Protease inhibitor: protease sensitivity expression system and method improving the therapeutic activity and specificity of proteins and phage and phagemids delivered by bacteria

摘要

The present invention uses co-expression of protease inhibitors and protease sensitive therapeutic agents that results in their localized production within the target tissue and inactivation outside of the target tissue, thereby increasing therapeutic activity and reducing the systemic toxicity. Inactivation is also accomplished by engineering protease degradation sites within the therapeutic construct for proteases, preferably those that are under-expressed within the target tissue yet present in non-target tissues within the body, resulting in therapeutic activity within the target tissue and inactivation outside of the target tissue. Novel chimeric proteins secreted by bacteria are also described. The chimeric proteins include chimeric toxins targeted to neoplastic cells and cells of the immune system. Novel combination therapies of these protease inhibitor:chimeric toxin-expressing bacteria together with small-molecule and biologic agents are also described. Non-conjugative bacteria capable of delivering phage/phagemids expression cassettes for DNA and RNA-based therapeutics are also described.

主权项

1. A method comprising:
coexpressing within, and secreting from, a live genetically engineered bacterium: (a) at least one heterologous protease sensitive molecule having a protease cleavage site susceptible to cleavage by a non-bacterial protease outside of the bacterium, produced based on a first nucleic acid sequence genetically engineered within the genetically engineered bacterium, and (b) at least one heterologous protease inhibitor peptide, the at least one heterologous protease inhibitor peptide being effective for inhibiting the non-bacterial protease outside of the bacterium from proteolytically degrading the at least one protease sensitive molecule, produced based on a second nucleic acid sequence genetically engineered within the genetically engineered bacterium.