Compositions for the Treatment of Cancer, and Methods for Testing and Using the Same
a technology of cancer and compositions, applied in the direction of antibody medical ingredients, peptide/protein ingredients, instruments, etc., can solve the problems of high risk of human recurrence, and all too common devastating side effects, so as to reduce the level of cells expressing activated lfa-1
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example 1
Purification of LtxA from the NJ4500 Strain of A. actinomycetemcomitans
[0092]The JP2 strain of A. actinomycetemcomitans produces abundant LtxA, but it does not represent a fresh clinical isolate. Here, LtxA was purified from the clinical isolate NJ4500 of A. actinomycetemcomitans. This strain also produces and secretes a large amount of LtxA, but the cells adhere to surfaces instead of growing planktonically. This type of adherent growth results in a relatively low number of cells per volume. The cell density of adherent cells was increased by increasing the surface area on which the cells can grow through the addition of spherical glass beads. Soda lime beads provided the greatest amount of LtxA when compared to Pyrex glass beads. The amount of LtxA that was purified from NJ4500 in the presence of soda lime beads was approximately twice that of JP2.
[0093]It is important to note that growth of A. actinomycetemcomitans in the presence of both types of glass beads was similar suggest...
example 2
LtxA Specificity Towards WBCs Expressing Activated Surface LFA-1
[0098]One hypothesis why some cells are more sensitive to LtxA than others is that LtxA recognizes the activated form of LFA-1 better than LFA-1 in the resting state. To test this, an assay was carried out using Jurkat T-cell line that expresses a high level of constitutively active LFA-1 (J-β2.7 / LFA-1Δ). As controls, isogenic cell lines that either express the wild type form of LFA-1 (J-β2.7 / LFA-1 wt) or lack LFA-1 expression completely (J-β2.7 / mock) were used. It was found that cells with activated LFA-1 were ten times more sensitive to LtxA-mediated toxicity than cells with resting state LFA-1 and LFA-1-deficient cells were not affected by the toxin (FIG. 1A). Thus, LtxA is more toxic towards WBCs expressing activated form of LFA-1, which are the same type of cells that are predominantly present in a lymphoma tumor.
[0099]In another experiment, the specificity of LtxA for activated PMBCs was examined. Malignant human ...
example 3
LtxA Activity Towards Lymphoma Cell Lines
[0103]It was known that LFA-1 plays a crucial role in lymphoma cell proliferation. LtxA may therefore be a valuable therapy for the treatment of lymphoma. In this example, in vitro specificity and activity of LtxA towards various hematological cancer cell lines was demonstrated. Various assays were carried out to determine the concentration of LtxA required to kill 50% of the cells (IC50 values) after a 24-hour incubation. To determine IC50 values, human cells (˜106 cells / ml) were mixed with purified LtxA at various concentrations. The mixture was incubated at 37° C., 5% CO2 for 24 hours. Cellular viability (ATP production) was then determined using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, Wis.) according to the manufacturer's instructions. Plates were read in a Synergy HT plate reader in the luminescence mode (Bio-Tek, Winooski, Vt.). Cytotoxicity assays were performed at least three different times. The fraction...
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