Check this out!!!! Guess who furnished the human anti-DNA antibody. :)
Abstract Number: 654
Engineering of an IL-2 immunocytokine with very low toxicity that retains potent anti-tumor activity in immune competent and immune deficient mouse tumor models
Stephen D. Gillies, Yan Lan, Scott Lauder, Beatrice Brunkhorst, Yaping Sun, Kin-Ming Lo. EMD-Lexigen Research Center, Billerica, MA.
The use of IL-2 for cancer therapy has been limited by its side effect profile that primarily includes effects in the vascular compartment such as vascular leak syndrome and hypotension. Many theories have been proposed to explain the mechanism of IL-2 toxicity including direct binding to endothelial cells as well as over-stimulation of intermediate affinity IL-2 receptor (IL-2R) bearing cells (e.g. NK cells) in the bloodstream. In our studies of IL-2 toxicity we have identified a specific IL-2 mutant, D20T, which has little selectivity for high over intermediate affinity IL-2 receptors as a free IL-2 molecule but which has profound selectivity when it is fused to the carboxyl terminus of an antibody to form an immunocytokine. Measurements in vitro show it to have near normal biological activity using T cell lines expressing the high affinity IL-2R but little or no activity using lines expressing only the intermediate IL-2R. This mutation also removes a sequence motif proposed earlier to resemble a binding site for endothelial cells found in many bacterial exotoxins. Surprisingly, this mutation within IL-2 had a detrimental effect on the circulating half-life of the immunocytokine that could only be reversed by removing the N-linked glycosylation site within the IgG constant region (CH2 domain). The final molecule was engineered using the V regions from a human antibody, NHS76, with high affinity for the necrotic core of solid tumors, thus allowing for selective targeting of this modified IL-2 to virtually any tumor with necrosis. Our in vitro studies with this molecule show that the binding to necrotic tissue is mediated through its high affinity for single or double-stranded DNA, despite original reports that the NHS76 antibody recognized DNA-histone complexes. Studies in several mouse tumor models demonstrate that this immunocytokine, NHS-IL2(D20T), is extremely well tolerated in immune competent mice and retains most of its anti-tumor activity against established metastases. It also retains significant anti-tumor activity in immune deficient SCID mice lacking functional T cells, although somewhat higher doses are required to achieve equivalent effects. These results suggest additional effector cells expressing high-affinity IL-2R, besides T cells, are functioning in these tumor models. They also show that small metastatic tumors can be targeted using an immunocytokine with specificity for a necrotic marker such as DNA.
Presenter: Stephen D. Gillies
Affiliation: EMD-Lexigen Research CenterBillerica, MA |
