Human-derived glycoprotein, biologically active factor which...

Details Human-derived glycoprotein, biologically active factor which... Human-derived glycoprotein, biologically active factor which...

1996-03-19

2001-12-25

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S350000, C530S399000

Reexamination Certificate

active

06333309

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a glycoprotein obtained from the culture broth of the human-derived fibroblasts, biologically active factor which includes glycoprotein, and pharmaceutical composition which comprises biologically active factor as active component.
The glycoprotein in the present invention, which shows cytotoxic activity to various tumor cell lines but not to normal cells, is a new tumor cytotoxic factor, leukemic cell differentiation inducing factor, cellular immunology enhancing factor, vascular endothelial cell growth factor and hepatocyte growth factor. This material is useful as an anti-tumor drug, an anti leukemia drug, a cellular immunology enhancing drug, a wound healing drug and a liver regenerating drug, etc. or a biochemical or pharmacological reagent.
DESCRIPTION OF THE RELATED ART
&bgr;-Interferon is a representative factor as the biologically active factor, for example, the tumor cytotoxic factor which is produced by human-derived fibroblasts. This is a glycoprotein which is secreted by the fibroblasts, when after the culture, the cells are harvested and stimulated by poly I-polyC or sendai viruses. It has been clarified that the protein has various physiological activities in addition to its anti virus or anti-tumor action. A fibroblast-derived tumor cytotoxic glycoprotein called as CBF is represented in Japanese Laid-Open No. 58-146293. A tumor growth inhibitory factor (INF) with a molecular weight of 35,000 to 45,000 which is purified from the culture broth of human tissue-derived fibroblasts is disclosed in Japanese Laid Open No. 671-33120. Also, a tumor necrosis factor-like material which is purified from the culture broth of fibroblasts, a fibroblast-derived necrosis factor, FNF, and a biologically active material with the cytotoxic activity, which is produced by animal fibroblast cells and has a molecular weight of 40,000 to 50,000 and an isoelectric point of 5.0±0.5, are disclosed in Japanese Laid Open No. 61-56131, Japanese Laid Open No. 61-1872, and Japanese Laid Open No. 62-103021, respectively. Furthermore, all amino acid sequence and cDNA sequence coding for the amino acid sequence of a tumor cytotoxic factor, which is obtained from the culture broth of human-derived fibroblasts, with a molecular weight of 36,000±1,000 and an isoelectric point more than 10.5, are disclosed in Japanese Laid Open No. 64-10998.
SUMMARY OF THE INVENTION
The present inventors have investigated a biologically active material which is contained in the culture broth of human-derived fibroblasts and have found a glycoprotein with various biological activities and, which is different from the materials reported previously with respect to the molecular weight and the isoelectric point etc.
Therefore, the main aim of the present invention is to offer 1 new glycoprotein, biologically active factor which includes said glycoprotein, and pharmaceutical product which comprises said biologically active factor.
A new human fibroblast-derived glycoprotein in the present invention (it is referred to as TCF-II hereafter) is characterized by the following physichochemical properties.
a. Molecular weight; On SDS gel electrophoresis, 78,000±2,000 or 74,000±2,000 under the nonreduced conditions and a common band A with 52,000±2,000 and band B with 30,000±2,000 or band C with 26,000±2,000 under the reduced conditions.
b. Isoelectric point; 7.4 to 8.6.
c. Heat stability; Stable in the heating at 60° C. for 10 min.
d. pH stability; Stable in the range of pH6 to 9.
e. Carbohydrate chain; Adsorbed to a Concanavalin A (Con A)-Sepharose column.
f. Biological activity; Inhibit the growth of KB cells, HeLa cells, and L-929 cells but not IMR-90 cells.
g. Reactivity to antibodies; The cytotoxic activity is not neutralized by anti-TNF antibody, anti-lymphotoxin antibody, and anti-interferon-&bgr; antibody.
Furthermore, the suitable lot of TCF-II in the present invention has the following properties in addition to the above physicochemical characteristics (a~g):
h. N-terminal amino acid sequence; The above mentioned band B and band C are subchains of band A, respectively. N-terminus of band A is blocked. Band B and band C has a common N-terminal amino acid sequence as follows;
Val-Val-Asn-Gly-Ile-Pro-Thr- or
Val-Val-Asn-Gly-Ile-Pro-Thr-X-Thr-Asn-Ile-Gly-X-Met-Val-Ser-Leu-
X means an unidentified amino acid.
i. Amino acid composition; When it is hydrolyzed with HCl it exhibits the following amino acid composition.
A.A
nmol
mol %
Asp
10.375
12.97
Glu
7.750
9.69
Ser
5.000
6.25
Gly
7.250
9.06
His
3.000
3.75
Arg
5.375
6.72
Thr
5.125
6.41
Ala
2.625
3.28
Pro
5.625
7.03
Tyr
3.875
4.84
Val
4.125
5.16
Met
1.875
2.34
Cys
ND
—
Ile
5.000
6.25
Leu
4.875
6.09
Phe
2.250
2.81
Trp
ND
—
Lys
5.875
7.34
total
80.000
100(99.99)
Furthermore, all the primary sequence of TCF-II in the present invention was deduced from its complementary DNA (cDNA). TCF-II cDNA was cloned by screening cDNA library which was prepared by using mRNA purified from total RNA which was extracted from human embryonic fibroblast cells (IMR-90), according to the following method.
(1) Extraction of Poly(A)
+
RNA from IMR-90 Cells
Total RNA was prepared by guanidine thiocyanatecesium chloride method (Biochemistry 18 5294-5299(1979)) from 2×10
8
IMR-90 cells which were cultured in the Dulbecco's modified eagle (DME) medium containing 5% of new born calf serum (NBCS). The IMR-90 cells were suspended in 28 ml of 6M guanidine thiocyanate containing BmM sodium citrate, 0.5% Sarcoseal and 0.1M &bgr;-mercaptoethanol, and were homogenized. 5.7M cesium chroride solution, 4 ml containing 0.1M EDTA was put into polyallomer centrifuge tubes. The homogenized solution, 7 ml was loaded on the cesium chloride solution and then centrifuged at 35,000 rpm, 20° C. for 16 hours, using 40T1 rotor of Beckman centrifugator. After centrifugation, the pellets were washed twice with 95% ethanol and dissolved in 200 &mgr;l of 10 mM Tris HCl buffer (pH7.5) solution containing 1 mM EDTA by heating at 65° C. for min, designated as total RNA solution. Poly (A)
+
RNA was purified from the total RNA by the method of oligo (dT)cellulose-column chromatography. The total RNA solution was loaded on the oligo (dT) cellulose-column which was equilibrated with 10 mM Tris HCl buffer(pH7.4) solution containing 1 mM EDTA, 0.5M NaCl and 0.05% SDS. The adsorbed fraction was eluted with 10 mM Tris HCl buffer, pH7.4, containing 1 mM EDTA and 0.05% SDS , and designated as poly(A)
+
RNA solution.
(2)Synthesis of cDNA Library
Double strand cDNA was synthesized by using poly (A)
+
RNA from (1) as a template and by using cDNA synthesis kit (Pharmacia Co. Ltd), and was attached EcoRI adaptor was attached. The method of synthesis was performed according to the protocol of Pharmacia Co.Ltd, except addition of reverse transcriptase (40 units/reaction mixture, Life Science Co. Ltd ) derived from non-sphere disease virus of avian bone marrow at the synthesis of single strand DNA.
(3) Preparation of cDNA Library
The cDNA obtained from (2) was inserted in EcoRI arm (Promega Co. Ltd) of phage vector &lgr; gt10. 3.3 &mgr;g of cDNA synthesized from poly(A)
+
RNA was dissolved in 150 1 of column buffer, 66 mM Tris-HCl buffer (pH7.6) containing 1 mM spermidine, 10 mM magnesium chloride, 15 mM dithiothreitol and bovine serum albumin (0.2 mg/ml). 5.2 &mgr;l of the above solution was mixed with 1 &mgr;g of &lgr; gt 10 EcoRI arm, and then precipitated with ethanol. Recombinant phage DNA including both &lgr; gt 10 and cDNA was prepared as follows. The above precipitate was reconstituted in 9 &mgr;l of the column buffer and was incubated at 16° C. overnight by adding 1 &mgr;l of 10 mM adenosine triphosphate and 1 &mgr;l of T4 DNA ligase (350 units/&mgr;l).
(4)Screening of cDNA Library
(i) Preparation of Oligonucleotide Probe
For preparation of the probe, a mixture of complementary oligonucleotide of 17 mer (384 species mix) corresponding to the amino acid sequence from Val
1
to Pro
6
in N-terminal

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