(485aq) Cell-Free Production of Gaussia Princeps Luciferase – Antibody Fragment Bioconjugates for Personalized Cancer Diagnostics

Antibody fragments (scFvs) specific for unique cell surface markers can be used to detect cells that bear this marker (e.g. tumor cells). Fusion of a Gaussia princeps luciferase (GLuc) reporter protein to these antibody fragments allows sensitive detection of certain tumors in vivo and ex vivo. However, production of complex disulfide bonded proteins like scFvs as well as GLuc is challenging and production of GLuc-scFv fusions is even harder. We produce GLuc and antibody fragments individually in a properly folded and active form, followed by coupling to produce the desired conjugate. Cell-free protein synthesis (CFPS) has been used to produce GLuc and scFv proteins in soluble and active form with high yields. We can modify the open production system in order to encourage the formation of disulfide bonds. The CFPS system also facilitates addition of orthogonal components for the production of proteins containing non-natural amino acids (nnAAs). We have successfully produced Gaussia luciferase and scFv fragments from antibodies to a B lymphoma cell surface receptor, both containing nnAAs. Unique azide and alkyne reactive groups in the two proteins were used for conjugation by ?click chemistry?. GLuc ? antibody fragment conjugates were shown to differentiate between cells bearing the surface idiotype and cells that did not carry this marker.