The applicant's long-term goal is to develop novel, targeted therapeutics for the treatment of human T cell malignancies. Immunotoxins (ITs) are a class of therapeutic agents with a high degree of specificity and a unique mechanism of action. An IT is a hybrid molecule consisting of a targeting moiety linked to a toxin. The targeting moiety selectively binds to a tumor cell and targets it for death via the attached toxin. Generally, ITs are specifically potent against cancer cells in vitro and in animal models of human malignancies. However, ITs are limited clinically by immunogenicity, toxicity, and/or instability. A clinical grade IT called DA7 was synthesized at the University of Minnesota by biochemically linking deglycosylated ricin toxin A chain (dgRTA) to a monoclonal antibody specific for the T cell-associated antigen CD7. A Phase I clinical study of DA7 revealed that its efficacy was primarily limited by instability and nonspecific vascular toxicity. Despite these limitations, DA7 achieved objective clinical responses at the maximal tolerated dose. If the instability and vascular toxicity of DA7 were reduced, then the applicant contends that DA7 could find a therapeutic niche in the treatment of refractory T cell disease, or as an adjuvant to surgery or chemoradiotherapy. The objective of this revised application is to use genetic engineering to enlarge the 'therapeutic window' of DA7 by increasing its stability and decreasing its toxicity. The first specific aim focuses on the construction and testing of recombinant ITs containing a derivative of Diphtheria toxin (DT) linked to CD7-specffic single chain Fv (sFv) fragments. DT will be used initially since it is a component of the only FDA-approved immunotoxin. Modifications of the sFv structure will be made to enhance stability. Novel approaches for the high-level expression of soluble fusion toxins, and for the direct visualization of IT-mediated tumor cell killing in vivo, are included in this aim. The second specific aim focuses on decreasing toxicity (and immunogenicity) by linking the most stable sFv structure identified in Specific Aim l to human RNAses. The RNAses will be engineered to be both resistant to RNAse inhibitors and to be optimally cytotoxic once internalized. All immunotoxins proposed in Specific Aim 1 and Specific Aim 2 will be tested for their stability, relative affinity, pharmacokinetics, toxicity, and anti-tumor activity. These experiments will allow the applicant to determine more precisely the relationships between stability, toxicity, and efficacy. His ultimate goal is to return to the clinic with a more potent version of DA7.
| Erickson, Heidi A; Jund, Michelle D; Pennell, Christopher A (2006) Cytotoxicity of human RNase-based immunotoxins requires cytosolic access and resistance to ribonuclease inhibition. Protein Eng Des Sel 19:37-45 |
