Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1998-05-28
2001-04-17
Borin, Michael (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S412000, C530S350000
Reexamination Certificate
active
06218517
ABSTRACT:
TITLE OF THE INVENTION
A protein having a vascularization inhibitory effect and a method for production thereof and a method for producing angiostatin
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to vascularization inhibitors and a method for producing protein which is useful as a vascularization inhibitor.
2. Description of the Related Art
It is known in clinical field that, when a carcinoma is removed, other small tumors which were in a state of lull become activated to start growing or to raise their metastatic inclination. As a reason for this phenomena, the carcinoma itself is considered to produce some substances, which would inhibit the growth of other tumors. Angiostatin is known as one of such substances.
O'Reilley, et al. (Cell. Vol. 79, 315-328, Oct. 21, 1994, J. Folkman's Research Group) showed that urine and serum from mice having tumors inhibited metastasis of carcinoma in mice as well as growth of endothelial cells participating in vascularization. The substance showing such an inhibitory effect was purified and identified as a protein of 38 kDa by analysis of SDS-PAGE. This protein showed at least 98% homology to an internal fragment of 38 kDa plasminogen which fragment has N-terminal portion starting at mino acid
98
of plasminogen. (See the Discussion on the left column of page 323 of the above-mentioned reference.)
This protein called “angiostatin” has been found to coincides with a partial sequence of plasminogen. The plasminogen is a protein in plasma having 5 domains called “kringles”. The kringle is a domain of triple loops formed by disulfide bonds. The domain is considered to play some role in linkages in which membranes, proteins and phospholipids are involved, and in regulating the proteolytic activity of enzymes. Hereinafter the five kringles will be referred to as “K
1
” to “K
5
” starting with K
1
close to the N-terminus along the plasminogen molecule.
The structure of human plasminogen is shown in
FIGS. 1-3
.
FIG. 1
is a partial modification of the figure on page 276 of “Hemostasis, Thrombus, Fibrinogenolysis”, (Edited by Michio Matsuda, et al., p.276, First Edition published in 1994, Chugai-Igakusya Co. Tokyo, Japan). Plasminogen is a protein consisting of 791 amino acids and contains 5 kringles K
1
-K
5
. The amino acid sequence of plasminogen has been determined (FIG.
1
).
FIG. 1
shows this amino acid sequence, using one letter code. In
FIG. 1
, “A” means alanine, “C” means cysteine, “D” means asparagine acid, “E” means glutamic acid, “F” means phenylalanine, “G” means glycine, “H” means histidine, “I” means isoleucine, “K” means lysine, “L” means leucine, “M” means methionine, “N” means asparagine, “P” means proline, “Q” means glutamine, “R” means arginine, “S” means serine, “T” means threonine, “V” means valine, “W” means tryptophane, and “Y” means tyrosine. Human plasminogen has four an elastase hydrolytic sites. The peptide bonds at amino acids between position
78
and position
79
, position
338
and position
339
, position
354
and position
355
, and position
440
and position
441
(or position
442
and position
443
) can be hydrolyzed with an elastase. (These bonds are indicated by
in FIG.
1
and ▴ in
FIGS. 2 and 3
.)
Glycosylation sites exist at
289
and
346
amino acids of plasminogen (as shown by ♦ in
FIG. 1
, &Circlesolid; in FIG.
2
and ∘ in FIG.
3
). Plasminogen has two kinds of isoforms. One is attached to a sugar chain at amino acid
289
(Form
1
), and the other has no sugar chain at amino acid
289
(Form
2
).
Plasminogen has a lysine-binding site
1
in K
1
-K
3
and another lysine-binding site
2
in K
4
(marked by ☆ in FIG.
3
), and so it can bind with lysine at these sites. In purifying plasminogen from plasma, Lysine-Sepharose chromatography can be used because of its property of binding with lysine. K
5
has an aminohexal-binding site (marked by ⊚ in FIG.
3
).
The hydrolysis of human plasminogen with an elastase by O'Reilly, et al. was carried out under conditions so as to hydrolyze the peptide bonds at amino acids between positions
78
and
79
, positions
338
and
339
, positions
354
and
355
, and positions
440
and
441
(or positions
442
and
443
). Therefore, the resultant fragments are those from amino acids
1
-
78
(N-terminal potion of plasminogen), amino acids
79
-
338
(fragments containing K
1
-K
3
considered to be a mixture of those derived from plasminogen in Form
1
and those derived from that in Form
2
), amino acids
339
-
354
(a short fragment between K
3
and K
4
), amino acids
355
-
440
(a fragment containing K
4
), and amino acids
441
(or
443
)-
791
(the portion containing K
5
and C-terminal portion of plasminogen, called “miniplasminogen”).
Using the fragment containing K
1
-K
3
, the fragment containing K
4
and miniplasminogen, O'Reilly, et al. carried out some experiments for the inhibitory effect on the growth of endothelial cells and on the metastasis of carcinoma. As the result, the inhibitory effects on the growth of endothelial cells and metastasis of carcinoma was observed only in the fragments containing K
1
-K
3
. On the contrary, no effect was observed either in the fragment containing K
4
only or in human plasminogen itself.
O'Reilly, et al. named the fragments containing K
1
-K
3
“purified angiostatin”. Purified angiostatin is considered to inhibit the growth and metastasis of carcinoma cells by preventing vascularization.
Angiostatin can control the metastatic inclination of carcinoma, which would otherwise extremely increase after removal of the carcinoma, to the same level as before the carcinoma removal. In other words, the metastasis of carcinoma is inhibited after its removal by administering the same amount of angiostatin as produced by the carcinoma itself. Furthermore, angiostatin is obtained as a result of hydrolysis of plasminogen, which existing abundantly in the form of protein in plasma, as a substrate with an elastase which is a protease produced by vascular endothelial cells. As angiostatin itself is a protein existing in blood vessels, it is digested by a protease like plasmin. Therefore, it is considered that angiostatin can be used as an anticancer agent naturally occurring in a living body with almost no side effect. In addition, plasminogen is abundantly present in plasma, it is convenient to use plasminogen as a starting raw material for the production and purification of angiostatin. These suggest that angiostatin is excellent as one of known anti-metastatic agents.
In addition to angiostatin, fumagillin is also known as a vascularization inhibitor. Fumagillin is a kind of antibiotics produced by microorganisms that is different from angiostatin. Japanese Patent Publication No. 6-60095 discloses that fumagillin and its derivatives can be used as a vascularization inhibitor.
For using angiostatin as a carcinoma metastasis inhibitor, it is necessary to hydrolyze plasminogen extracted from plasma with an elastase and fractionate on columns. This fractionation can be presently effected only by HPLC. If angiostatin is to be extracted and purified according to such procedures, not only it is time-consuming and costly but also it shows low recovery. Besides, it is difficult to store angiostatin because of its strong inclination to decompose.
It is problematic to use fumagillin in clinical aspects because of its strong adverse side effects, though it is also effective like angiostatin in inhibiting vascularization and metastasis of carcinoma.
Moreover, it is disclosed in BBRC, Vol. 102, 1, 46-52, 1981 and Chemical Reviews, Vol. 81, Oct. 5, 1981 that the fragment consisting of amino acids
355
-
791
was obtained by limiting hydrolysis with an elastase. It is stated in the references that, after separating plasminogen into Form
1
and Form
2
, the fragment bound on the Lysine-Sepharose column is eluted at gradient concentrations of -aminocaproic acid (hereinafter referred to as “EACA”), and that the several eluted peaks ar
Borin Michael
Burns Doane Swecker & Mathis L.L.P.
Suzuki Motor Coporation
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