Surgery – Diagnostic testing – Detecting nuclear – electromagnetic – or ultrasonic radiation
Reexamination Certificate
2001-06-29
2003-10-07
Walberg, Teresa (Department: 3742)
Surgery
Diagnostic testing
Detecting nuclear, electromagnetic, or ultrasonic radiation
C600S001000, C600S003000, C128S898000
Reexamination Certificate
active
06631283
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to the use of a targeting fragment of a toxin or lectin molecule for the delivery of a substance of interest to cells. In particular, the invention provides a composition comprising a targeting fragment of a toxin molecule and a substance of interest, and methods for use of the composition. More particularly, the substance of interest may be a photosensitizing agent for use in targeted cell killing, or a visualizing agent for use in identifying cell surface receptors of interest.
2. Description of Related Art
The “holy grail” of research in the battle against cancer has been the development of a magic bullet to selectively kill cancerous cells while leaving normal cells untouched. Standard, therapeutic approaches to the treatment of cancer include surgery to remove the cancerous tissue (if the tumor is well defined and localized), radiotherapy, chemotherapy or combinations of these methods. Frequently, when a cancerous tumor is removed, a significant portion of surrounding tissue is also removed to ensure that the majority of cancerous cells are eliminated. In some cases, an entire organ is removed, even though portions of the organ are still healthy. In spite of such radical procedures, cancer cells may spill into body cavities and remain behind to proliferate. Further, portions of a tumor may be difficult to discern or difficult to access. The ability to accurately target cancerous cells for destruction while leaving normal, healthy tissue intact would be a major step forward in the treatment of this disease.
One form of therapy that is currently gaining acceptance for the treatment of hyperproliferating tissues is photodynamic therapy (PDT). Based on the discovery made over years ago that rapidly growing cells treated with certain chemicals will die when exposed to light, PDT is currently being used to treat several different types of cancers and non-malignant lesions. There appears to be a selective affinity and retention of photosensitizers in hyperproliferating tissue. Commonly, a patient is injected with a photosensitizer (PS) molecule that spreads throughout the body. There is then a waiting period during which the PS molecules accumulate in the target tissue, and are eliminated from most non-target tissue. Light is then used to illuminate a mass of tumor cells and activate the PS molecules to produce singlet oxygen, thereby killing cells and tissue in the area. The use of PDT to treat esophageal cancer, lung cancer and macular degeneration is currently being evaluated in clinical trials. One of the theoretical advantages of PDT is that tissues unexposed to light will not be affected. However, in reality, the rate of clearance of the photosensitizer from normal tissue is highly variable. Thus, while success rates of treatment with PDT are so far impressive, deleterious side effects such as skin sensitivity to light for four to six weeks have been observed. In addition, inflammation of the treatment site resulting in shortness of breath and coughing has been observed as a result of PDT treatment of lung and esophageal cancers.
Attempts have been made to optimize PDT treatments. U.S. Pat. No. 6,058,937 to Dorion et al., the disclosure of which is incorporated herein by reference, presents a method for shortening the waiting period after administration of PDT to a tissue. The technique is limited, however, to highly vascularized tissue. Rather than destroying the tissue itself, PDT is used to destroy the vasculature that nourishes the tissue and thus indirectly causes tissue death. The majority of molecules do not readily penetrate cell membranes. Methods for introducing molecules of interest into the cytosol of living cells are disclosed in U.S. Pat. No. 5,876,989 to Berg et al., the disclosure of which is incorporated herein by reference. The molecules to be released into the cell's cytosol are first taken up in endosomes, lysosomes or other cell compartments together with a photosensitizing compound. Light activation of the photosensitizing compound is then used to rupture the membranes of the cell compartments. The contents of the ruptured compartments (including the molecule of interest) are released into the cell cytosol without killing the majority of the cells. This invention thus utilizes PDT as a mechanism for releasing a drug (such as gelonin, a ribosome inactivating protein) from endosomes/lysomes to the cell compartment where the drug is effective (cytosol). The method does not provide a method of cell killing by PDT.
Kraus et al. in U.S. Pat. No. 6,160,024 describe chemical linkers to connect an energy emitting compound to a photosensitizing molecule, thereby providing an internal chemically-activated light source for activation of the photosensitizer. The method was employed to destroy virus-infected or tumor cells. However, this and other known PDT methods generally lack specificity. Their use results in whole body sensitization to illumination and the attendant side effects.
A need, therefore, exists for a way to enhance the specificity of PDT in order to avoid whole-body light sensitivity.
In order to enhance the specificity of cancer therapies, researchers have attempted to take advantage of the many biochemical and physiological changes that occur during cancer cell transformation. Some of these changes include the presence of cell-surface molecules when cells become cancerous. This has led to the use of antibodies coupled with toxic compounds to selectively bind to the surface of cancerous cells thereby killing those cells. However, this approach also has limitations that include the heterogeneous uptake of the toxin by the tumor cells, the slow elimination of the antibody-toxin complex from the blood system, and the cross-reactivity of the antibody with normal tissue.
The differential expression of many cell-surface molecules in human cancerous cells has been well studied and thoroughly documented. One such molecule is globotriaosylceramide (also known as Gb
3
, CD77 and p
k
antigen). The Gb
3
glycosphingolipid is normally expressed in several tissues including intestinal epithelium, kidney epithelium, and endothelial cells, in addition to being found in human milk as a glycolipid. Gb
3
is also expressed in a fraction of germinal center B lymphocytes, and traces of Gb
3
are found in red blood cell membranes of most individuals. Gb
3
is strongly expressed in the red blood cell membranes of p
k
blood type individuals (0.01% of the population). The over-expression of this cell-surface receptor has been documented in ovarian cancer, Burkitt's lymphoma (non-Hodgkin's lymphoma), breast cancer, brain cancer, gastric cancer, and testicular cancer. It would not be unreasonable to predict that the over-expression of Gb
3
may occur in many other types of cancer, as well.
The Gb
3
receptor (intestinal) is targeted by the bacterial toxin proteins belonging to the verotoxin family of bacteriotoxins that includes the Shiga toxins and Shiga-like toxins. Bacterial (
Shigella dysenteriae
and
Escherichia coli
) production of these toxins leads to disorders such as food poisoning, dysentery, hemorrhagic colitis, and hemolytic uremic syndrome. It is not the actual bacterial infection, but the production of the toxin molecules that leads to the disease symptoms. The bacteriotoxins, both Shiga and Shiga-like, are comprised of two protein components, a catalytic A subunit and a pentameric B subunit. The catalytic A subunit is a potent N-glycosidase that inhibits protein synthesis once inside a cell. The B subunit array is responsible for targeting specific cells expressing Gb
3
on their surface by recognizing and binding the Gb
3
receptor. Several issued patents take advantage of the targeted specificity of verotoxins to localized positions on cancerous cells.
Verotoxin 1, or the pentameric B subunit of verotoxin 1, administered in a non-lethal amount has been effective in treating mammalian neoplasia, including ovarian, brain, breast, and skin cancers as descri
English Sam
Storrie Brian
Tarrago-Trani Maria
Dahbour Fadi H.
Virginia Tech Intellectual Properties Inc.
Walberg Teresa
Whitham Curtis & Christofferson, P.C.
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