Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
1999-07-12
2002-12-31
Caputa, Anthony C. (Department: 1642)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S185100, C530S300000
Reexamination Certificate
active
06500431
ABSTRACT:
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not Applicable.
BACKGROUND OF THE INVENTION
Angiogenesis is the formation of new blood vessels from existing blood vessels. To initiate the angiogenic process, biochemical signals stimulate protease secretion from, among other cell types, endothelial cells lining the lumen of the vessel. The secreted proteases degrade the basement membrane and the endothelial cell layer protrudes through the hole created in the basement membrane. If the biochemical signals are continuously present, the migrating endothelial cells undergo mitosis and divide. The dividing cells form a sprout through the vessel wall. Again, if the angiogenic stimulus remains, the sprouts merge to form capillary loops which later mature into new blood vessels.
Under normal circumstances of wound healing, fetal and embryonic development and formation of the corpus luteum, endometrium and placenta, the initial angiogenic signals subside and other, secondary, signals predominate to turn off the angiogenic process. However in disease states such as cancer, angiofibroma, neovascular glaucoma, arteriovenous malformations, nonunion fractures, arthritis and other connective tissue disorders, Osler-Weber syndrome, atherosclerotic plaques, psoriasis, corneal graft neovascularization, pyogenic granuloma, retrolental fibroplasia, diabetic retinopathy, scleroderma, hemangioma, trachoma, vascular adhesions and hypertrophic scars, the local concentration of angiogenic signals never decreases and new blood vessels continuously form, supplying the diseased tissue with nutrients. This allows the tumor or diseased tissue to grow.
In cancer, undesired angiogenesis provides a steady supply of nutrients to the tumor. This allows the tumor to grow as well as metastasize. However, in addition to a general tumor growth-supporting role, some tumors are highly angiogenic. For example, Kaposi's Sarcoma (KS) is a tumor characterized by unregulated growth of blood vessels. It is, in fact, an angiogenic tumor. Currently, the treatment of Kaposi's Sarcoma, like most tumors, is based on chemotherapy. However, most chemotherapeutic agents are universally harmful to all dividing cells; cancerous or not. Thus, there is a need for compounds that will reduce the angiogenesis required for many disease states, including cancer and specifically, Kaposi's Sarcoma. This invention surprising meets these and other needs.
BRIEF SUMMARY OF THE INVENTION
A surprising discovery of this invention is that Saposin B, previously known as a protein involved in the hydrolysis of sphingolipids has potent anti-angiogenic and antitumoral activity. In addition, this protein has been found to have anti-proliferative and anti-migratory activity against endothelial cells. Even more surprising was the discovery that the activity against tumor and endothelial cells was conserved in cryptic polypeptides as small as five amino acids. These small polypeptides can now be used either in vitro as well as in vivo as anti-angiogenic and anti-tumor agents.
One embodiment of this invention is an isolated polypeptide of about 5 to about 80 amino acids in length and comprising a contiguous amino acid sequence DX
1
CX
2
D. X
1
and X
2
can be any amino acid. In one aspect of this embodiment, the isolated polypeptide is between 7 and 50 amino acids in length. In another embodiment, the isolated polypeptide is between 11 and 50 amino acids in length. In yet another embodiment, the isolated polypeptide is between 5 and 40 amino acids in length. In yet another embodiment, the isolated polypeptide is between 7 and 40 amino acids in length. In yet another embodiment, the isolated polypeptide is between 11 and 40 amino acids in length. In yet another embodiment, the isolated polypeptide is between 5 and 30 amino acids in length. In yet another embodiment, the isolated polypeptide is between 7 and 30 amino acids in length. In yet another embodiment, the isolated polypeptide is between 11 and 30 amino acids in length. In yet another embodiment, the isolated polypeptide is between 5 and 20 amino acids in length. In yet another embodiment, he isolated polypeptide is between 7 and 20 amino acids in length. In yet another embodiment, the isolated polypeptide is between 11 and 20 amino acids in length.
In further embodiments, X
1
is a valine or a conservatively modified variant thereof or X
2
is a glutamine or a conservatively modified variant thereof. In a preferred embodiment, the polypeptide will comprise the contiguous amino acid sequence DVCQD (SEQ ID NO:28).
In yet another embodiment, the isolated polypeptide specifically binds to an antibody raised against Saposin B. In a preferred embodiment, the polypeptide comprises an amino acid sequence substantially identical to that shown in SEQ ID NO:2 beginning at position 2. In a most preferred embodiment, the polypeptide comprises at least 5 contiguous amino acids, or conservatively modified variants thereof, said contiguous amino acids having an amino acid sequence as shown in SEQ ID NO:2, beginning at position 2.
In still another embodiment, the isolated polypeptide comprises R-DVCQD-R′; wherein R is from 0 to about 6 contiguous amino acids; and wherein R′ is from 0 to about 59 contiguous amino acids (SEQ ID NO:49). In a preferred embodiment, the polypeptide comprises R-XDVCQD-R′; wherein R is selected from the group consisting of Aa
1
-Aa
2
-Aa
3
-Aa
4
-Aa
5
, Aa
2
-Aa
3
-Aa
4
-Aa
5
, Aa
3
-Aa
4
-Aa
5
, Aa
4
-Aa
5
and Aa
5
. Aa
1
, Aa
2
, Aa
3
, Aa
4
and Aa
5
or no amino acid are selected from the group consisting of amino acids; X is selected from the group consisting of G, A, S and T; and wherein R′ is from 0 to about 59 contiguous amino (SEQ ID NO:45) acids. In a more preferred embodiment (SEQ ID NO:46), Aa
1
is a glutamine or a conservative substitution thereof, Aa
2
in a proline or a conservative substitution thereof, Aa
3
in a lysine or a conservative substitution thereof, Aa
4
in an aspartic acid or a conservative substitution thereof, or Aa
5
in a asparagine or a conservative substitution thereof.
In another embodiment, R′ is selected from the group consisting of Aa
12
-Aa
13
-Aa
14
-Aa
15
-Aa
16
, Aa
12
-Aa
13
-Aa
14
-Aa
15
, Aa
12
-Aa
13
-Aa
14
, Aa
12
-Aa
13
and Aa
12
, or no amino acid, wherein Aa
12
, Aa
13
, Aa
14
, Aa
15
and Aa
16
are selected from the group consisting of amino acids (SEQ ID NO:47). In a preferred embodiment (SEQ ID NO:48), Aa
12
is a cysteine or a conservative substitution thereof, Aa
13
is an isoleucine or a conservative substitution thereof, Aa
14
is an glutamine or a conservative substitution thereof, Aa
15
is a methionine or a conservative substitution thereof, or Aa
16
is a valine or a conservative substitution thereof.
In a most preferred embodiment, the isolated polypeptide has the amino acid sequence GDVCQDCIQMV (SEQ ID NO:19).
In another embodiment of this invention, a receptor is provided wherein the receptor specifically binds to Saposin B and is found on the surface of cells selected from the group consisting of KS Y-1, SLK, HUVEC and murine endothelial cells. In a preferred embodiment, the receptor is recombinantly expressed.
In another embodiment, a method of treating a mammal is provided, wherein said organism has a pathological condition associated with undesired angiogenesis. The method comprises administering to the mammal an amount of an isolated polypeptide comprising a contiguous amino acid sequence DX
1
CX
2
D, wherein X
1
and X
2
are selected from the group consisting of amino acids, wherein the amount of polypeptide effective to reduce angiogenesis. In a most preferred embodiment, the mammal is human and the isolated polypeptide is Saposin B.
In a more preferred embodiment, the pathological condition to be treated is cancer. In the most preferred embodiment, the cancer is Kaposi's Sarcoma. Administration of the isolated polypeptide is selected from the group consisting of subcutaneous, intramuscular, intravenous, intra-arterial, in
Bingham & McCutchen LLP
Caputa Anthony C.
Nickol Gary B.
University of Southern California
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