Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

The use of therapies based on antibody fusion proteins for the selective elimination of tumor cells has increased markedly over the last two decades because the severe side effects associated with conventional chemotherapy and radiotherapy are reduced or even eliminated. However, the initial development of immunotoxins suffered from a number of drawbacks such as nonspecific cytotoxicity and the induction of immune responses because the components were non-human in origin. The most recent iteration of this approach is a new class of targeted human cytolytic fusion proteins (hCFPs) comprising a tumor-specific targeting component such as a human antibody fragment fused to a human effector domain with pro-apoptotic activity. Certain tumors resist the activity of hCFPs by upregulating the intracellular expression of native inhibitors, which rapidly bind and inactivate the human effector domains. Higher doses of the hCFPs are, therefore, required to improve therapeutic efficacy. To circumvent these inhibitory processes, novel isoforms of the enzymes granzyme B and angiogenin have been designed to increase their intrinsic activity and reduce their interactions with native inhibitors resulting in more potent hCFPs that can be applied at lower doses. This chapter summarizes the basic scientific knowledge that can facilitate the rational development of human enzymes with novel and beneficial characteristics, including the ability to avoid neutralization by native inhibitors

Enzymes used in molecular biology: a useful guide

Since molecular cloning has become routine laboratory technique, manufacturers offer countless sources of enzymes to generate and manipulate nucleic acids. Thus, selecting the appropriate enzyme for a specific task may seem difficult to the novice. This review aims at providing the readers with some cues for understanding the function and specificities of the different sources of polymerases, ligases, nucleases, phosphatases, methylases, and topoisomerases used for molecular cloning. We provide a description of the most commonly used enzymes of each group, and explain their properties and mechanism of action. By pointing out key requirements for each enzymatic activity and clarifying their limitations, we aim at guiding the reader in selecting appropriate enzymatic source and optimal experimental conditions for molecular cloning experiments