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Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V(H)-71, previously described as a key residue for maintaining the CDR-H2 main-chain conformation and thus important for antigen binding, markedly stabilised the scFv while having only a minor effect on the binding affinity of the molecule. Because of its improved stability, the engineered fragment exhibited immunoreactivity with tumour cells even after 7 days of incubation in human serum at 37 degrees C. It also showed, in contrast to the wild-type scFv, a concentration-dependent binding to the target antigen when displayed on phage. When fusing the scFv to the recombinant ribonuclease rapLRI, only the fusion protein generated with the stable mutant scFv was able to kill MUC1(+) tumour cells with an IC(50) of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUC1(+) malignancies. [ABSTRACT FROM AUTHOR]

Introduction: Photochemical Internalization is a novel drug delivery technology for cancer treatment based on the principle of Photodynamic Treatment. Using a photosensitizer that locates in endocytic vesicles membranes of tumor cells, Photochemical internalization enables cytosolic release of endocytosed antitumor agents in a site-specific manner. The purpose of the present in-vitro study was to explore whether Photochemical Internalization is able to enhance the efficacy of Ranpirnase, a cytotoxic amphibian ribonuclease, for eradication of squamous cell carcinoma of the head and neck., Methods: Cell viability was measured in 8 primary human cell lines of squamous cell carcinoma of the head and neck after treatment with Ranpirnase and Photochemical Internalization. For Photochemical Internalization the photosensitizer disulfonated tetraphenyl porphine was incubated with tumor cells followed by exposure to blue light (435 nm)., Results: Our study demonstrates significant enhancement of antitumor activity of Ranpirnase by Photochemical Internalization. Treatment responses were heterogeneous between the primary cancer cell lines. Combining Photochemical Internalization with Ranpirnase resulted in 4.6 to 1,940-fold increased cytotoxicity when compared with the ribonuclease alone (P < 0.05)., Conclusion: Cytotoxicity of Ranpirnase can be markedly enhanced by Photochemical Internalization in squamous cell carcinoma of the head and neck.

Cytotoxic ribonucleases such as the leopard frog derivative Ranpirnase (Onconase(®)) have emerged as a valuable new class of cancer therapeutics. Clinical trials employing single agent Ranpirnase in cancer patients have demonstrated significant clinical activity and surprisingly low immunogenicity. However, dose-limiting toxicity due to unspecific uptake of the RNase into non-cancerous cells is reached at relatively low concentrations of > 1 mg/m(2). We have in the present study generated a dimeric anti-EGFR Ranpirnase-diabody fusion protein capable to deliver two Ranpirnase moieties per molecule to EGFR-positive tumour cells. We show that this compound mediated far superior efficacy for killing EGFR-positive tumour cells than a monomeric counterpart. Most importantly, cell killing was restricted to EGFR-positive target cells and no dose-limiting toxicity of Ranpirnase-diabody was observed in mice. These data indicate that by targeted delivery of Ranpirnase non-selective toxicity can be abolished and suggests Ranpirnase-diabody as a promising new drug for therapeutic interventions in EGFR-positive cancers., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)