Robert Immormino, Ph.D.

My research focuses on developing a method for rapidly generating new antibodies. Antibodies are extraordinarily useful research, diagnostic, and therapeutic reagents. Their utility depends greatly on their affinity and specificity, which in vivo would be selected in the course of the natural immune response. Most approaches to generating high affinity and specific antibodies depend on immunizing animals with recombinant proteins or synthetic peptides. Some approaches depend on in vitro mutagenesis and selection of antibodies displayed on microorganisms such as phage or yeast. These approaches can be limited by reproducibility and by immune regulation, which can make it difficult to raise antibodies against some targets. Our system for rapidly generating high affinity antibodies lies dogmatically in the middle of the two aforementioned approaches, and uses the rapidly proliferating and constitutively hypermutating chicken B cell line, DT40 as a vehicle. To increase the rate of antibody evolution we have introduced polymerized lac operator (PolyLacO) at the rearranged and expressed Ig light and Ig heavy chain alleles. The addition of these targeting sequences allows LacI fusions to be reversibly tethered to the immunoglobulin genes. Such LacI fusion proteins, including the chromatin modifying effectors HP1, VP16, and HIRA, or the Ig diversification regulator E47, accelerate Ig gene diversification using either templated or non-templated mutagenic pathways. Once antigen specific cells have been selected, culture with IPTG will release the LacI fusion protein from PolyLacO, reducing the rate of diversification and allowing for the stable expression of an antibody of interest.


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