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Our work focuses on costimulatory signals for T cell activation. Costimulatory signals determine the strength and direction of an immune response. Over the last few years we have found that despite the great effort needed to start an immune response, the immune system also has pathways, such as the PD-1/PD-1 ligand pathway, that actively inhibit and stop immune responses. This inhibition is important for peripheral T cell tolerance and also prevents immune responses from being too strong and damaging the host. We have found that in chronic infections, such as AIDS and hepatitis, the antigen-specific T cells highly express the PD-1 receptor for inhibitory signals, contributing to T cell exhaustion in chronic viral diseases. Many cancer cells express PD-1 ligands that inhibit the anti-tumor immune response. Our goal is to make antibodies or fusion proteins that block this inhibitory pathway, thereby releasing the brakes on the immune response and re-invigorating the immune response against cancer and chronic viral diseases.
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The Technique
We read the In-Fusion™ technical brochure describing the joining of a PCR product to a restriction enzyme-digested vector via 15-bp overlaps at the ends of the DNA segments, and thought, why not use In-Fusion to join three pieces of DNA together? So we designed a new construct on the computer and made three PCR products with 15-bp overlaps at their ends. In-Fusion joined them together correctly, and we quickly became enamored with the possibilities of joining multiple pieces of DNA at any point desired. We designed an expression vector in which each functional segment was generated by PCR with 15-bp overlaps—including a unique restriction enzyme sequence—and joined them in a series of In-Fusion reactions. This made a modular vector where we could readily swap in and out any particular segment, such as a selectable marker or promoter. We found that we could design fusion proteins or recombinant antibodies composed of three PCR products and then assemble them with the fourth piece—our modular vector. We also adapted the technique to introduce mutations and, in fact, to replace a DNA segment with any desired DNA segment. Using these seamless fusion proteins designed via In-Fusion, we are able to more rapidly understand costimulatory pathways and how to best therapeutically manipulate the immune response.
See “In-Fusion™ assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations” on page 354.
