Daniel S. Allison

The promoter sequence of a yeast tRNAtyr gene

Thirty-one base substitution mutations within the yeast SUP4 tRNAtyr gene were used to probe the effects of different intragenic sequences on promoter activity. The various mutant plasmids were tested quantitatively for their in vitro template activity and for their ability to block competitively the transcription of a reference gene. Five mutations within the coding sequence of SUP4 decreased template activity for pre-tRNAtyr synthesis. The competition assays revealed 11 mutant genes that behaved differently than SUP4-o. Six were weaker competitors and five were stronger. The 12 mutations affecting template activity or competition are clustered in three regions: those encoding the dihydrouracil (D) arm, the extra loop, and the T psi arm of the tRNA. All of the mutations that reduce competition involve base changes that decrease homology to a eucaryotic tRNA consensus sequence in the highly conserved D and T psi regions. Three of the five up mutations increased homology to the tRNA consensus sequence.

A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.

Antibody discovery ex vivo accelerated by the LacO/LacI regulatory network.

Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo. This DTLacO platform derives from a chicken B cell line that has been engineered to enable rapid selection and seamless maturation of high affinity mAbs. We have validated the DTLacO platform by generation of high affinity and specific mAbs to five cell surface targets, the receptor tyrosine kinases VEGFR2 and TIE2, the glycoprotein TROP2, the small TNF receptor family member FN14, and the G protein-coupled receptor FZD10. mAb discovery is rapid and humanization is straightforward, establishing the utility of the DTLacO platform for identification of mAbs for therapeutic and other applications.