Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
1999-10-08
2003-08-19
Capura, Anthony C. (Department: 1642)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S192100, C424S193100, C514S002600
Reexamination Certificate
active
06607724
ABSTRACT:
Blood vessels are constructed by two processes: vasculogenesis, whereby a primitive vascular network is established during embryogenesis from multipotential mesenchymal progenitors; and angiogenesis, in which preexisting vessels send out capillary sprouts to produce new vessels. Endothelial cells are centrally involved in each process. They migrate, proliferate and then assemble into tubes with tight cell-cell connections to contain the blood (Hanahan,
Science
277:48-50 (1997)). Angiogenesis occurs when enzymes, released by endothelial cells, and leukocytes begin to erode the basement membrane, which surrounds the endothelial cells, allowing the endothelial cells to protrude through the membrane. These endothelial cells then begin to migrate in response to angiogenic stimuli, forming off-shoots of the blood vessels, and continue to proliferate until the off-shoots merge with each other to form the new vessels.
Normally angiogenesis occurs in humans and animals in a very limited set of circumstances, such as embryonic development, wound healing, and formation of the corpus luteum, endometrium and placenta. However, aberrant angiogenesis is associated with a number of disorders, including, tumor metastasis. In fact, it is commonly believed that tumor growth is dependent upon angiogenic processes. Thus, the ability to increase or decrease angiogenesis has significant implications for clinical situations, such as wound healing (e.g., graft survival) or cancer therapy, respectively.
Antithrombin or Antithrombin III (AT3) is a single chain glycoprotein involved in the coagulation process. It is synthesized primarily in the liver with a signal peptide of 32 amino acids necessary for its intracellular transport through the endoplasmic reticulum; the peptide is then cleaved prior to secretion. Mourey et al.,
Biochimie
72:599-608 (1990).
AT3 is a member of the serpin family of proteins and functions as an inhibitor of thrombin and other enzymes involved in the clotting cascade. As used herein, the active native intact form of AT3 is designated the S (stressed) form (S-AT3). S-AT3 forms a tight binding complex with thrombin (markedly enhanced by the presence of heparin) and other enzymes (not all serpins have heparin affinity).
S-AT3 can be cleaved to the relaxed (R)-conformation (R-AT3) by a variety of enzymes, including thrombin. Evans et al.,
Biochemistry
31:1262912642 (1992). For example, it has been thought that thrombin binds to a reactive C-terminal loop of AT3 and the resultant complex slowly dissociates releasing thrombin and cleaving off the C-terminal loop of inactive AT3, resulting in R-AT3. R-AT3 is unable to bind thrombin and has a conformation that is quite different from that of S-AT3. The role of R-AT3 had only been known to facilitate hepatic clearance of the molecule.
Other forms of AT3, such as L-AT3, which is the group of forms of ATIII that includes both the latent form and the locked form, are similar in conformation to R-AT3, and are also known in the art. Carrell et al.,
Nature
353, 576-578 (1991); Wardell et al.,
Biochemistry
36, 13133-13142 (1997). L-AT3, for example, can be produced by limited denaturing and renaturing the AT3 protein under specific temperature conditions, e.g., with guanidium chloride.
Prior to the present invention, AT3 was not known to be associated with angiogenesis. The present invention is, in one embodiment, drawn to a fragment, conformation, derivative or biological equivalent of AT3 that inhibits endothelial cell proliferation, angiogenesis and/or tumor growth in vivo.
In one embodiment, the invention relates to a method of inhibiting tumor growth by delivering or administering a composition comprising a fragment, conformation, biological equivalent, or derivative of AT3. In a preferred embodiment, the fragment, conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3, the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3 may also be chosen from a synthesized fragment of AT3 that inhibits tumor growth, conformational variations of other serpins that inhibit tumor growth, an aggregate form of AT3 that inhibits tumor growth, or a fusion protein of AT3 that inhibits tumor growth. The composition may further comprise a physiologically acceptable vehicle.
The invention further relates to a method of inhibiting endothelial cell proliferation comprising delivering or administering a composition comprising a fragment, conformation, biological equivalent, or derivative of AT3. In a preferred embodiment, the fragment, conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3, the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3 may also be chosen from a synthesized fragment of AT3 that inhibits endothelial cell proliferation, conformational variations of other serpins that inhibit endothelial cell proliferation, an aggregate form of AT3 that inhibits endothelial cell proliferation, or a fusion protein of AT3 that inhibits endothelial cell proliferation. The composition may further comprise a physiologically acceptable vehicle.
The invention also relates to a method of reducing or inhibiting angiogenesis comprising delivering or administering a composition comprising a fragment, conformation, biological equivalent, or derivative of AT3. In a preferred embodiment, the fragment, conformation, biological equivalent, or derivative of AT3 is chosen from the L form of AT3, the R form of AT3 and fragments that include the active sites of the L form of AT3 and/or the R form of AT3. The fragment, conformation, biological equivalent, or derivative of AT3 may also be chosen from a synthesized fragment of AT3 that reduces angiogenesis, conformational variations of other serpins that reduce angiogenesis, an aggregate form of AT3 that reduces angiogenesis, or a fusion protein of AT3 that reduces angiogenesis. The composition may further comprise a physiologically acceptable vehicle.
In another embodiment, the invention pertains to a method for identifying an inhibitor of tumor growth or an agent that reduces tumor growth, comprising the steps of inoculating an animal with an appropriate innoculum of tumor cells in each of two suitable inoculation sites; identifying inhibition of growth of a tumor, known as the subordinate tumor, at one inoculation site with concomitant growth of a tumor, known as the dominant tumor, at the other inoculation site; isolating cells from the dominant tumor; and purifying a component which inhibits endothelial cell proliferation and/or angiogenesis from the isolated cells. For example, the component may be purified from conditioned media from the cells. In one embodiment of the invention, the tumor cells are derived from tumors selected from the group consisting of small cell lung cancers and hepatocellular carcinomas. In a particular embodiment, the inoculation sites are the flanks of the animal. In one embodiment the inhibitor of tumor growth is an inhibitor of endothelial cell proliferation. In another embodiment the inhibitor of tumor growth is an inhibitor of angiogenesis. In a further embodiment of the invention, the method further comprises a step of selecting for an animal in which inhibition of the growth of the subordinate tumor by the dominant tumor is substantially complete.
The invention further relates to a method of inhibiting tumor growth comprising delivering or administering an inhibitor of tumor growth identified by the methods described herein to a mammal. In a preferred embodiment the inhibitor of tumor growth is a fragment, conformation, biological equivalent, or derivative of AT3.
It is also within the practice of the invention to use a similar method to identify an agent that reduces or an inhibitor of angiogenesis and/or endothelial cell proliferation. Such a method would also co
Folkman M. Judah
O'Reilly Michael S.
Pirie-Shepherd Steven
Capura Anthony C.
Children's Hospital
Finnegan Henderson Farabow Garrett & Dunner LLP
Nickol Gary B.
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