Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
1999-08-19
2002-04-02
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C435S184000, C435S007230, C435S007710, C435S004000, C530S380000
Reexamination Certificate
active
06365364
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to inhibitors of blood vessel growth (angiogenesis) and particularly to amino acid sequences that can reduce or eliminate angiogenesis. In one aspect, the invention features methods for making angiogenesis inhibitors. The invention has a variety of applications including use in the treatment of angiogenesis-associated diseases such as cancer.
BACKGROUND
It is recognized that angiogenesis plays a role in diseases such as cancer. Therapies that can reduce or eliminate angiogenesis have been reported to be useful in the treatment of cancer and other angiogenesis-related diseases. See generally, Folkman J., (1971)
N. Engl. Jour. Med
. 285:1182; Folkman, J. (1989)
J. Natl. Cancer Inst
. 82: 4; O'Reilly, M. S. et al. (1994)
Cell
, 79: 315 and references cited therein.
There has been significant effort toward developing therapies that can reduce or eliminate angiogenesis. One approach has been to identify specific compounds that block or reduce growth of new blood vessels (neovascularization). See e.g., Ingber D, et al. (1990)
Nature
, 48:555; Clapp, C. et al. (1993)
Endocrinology
133: 1292; and Folkman, J. (1995)
N. Engl. J Med
. 333: 1757.
Particular attention has focused on a compound called “angiostatin”. This compound has been reported to be a 38 kDa to 45 kDa fragment from plasminogen. See generally O'Reilly, M. S. (1997) in:
Regulation of Angiogenesis
(I D Goldberg and E M Rosen, eds) Birkhauser Velag (Basel, Switzerland), pgs. 273-294.
Plasminogen is a protein involved in many physiological processes such as clot lysis. The protein includes five linked domains that are sometimes called “kringles”. Each kringle can be referred to as K1, K2, K3, K4 and K5, respectively. The K5 kringle is fused to a plasmin domain to form what is sometimes called a B chain. See e.g., Kelm R. J. et al (1994)
J. Biol. Chem
. 269:30147 and references cited therein.
Angiostatin has been disclosed as consisting of plasminogen kringles K1-4 Nucleic acid and protein sequences for angiostatin and various plasminogens have been reported. See e.g., Cao, Y. (1996)
J. Biol. Chem
. 271:29461; O'Reilley, M. S. et al. (1994, 1997) supra; and U.S. Pat No. 5,639,725.
Specific fragments of angiostatin have been reported to have anti-angiogenic activity. Sometimes the fragments are referred to as “angiostatin-like” to denote capacity to block blood vessel growth. Examples of such fragments include the K1and K1-3 Kringles. The K5 fragment has been reported to have minimal anti-angiogenic activity. See e.g., Cao, Y. supra.
There is increasing recognition that angiostatin is a strong inhibitor of neovascularization in vitro and in vivo. Thus, there has been an emerging need to develop effective methods for producing angiostatin and angiostatin-like fragments.
Several methods for producing angiostatin use proteases to cleave plasminogen into specific fragments. For example, one reported way to make angiostatin involves treating plasminogen with a protease called elastase. The fragments produced by the elastase are subsequently purified by conventional techniques.
However, the prior methods for making angiostatin are associated with significant problems. For example, many proteases produce disrupted kringle domains. That is, the proteases cleave plasminogen at more than one site. As a specific example, elastase has been reported to produce not only K1-4 (angiostatin) but also the K1-3 and K4 fragments. Although these methods can produce some angiostatin, isolation of significant amounts of the protein can be complicated by the other kringles. More generally, production of multiple fragments from plasminogen can substantially reduce yields and make purification of angiostatin and angiostatin-like fragments more difficult.
Additionally, many of the prior methods do not always completely remove the K5 fragment, thereby reducing the activity of certain angiostatin-like fragments.
One attempt to remedy this problem has been to minimize plasminogen cleavage. For example, one specific attempt has tried to reduce the amount of elastase used to cleave plasminogen so that larger kringle fragments can be obtained. However, this approach suffers from several drawbacks. For example, the need to reduce the amount or activity of the elastase makes assays more error prone and resistant to standardization and quality control. Use of other proteases has given rise to related problems. These shortcomings can be magnified by a variety of parameters, particularly during attempts to scale-up the methods.
The general availability of plasminogen and related molecules has supported efforts to isolate angiostatin by methods that include proteolytic cleavage. However, other approaches have been used to make angiostatin, e.g., recombinant DNA techniques. Use of the recombinant DNA techniques can be frustrated however by inability of some host cells to express suitable quantities of soluble protein. Additionally, there may be resistance to using recombinant DNA products in some settings.
It would be desirable to have angiogenesis inhibitors and more effective methods for producing same. It would be particularly desirable to have effective methods for making angiogenesis inhibitors from plasminogen that reduce or eliminate disruption of K1-4.
SUMMARY OF THE INVENTION
The present invention relates to angiogenesis inhibitors and effective methods for producing same from plasminogen. In general, we have discovered that by treating plasminogen with a specific cobra protein it is possible to enhance isolation of the angiogenesis inhibitors from plasminogen while minimizing or eliminating disruption of the first four plasminogen kringles (K1-4). The present invention has a variety of useful applications including use in the treatment of angiogenesis-associated diseases such as cancer.
In one aspect, we have found that cobra venom, particularly from the spitting cobra (
Naja Nigricollis Nigricollis
, hereinafter
Naja Nigricollis
), includes a protein and particularly a protease that is especially useful for producing certain angiogenesis inhibitors. More particularly, we have found that a protease (hereinafter “K-4 protease”) found in cobra venom specifically cleaves plasminogen at a single site near the K4 and K5 kringles, thereby isolating, in a single fragment, nearly all of the K1-4 fragment. Disruption of the K1-4 fragment is reduced or eliminated by use of the K-4 protease. Additionally, we have found that use of the K-4 protease can enhance activity of the present angiogenesis inhibitors by removing the K5 fragment therefrom. Practice of the present invention can enhance the preparation and use of the angiogenesis inhibitors by significantly boosting yields of nearly intact K1-4 fragment.
In contrast to the present invention, prior methods for making anti-angiogenic compounds from plasminogen often use methods that disrupt the K1-4 fragment and do not always efficiently remove K5 therefrom. Thus, yields of intact K1-4 are often decreased by the prior methods.
FIG. 1
provides a schematic representation of human plasminogen including the plasmin catalytic domain (catalytic domain), the first five plasminogen kringles (K1-5) attached to the plasmin domain (5B chain), and the
Naja Nigricollis
(K-4) protease cleavage site.
The present angiogenesis inhibitors can be made by one or a combination of different strategies in accord with the invention. In one approach, plasminogen or other suitable plasminogen-related molecule is treated with an amount of the K-4 protease sufficient to cleave the plasminogen or related molecule at a single specific site. As will be discussed, that site has been determined to be near the border between of the K4 and K5 fragments of plasminogen. More particularly, the K-4 protease has been found to cleave plasminogen specifically between amino acid position 451 and 452. Thus, unless specified otherwise, the angiogenesis inhibitors of the invention include a C-terminal amino acid that corresponds to the cleavage of plasminogen at or near amino ac
Jenny Nancy Swords
Mann Kenneth G.
Buchanan Robert L.
Caputa Anthony C.
Conlin David G.
Edwards & Angell LLP
Nickol Gary B.
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