A deimmunized and pharmacologically optimized Toll-like receptor 5 agonist for therapeutic applications.

The Toll-like receptor 5 (TLR5) agonist entolimod, a derivative of Salmonella flagellin, has therapeutic potential for several indications including radioprotection and cancer immunotherapy. However, in Phase 1 human studies, entolimod induced a rapid neutralizing immune response, presumably due to immune memory from prior exposure to flagellated enterobacteria. To enable multi-dose applications, we used structure-guided reengineering to develop a next-generation, substantially deimmunized entolimod variant, GP532. GP532 induces TLR5-dependent NF-κB activation like entolimod but is smaller and has mutations eliminating an inflammasome-activating domain and key B- and T-cell epitopes. GP532 is resistant to human entolimod-neutralizing antibodies and shows reduced de novo immunogenicity. GP532 also has improved bioavailability, a stronger effect on key cytokine biomarkers, and a longer-lasting effect on NF-κB. Like entolimod, GP532 demonstrated potent prophylactic and therapeutic efficacy in mouse models of radiation-induced death and tissue damage. These results establish GP532 as an optimized TLR5 agonist suitable for multi-dose therapies and for patients with high titers of preexisting flagellin-neutralizing antibodies.

Subtractive cloning: new genes for studying inflammatory disorders.

Understanding of the biology of interaction between pathogens and host is the central question in studying inflammatory disorders. Subtractive DNA cloning is one of the most efficient and comprehensive methods available for identifying eukaryotic genes regulated under specific physiological conditions, including inflammation and host response. Here we explore the utility of subtractive DNA cloning and describe suppression subtractive hybridization (SSH), a polymerase chain reaction (PCR)-based DNA subtraction method that has been developed and evolved in our labs over several years. The SSH method possesses a number of advantages as compared to other subtractive cloning techniques, making it one of the most adventitious methods for cloning differentially expressed genes. Besides isolation of differentially expressed eukaryotic mRNAs, subtractive DNA cloning can be used to identify genes that are differentially expressed between diverse bacterial species. These genes can be of great interest, as some may encode strain-specific traits such as drug resistance, or bacterial surface proteins involved in determining the virulence of a particular strain. Other genes may be useful as markers for epidemiological or evolutionary studies. To demonstrate the potential of the SSH technique, we describe here the comprehensive characterization of 2 SSH subtracted libraries constructed in our laboratories. One library was created using eukaryotic cDNA subtraction and is specific for mRNAs up-regulated in CD25 positive cells from mouse lymph nodes as compared to CD25 negative cells. The second subtracted library is specific for a methicillin-resistant Staphylococcus aureus bacterial strain, but not in a methicillin-sensitive strain. The bacterial genomes of these 2 strains have been completely sequenced and this second library provides an excellent reference for testing the ability of SSH to recover all strain-specific gene content. The analysis of these 2 subtracted libraries serves as the basis for a discussion of the strength and limitations of the SSH technique. We will also compare and contrast subtractive DNA cloning to other current technologies used to isolate differentially expressed genes.