A 621 bp hCG.alpha. cDNA (obtained from J. Fiddes,
California Biotechnology Inc., Palo Alto, Calif.) was inserted
downstream of the early promoter of SV40 in pSV2.G (obtained from
B. Howard, NIH, Bethesda, Md.) (Gorman et al. 1982, Mol Cell Biol
2:1044-1051) or the P40 promoter of adeno-associated virus in pAV2
(Laughlin et al. 1983, Gene 23:65-73) at the HindIII site forming
pSV2.G-hCG.alpha. and pAV2-hCG.alpha. (FIG. 1). HindIIII linkers
were ligated to a 2.0 kb PvuII fragment of the hTSH.beta. gene
containing 277 bp of 5'-intron, both coding exons, a 450 bp intron,
and approximately 800 bp of 3'-flanking DNA. It was inserted into
the same HindIII sites as hCG.alpha. forming pSV2.G-hTSH.beta. and
pAV2-hTSH.beta. (FIG. 1). All plasmids were subjected to multiple
restriction enzyme digestions to confirm the presence of only one
insert in the proper orientation.
Cell Culture
[0023] Adenovirus transformed human embryonal kidney cells (293
cells) and SV40 transformed monkey kidney cells (COS cells) were
grown in a modified minimal essential (MEM) and Dulbecco's modified
Eagle's medium, respectively. Both media were supplemented with 10%
fetal bovine serum, 4.4 mM L-glutamine, 100 U/ml penicillin, 100
.mu.g/ml streptomycin, and 250 ng/ml amphotericin B. Twenty-four
hours before transfection, the cells were replated on 100-mm dishes
at the same density (5.times.10.sup.5). On the day of transfection
fresh medium was added to the cells 4 h before transfection.
Transfection
[0024] All transfections were performed using the calcium phosphate
precipitation method (Graham et al. 1973, Virology 52:456-467). The
precipitate was applied for 4 h, the cells were washed, and fresh
medium was added. Total RNA was isolated according to the method of
Cathala et al. 1983, DNA 2:329-335. The pAV2 plasmids were
transfected into both 293 and COS cells in Exp 1 and into only 293
cells in Exp 2. The pSV2.G plasmids were only transfected into COS
cells. When either the .alpha.- or .beta.-subunit was transfected
alone into cells, 15 .mu.g purified plasmid were applied to each
plate. When both the .alpha.- and .beta.-subunit were
cotransfected, 9 .mu.g each purified plasmid were applied to
one-plate. In some cases, cells were cotransfected with pVARNA.
pVARNA consists of an adenovirus type 2 DNA HindIII B fragment
containing the genes for VA.sub.x and VA.sub.xx inserted into the
HindIII site of pBR322 (obtained from Ketner, Department of
Biology, Johns Hopkins University, Baltimore, Md.). VA.sub.x RNA
stimulates translation by inhibiting phosphorylation and
inactivation of the a subunit of eucaryotic initiation factor 2
(Akusjarvi et al. 1987, Mol Cell Biol 7:549-551).
RNA Analysis, RIA, and IRMA
[0025] Northern blot analysis of total RNA from transfections was
performed using standard methods (Maniatis et al. 1982, Molecular
Cloning, ed 1, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., p 202) and manufacturer's specifications. Common human
.alpha.-subunit RIA, hTSH.beta.-subunit RIA, and hTSH IRMA were
performed in duplicate on the medium from each transfected culture
(McBride et al. 1985 Clin Chem 31:1865-1867; Kourides et al. 1974
Endocrinology 94:1411-1421). The sensitivities of the assays were
less than 0.03 ng/ml, less than 0.03 ng/ml, and less than 0.06
ng/ml, respectively. Cross-reactivity between the corresponding
subunit and hTSH, at the measured concentration, was less than 5%
in the common .alpha. and less than 2% in the hTSH.beta. RIA
(Kourides et al., supra). In addition, at the measured free subunit
concentrations, the hTSH IRMA exhibited less than 1%
cross-reactivity (data not shown).
Gel and Lectin Affinity Chromatography
[0026] The apparent mol wt of hTSH synthesized in 293 cells was
determined by gel chromatography on a 1.5.times.90-cm Sephadex
G-200 fine column calibrated during each chromatography run with
five protein standards (bovine thyroglobulin, BSA, ovalbumin,
bovine chymotrypsinogen A, and whale myoglobin). The column was
equilibrated and run at 4.degree. C. in a buffer containing 0.12 M
sodium chloride, 0.1 M borate, and 0.02% (wt/vol) sodium azide, pH
7.4. Two milliliters of fresh MEM medium containing 100 .mu.UhTSH
(WHO 80/558). 100 ng hCG.alpha. (CR-119), and 100 ng hTSH.beta.
(APP-3929.beta.) were applied during chromatography of standard
preparations. The column was washed and then 2 ml MEM medium from
293 cells transfected with pAV2-hCG.alpha./pAV2-hTSH.beta./pVARNA
were applied. Fractions of 1.5 ml were collected at a flow rate of
6 ml/h.
[0027] The binding of hTSH synthesized in 293 cells to concanavalin
A-Sepharose was also determined using methods previously described
(Gesundheit et al. 1987, J Biol Chem 262:5197-5203). Samples were
applied to lectin column, and 2-ml fractions were collected at a
flow rate of 10 ml/h. Human TSH IRMA was common human .alpha.- and
hTSH.beta.-subunit RIAs were performed on fractions from gel and
lectin chromatography. Recovery of hTSH and its free subunits was
generally greater than 90% from chromatography.
TSH Bioassay
[0028] Thyrotropic bioactivity was measured as the ability to
stimulate the uptake of .sup.125I into rat thyroid cells (FRTL5) in
accordance with the procedure of Dahlberg et al. 1987 J Clin Invest
79:1388-1394. This assay measures human, rat, and bovine
thyrotropin but not gonadotropins or free .alpha.- or
TSH.beta.-subunits. Sample determinations were performed in
duplicate and compared to two pituitary hTSH standards (World
Health Organization 80/558 and National Institutes of Health 1-6).
Results are expressed as microunits per ml; one microunit of WHO
80/558 is equivalent to 0.09 ng of NIH 1-6 purified hTSH
(unpublished data).
Statistics
[0029] Significant differences in immunoassay of cell media from
various control and transfected cultures were determined using
Student's t test.
RESULTS
Human TSH.beta. Gene
[0030] A 17 kb genomic fragment was isolated by screening
1.times.10.sup.6 recombinant phage clones. A restriction map (FIG.
1) was constructed using Southern blots of phage DNA hybridized
with both a mouse TSH.beta. cDNA probe (lacking 5'-untranslated
sequences) and a 34 bp bovine 51-untranslated sequence probe. Two
coding exons are separated by a 450 bp intron and the sequence is
identical to the published partial sequence (Hayashizaki et al.
1985 FEBS Lett 188:394-400) (data not shown). However, the complete
coding sequence was not heretofore known.
Transfection
[0031] Two experiments were performed to compare the level of mRNA
and protein production between the most active adeno-associated
virus promoter, P40, and the early promoter of SV40.
[0032] Table 1 shows the RIA and IRMA assay results from these two
experiments. Interestingly, the 293 cells synthesized small amounts
of free .alpha.-subunit (control) whose levels were increased
approximately 10-fold in a transfection with the calcium phosphate
precipitate but without DNA (mock) (P<0.0005). While neither
transfection with pAV2-hCG.alpha. nor pVARNA increased
.alpha.-production above the level of a mock transfection, the
combination increased free .alpha.-levels 3- to 5-fold
(P<0.0005). Thus exogenous sources of the human .alpha.-subunit
were clearly important in mediating this increase. The same pattern
of pVARNA increasing protein production was seen when
pAV2-hTSH.beta. was transfected. 293 cells do not produce
hTSH.beta. so that the medium of cells exposed to a mock or pVARNA
transfection did not have measurable hTSH.beta.. Only the 293 cells
exposed to pAV2-hTSH.beta. produced hTSH. When the .beta. plasmid
was transfected alone the hTSH formed was due to combination with
endogenous .alpha.. Cotransfection with both .alpha.- and
.beta.-plasmids, though, increased hTSH levels 1.5- to 2-fold
(P<0.05).
[0033] COS cells synthesized neither free .alpha. nor .beta. but
could synthesize hTSH.beta. and hTSH when transfected with the
appropriate plasmids. The levels of protein production were 10- to
100-fold less than in 293 cells and were only measurable when the
pVARNA plasmid was also transfected. Regardless of whether pAV2 or
PSV2.G was used, protein levels were barely if at all measurable
without the pVARNA plasmid.
[0034] FIG. 2 shows a Northern blot of total cellular RNA
hybridized with the mouse TSH.beta. cDNA probe. In 293 cells,
hTSH.beta. message was not detected from nontransfected (control)
or from mock transfected cells. However, three RNA species of 2.3
kb. 1.6 kb, and 650 bases were noted after transfection of
pAV2-hTSH.beta. and pVARNA (lane 3). These three bands are the same
size as those predicted from pAV2-hTSH.beta. if transcription began
at all three adeno-associated viral promoters (FIG. 2). The mRNA of
650 bases presumably represent a properly spliced hTSH.beta.
message. Lane 4 shows the same three bands but at lesser intensity
when pAV2-hCG.alpha., pAV2-hTSH.beta., and pVARNA were
cotransfected. This reduction in signal intensity seen in lane 4
may have been due to the reduction in the amount of pAV2-hTSH.beta.
transfected from 15 .mu.g to 9 .mu.g.
[0035] Control and mock transfected COS cells also did not contain
hTSH.beta. message. When pSV2.G-hTSH.beta. was transfected (lanes
7-9), a major band of 900 bases and a minor band of 3.0 kb were
seen. Without being bound to any theory, it is postulated that the
900 base species could represent a mRNA with the 277 bases of
5'-intron remaining, while the 3.0 kb species could represent read
through of the hTSH.beta. poly-adenylation signal-site and use of
the polyadenylation signal-site of pSV2.G (See FIG. 2).
[0036] Specific human .alpha. mRNA transcripts of appropriate size
analogous to the hTSH.beta. mRNA above were observed in cells
transfected with pAV2-hCG.alpha. (data not shown). Since the main
object of this invention is protein expression, the relative
contribution of human a mRNA from endogenous vs. exogenous
(pAV2-hCG.alpha.) sources in 293 cells was not determined. However,
the data suggest that the high level of free .alpha.-subunit
observed after transfection with pAV2-hCG.alpha./pVARNA is most
likely due to mRNA from exogenous sources.
Gel and Lectin Affinity Chromatography
[0037] The apparent molecular weight of hTSH and its subunits
synthesized in 293 cells after transfection with
pAV2-hCG.alpha./pAV2-hTSH.beta./pVARNA was determined on a G-200
Sephadex column (FIG. 3). In addition, standard preparations of
hTSH, hCG.alpha., and hTSH.beta. were chromatographed on the same
column. Internal protein standards had identical elution patterns
between runs as determined by optical density at 280 nm. In each
case, the apparent mol wt of synthetic hTSH and its subunits was
larger than its corresponding standard. Specifically, synthetic
hTSH displayed an apparent mol wt of 45.000 and was larger than
standard pituitary hTSH (apparent M.sub.r=40,000). This clearly
indicates that the recombinant TSH is not constitutively identical
to the natural product. The human .alpha. and hTSH.beta. from
transfection coeluted with the hTSH pituitary standard, and both
were larger than their respective standard subunit preparation.
However, in the case of free human .alpha.-subunit, the relative
contribution to this chromatography pattern of endogenous .alpha.
as compared to exogenous .alpha. from pAV2-hCG.alpha. cannot be
determined.
[0038] The binding pattern to concanavalin A-Sepharose of synthetic
hTSH from 293 cells as, compared to standard human pituitary hTSH
is shown in Table 2. Synthetic hTSH was glycosylated as indicated
by complete binding to concanavalin A-Sepharose. The different
elution pattern of standard vs. synthetic hTSH from the lectin
columns is indicative of a difference at least in carbohydrate
structure, again showing that the recombinant TSH (rTSH) is
distinctly different from the naturally occurring TSH.
Immunoactivity and Bioactivity
[0039] FIG. 4 shows that the hTSH produced in cell culture was
indistinguishable from two pituitary hTSH standards in an assay
involving two antibodies directed at different epitopes of the hTSH
heterodimer (McBride et al., supra). The slopes were parallel over
the entire range of values. FIG. 5 shows the same hTSH in a
.sup.125I trapping in vitro TSH bioassay compared to the same
pituitary hTSH standards. The in vitro bioassay of standard
pituitary hTSH or, the cell culture product from 293 cells
(pAV2-hCG.alpha./pAV2-hTSH.beta./pVARNA) was normalized to
immunoreactivity in a hTSH immunoradiometric assay (IRMA) assay.
The dose response, and ED.sub.50 of the standards and cell culture
product were identical. In addition the cell culture product from
COS cells (pAV2-hCG.alpha./pSV2.beta./pVARNA) was biologically
active although the lower level of expression prevented
determination of a dose response curve.
[0040] In summary, a 17 kb genomic fragment of hTSH.beta. has been
isolated and both coding exons of this gene produced hTSH.beta. and
hTSH in a transient expression assay. This is the first report of
TSH from any species produced by gene transfection in cell culture.
The expression vectors of hTSH.beta. included only the two coding
exons, and not the 5'-untranslated exon of the gene (Wondisford et
al. Mol Endo 2(1):32-39, 1988).
[0041] Transient expression after gene transfection was used to
test both the early promoter of SV40 or the P40 promoter of
adeno-associated virus. The early promoter in COS cells produced
more mRNA than the P40 promoter in 293 cells regardless of whether
pVARNA was cotransfected. However, pVARNA clearly increased mRNA
levels in either vector system. This suggests that in addition to
increasing the rate of translation, pVARNA must either increase
transcriptional rate, RNA transport, or stability.
[0042] While the pSV2.G-hTSH.beta. expression vector produced
higher levels of hTSH.beta. mRNA than pAV2-hTSH.beta., this mRNA
was about 250 bases larger than that found in the human pituitary.
The 450 bp intron was certainly spliced out since this intron in
the mature message would have prevented hTSH.beta. protein
synthesis. Also, an mRNA of appropriate size was produced by
pAV2-hTSH.beta. indicating that the polyadenylation site in the
fragment must be active. Thus, the most likely reason for a larger
hTSH.beta. mRNA in COS cells was the lack of splicing of a 277 bp
intron fragment upstream of the first coding exon. Eighteen base
pairs downstream of the transcriptional start site of the P40
promoter is a consensus splice donor site which could explain why
the 277 bp intron fragment would be spliced out in the
adeno-associated virus vector.
[0043] The plasmid, pVARNA, increased protein production in either
vector system, but the P40 promoter in 293 cells led to expression
of between 10- to 100-fold more protein than the early promoter of
SV40 when cotransfected with pVARNA. This is most likely due to an
increased translational rate mediated by pVARNA as has been
previously demonstrated for expression of other mRNAs (Akusjarvi et
al. 1987 Mol Cell Biol 7:549-551). Of course, the possibility that
the larger mRNA from pSV2.G-hTSH.beta. contributed to the lower
protein levels from COS cells cannot be excluded
[0044] The hTSH produced in cell culture was functionally
indistinguishable from two pituitary hTSH standards in both a
highly specific IRMA and in vitro bioassay. It should be noted,
however, that the synthetic hTSH of the present invention was
larger in size than standard pituitary hTSH on gel chromatography.
Although it was glycosylated as indicated by complete binding to
concanavalin A, it displayed a somewhat different pattern on lectin
chromatography as compared to a standard hTSH preparation. The
larger mol wt of these synthetic glycoproteins as compared to
pituitary standards is most likely due to an altered glycosylation
pattern such as more sialylation. In the case of hTSH, this might
also reflect a .beta.-subunit containing the 118 amino acids
predicted from the nucleic acid sequence rather than the 112 found
in standard hTSH purified from postmortem human pituitaries.
[0045] Transient expression is more convenient than stable
integration in the analysis of a large number of expression
vectors. pAV2 and pVARNA now allow transient expression of hTSH in
293 cells at levels high enough to analyze protein and
glycosylation site structure-function relationships. Previously,
the only information about such relationships came from studies
involving chemical modifications of protein by iodination,
nitration, acetylation, and carboxymethylation (Pierce et al. 1981,
Annu Rev Biochem, Annual Reviews Inc., Palo Alto, Calif., pp
465-495) or inhibition of glycosylation by tunicamycin (Weintraub,
et al. 1980 J Biol Chem 255:5715-5723). The chemical groups could
themselves change protein conformation irrespective of the
alteration in amino acids they produce and inhibition of
glycosylation affects not only TSH but all cellular glycoproteins.
Site-directed mutagenesis of the hCG.alpha. cDNA and human
TSH.beta.-gene could directly address what regions are important
for protein conformation, subunit combination, receptor binding,
biological, activity, and metabolic clearance without introducing
chemical groups or unknown changes into the protein structure.
[0046] The availability of substantially pure rTSH now makes the
diagnosis and treatment of human thyroid cancer and the
determination of the level of TSH a reality.
[0047] Currently, the only available method to diagnose and treat
human thyroid cancer involves making patients hypothyroid and
allowing their own endogenous human TSH to rise after several weeks
to stimulate the uptake of .sup.131I into the cancer. Such
stimulation is used as a diagnostic test to localize the tumor by
scanning and is subsequently used to treat the cancer by giving
large doses of .sup.131I. All of the diagnostic tests and therapies
depend on high levels of human TSH. However, the technique of
producing endogenous hypothyroidism has disabling side effects
including lethargy, weakness, cardiac failure, and may also lead to
a rapid growth of the tumor over the several week period of
treatment. In contrast, if a desirable form of synthetic human TSH
were available, patients could be treated while they were euthyroid
by giving exogenous injection of the TSH. However, presently it is
not feasible to give exogenous TSH because there is not enough
natural product from available human pituitaries collected at
autopsies. Furthermore, even if available, the human pituitaries
have been found to be contaminated with viruses and the National
Pituitary Agency has forbidden the use of the natural product for
any human diagnostic or therapeutic studies. This is true for all
human pituitary hormones including human growth hormone which is
now exclusively marketed as a synthetic product. However, the
technology that was applicable for human growth hormone (a
non-glycoprotein) is not at all applicable for human TSH (a
glycoprotein hormone of two glycosylated subunits). As has been
described herein supra, only the methodology described herein
relating to transfection and proper glycosylation of each subunit
in mammalian cells produces a desirable biologically active rTSH
material. Moreover, it has been found that the altered
glycosylation pattern that can be achieved with the cells, as
described in the methodology of the present invention, produces a
longer acting human thyrotropin which is particularly suited for
the diagnosis and treatment. of thyroid cancer.
[0048] The diagnosis and treatment of thyroid cancer involves first
purifying the synthetic TSH from large volumes of tissue culture
media harvested from approximately ten billion cells over two to
four weeks. Using a chemically defined medium to reduce protein
contaminants as is well known in the art, synthetic human TSH,
which represents about five to ten percent of all the protein
secreted into the medium, can be obtained. The human TSH thus
obtained is then purified by a combination of standard techniques
including immunoaffinity chromatography, HPLC exclusion
chromatography (repeated two to three times) followed by dialysis
and concentration by ultrafiltration, lyophilization and the like.
The purified human TSH is then tested in animals to assure its
efficacy as well as freedom from any unexpected toxicity. The
synthetic TSH is then tested in patients in clinical trials using
different doses to determine the optimal doses to achieve maximal
uptake into the tumor for both diagnosis and treatment with
.sup.131I. During initial try-outs for diagnosis, one to two
administrations of about 100 .mu.g, while during therapy three to
six doses of about 100 to 200 .mu.g may be administered, but the
optimal dose schedule is determined by the results of the clinical
trials. It is noted that all of these procedures are accomplished
while the patient is still euthyroid without producing any of the
disabling side effects of hypothyroidism which are otherwise
encountered in the methods heretofore available.
[0049] When the optimal uptake of .sup.131I has been established,
patients may be treated with doses of about 50 to 400 mCi of
.sup.131I and the effect of therapy assessed by subsequent
.sup.131I diagnostic tests as well as conventional x-rays, CAT
scans, measurement of serum thyroglobulin and the like. Of course,
.sup.131I-labeled rTSH, which is produced by standard methodology
well known in the art, can be appropriately utilized in the
procedures mentioned above.
[0050] It is estimated that there will occur about ten thousand new
cases of thyroid cancer in the United States each year and a very
large prevalence of older cases of this cancer require repeated
diagnostic and therapeutic intervention which are currently
unsatisfactory. Availability of synthetic human TSH as taught
herein, even at a cost of $50.00 to $100.00 per injection will
still be a relatively inexpensive part of the complete evaluation
and therapy for this difficult, but curable cancer.
[0051] Another advantage of the product of the present invention is
to provide assay components for human thyrotropin using the
technique of radioimmunoassay. Certain immunoassay kits are
presently available, but the reagents therein are again derived
from a very short supply of natural product. Moreover, the natural
product varies greatly depending on the source of the human
pituitaries as well as the degree of degradation that occurs during
autopsy. This has led to considerable, variation among commercially
available kits with disagreements in results of the TSH testing
among various kits. In contrast, the present invention, for the
first time, provides a virtually unlimited supply of a stable
preparation of synthetic TSH allowing kit manufacturers to have a
universal standard preparation and a virtually identical and
inexhaustible supply of the reagents. This would allow world wide
consistency of dosage and lead to much needed standardization in
the measurement of human TSH which is vital in the assessment of
thyroid function in humans. This is accomplished by labeling the
rTSH with radioactive iodine. (.sup.131I, .sup.125I) or another
suitable labeling material such as chemiluminiscent or fluorescent
labels and by producing antibodies to the pure product by either
polyclonal or monoclonal techniques which are well known in the art
and providing inexhaustible supplies of immunoglobulins without
significant interfering cross-reactivity with other hormones. The
antibodies are then formulated in classic radioimmunoassay kits
which are supplied to the manufacturers to be used in a variety of
standard assay methodologies (RIA, IRMA, Sandwich Assays and the
like).
[0052] There are various other advantages of rTSH. Tests have
demonstrated that it is possible to modify the TSH by expressing
the hormone in various cell lines leading to altered glycosylation
patterns. Moreover, using the technique of site-directed
mutagenesis whereby individual bases in the DNA are changed,
products are obtained with altered biologic function such as
prolonged or decreased half life, as well as competitive
antagonists that bind to the TSH receptor and actually block TSH
function. Such competitive antagonists are useful in a novel way to
treat diseases such as TSH-induced byperthyroidism as well as
Graves' disease which is caused by auto-antibodies to the TSH
receptor. The function of these abnormal stimulators would be
blocked by the competitive antagonist that we have already shown to
be active at the cellular level. Moreover, using various
long-acting and short-acting preparations, superagonists can be
prepared which would be particularly stimulating to thyroid
function, and superantagonists can be prepared which would be
particularly inhibitory of thyroid function. In this manner,
thyroid function can be controlled in many different types of
disease of thyroid overactivity or underactivity. It should be
noted that these completely novel approaches are feasible only
because of the availability of the synthetic TSH by the methodology
of the present invention because, for the reasons mentioned above,
the natural product is prohibited from such in vivo use.
[0053] It has also been discovered that modifications of the
transfection process greatly enhances the amount of TSH production
by mammalian cells. For example, instead of using TSH-.beta. gene
constructs containing only the 2nd and 3rd exons (the 2 coding
exons), a new construct is made by adding the first untranslated
exon of TSH-.beta.. The inclusion of this untranslated TSH-.beta.
exon greatly increases TSH production. Without being bound to any
specific theory, it is postulated that the enhanced TSH production
occurs by increased transcription rate and/or mRNA stability.
Moreover, it has been discovered that an excess of the .alpha. gene
in a ratio of about 3 to 5 times greater than the .beta. gene,
yields high rate of TSH production (10-50 mg/month), close to
commercial scale production.
[0054] A standard concentration curve utilizing anti-rTSH
antibodies is established to determine the amount of TSH in a
sample by conventional immunological assays.
[0055] In summary, a recombinantly made synthetic TSH has been made
which, even though constitutively distinct from the natural
product, possesses functional properties similar to the natural
product and is useful for diagnostic as well as therapeutic
purposes.
[0056] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
TABLE-US-00001 TABLE 1 Immunoassay for Human TSH and its Subunits
in Cell Media from Control and Transfected Cultures. Human
TSH.beta. Human TSH Transfected Construct or Human .alpha.RIA
(ng/ml) RIA (ng/ml) IRMA (.mu.U/ml) Control (Exp no.) n Mean SEM
Mean SEM Mean SEM 293 cells Medium (1, 2) <0.03 <0.03 <0.6
Control (2) 4 0.31 .sup. 0.01.sup.a <0.03 <0.6 Mock (2) 4 3.4
.sup. 0.36.sup.b <0.03 <0.6 pVARNA (2) 4 5.0 0.68 <0.03
<0.6 pAV2 (2) 4 2.1 0.11 <0.03 <0.6 pAV2.alpha. (2) 4 1.6
0.31 <0.03 <0.6 pAV2.alpha./pVARNA (2) 4 17.3 1.0 <0.03
<0.6 PAV2.beta. (2) 4 3.4 0.37 <0.03 <0.6
pAV2.beta./pVARNA (1) 2 2.2 0.30 1.5 0.09.sup. 2.8 0.30 (2) 4 5.7
0.64 3.3 0.33.sup. 6.9 0.64.sup.a pAV2.alpha./.beta./pVARNA (1) 2
4.0 1.5 0.6 0.24.sup. 11.2 6.3 (2) 4 6.1 0.54 1.1 0.09.sup.d 15.1
3.6 COS Cells Medium (1, 2) <0.03 <0.03 <0.6 Control (2) 2
<0.03 <0.03 <0.6 Mock (2) 2 <0.03 <0.03 <0.6
pAV2.alpha./pVARNA (1) 2 0.06 0.sup.b <0.03 <0.6 pSV2.beta.
(1) 2 <0.03 0.04 0.sup.d <0.6 (2) 2 <0.03 <0.03 <0.6
pSV2.beta./pVARNA (2) 2 <0.03 0.16 0.01.sup.d <0.6
pSV2.alpha./.beta. (2) 2 <0.03 <0.03 <0.6
pAV2.alpha./pSV2.beta./pVARNA (1) 2 0.09 .sup. 0.02.sup.b 0.10
0.02.sup.d 0.9 0.06.sup.a Various constructs were transfected into
either 293 or COS cells in two separate experiments. The medium was
harvested after 2 days in Exp 1 and 3 days in Exp 2. Cell medium
was assayed for the human .alpha.-subunit, hTSH.beta. subunit, and
hTSH as shown. Human CG.alpha. and hTSH.beta. were labeled .alpha.
and .beta. in construct names. # n, Number of plates transfected.
IRMA, 1 .mu.U is immunologically equivalent to 0.09 ng NIH I-6
purified hTSH. Medium, Fresh medium before application to cells.
Control, Medium from nontransfected cells. Mock, Medium from cells
transfected with a calcium phosphate precipitate lacking DNA.
.sup.aP < 0.0005 compared to mock, 293 cells .sup.bP < 0.0005
compared to pAV2.alpha./pVARNA(2), 293 cells .sup.cP < 0.05
compared to pAV2.alpha./.beta./pVARNA(2), 293 cells .sup.dP <
0.005 compared to pAV2.beta./pVARNA(2), 293 cells.
[0057] TABLE-US-00002 TABLE 2 Lectin Chromatography of Synthetic
and Standard hTSH Column Unbound (%) Bound MG (%) Bound-MM 1 WHO
STD 0 12 88 2 pAV2.alpha./.beta./pVARNA 0 28 72 Binding of a
synthetic vs. a standard hTSH preparation, described in the legend
to FIG. 4, to concanavalin A-Sepharose, is shown. Results are
expressed as a percentage of the total hTSH immunoactivity eluted
from two identical columns. Unbound, Bound-MG, Bound-MM =
immunoactivity measured in 2 ml column fractions after elution with
Tris-buffered saline, .alpha.-methylglucoside (10 mm), and
.alpha.-methylmannoside (500 mM), respectively.
* * * * *