To tailor live viruses for cancer therapy (virotherapy), their tropisms and cytopathic activities should be redirected exclusively to tumor cells and/or tumor neovessels. We have recently demonstrated that attenuated measles virus (MV) is potently oncolytic and its tropism can be efficiently retargeted to alternative tumor-associated receptors through single-chain antibodies (scFvs). Given that generation and characterization of each retargeted MV requires considerable work and choosing the 'ideal' ligand-receptor pair can be difficult due to the vast number of possible ligands and tumor-associated receptors, it is pertinent that an informed choice regarding target selection can be made. In this study, we will define a set of parameters to facilitate target selection by investigating the influence of three critical variables for vector targeting: 1) cell surface density of the targeted receptor, 2) the presence and concentration of soluble receptors, shed from the cell surfaces and 3) the affinity with which the displayed ligand binds to the targeted receptor. Results from this study will help shape and refine concepts in viral vector targeting that will be applicable to other gene delivery systems. The measles display platform is highly versatile and tolerates display of a wide variety of ligands, making MV currently the only virus that can efficiently be retargeted via scFvs. The displayed scFv redirects MV infection and cytopathic effects of cell fusion exclusively to cells expressing the targeted receptor, with comparable efficiency to the parental virus, and without reversion to native receptor usage. In addition, oncolytic MV is able to discriminate between low and high densities of its cognate receptor (CD46), preferentially infecting and causing extensive cell fusion in high-CD46 expressing cells. This ability is highly desirable as few, if any, tumor targets are truly tumor specific. Most receptors are also expressed, albeit at low levels, on normal cells and the therapeutic agent must be able to discriminate between the relative receptor levels on tumor versus normal cells. Hence, the hypotheses to address the influence of receptor density, soluble receptors and affinity are as follows: 1) A threshold receptor density at which cell fusion occurs will exist for the receptors targeted by the anti-EGFR, anti-CD38 or anti-a-folate receptor scFvs on MV-H; 2) At clinically relevant concentration, soluble antigens will have minimal effect on virus entry and more importantly, will not significantly change the cell fusion threshold effect; and 3) Significant increases in scFv affinity for its receptor will have minimal impact on virus binding, but a large effect on virus-induced cell fusion.
The specific aims are: 1) To determine if receptor density discrimination by oncolytic MV can be applied to alternative tumor targets using virally displayed scFvs; 2) To determine the effect of soluble receptors on the infectivity and cell fusion of CD46-targeted or EGFR-targeted MV on receptor positive cells.; 3) To determine the relationship between affinity of displayed scFv and virus entry or cell fusion of retargeted MVs. ? ? ?
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