The ability to program cell function with biochemical tools has driven many recent breakthroughs in immunology, oncology, and medicine. Presently, the most effective method to change the behavior of a cell is to modify its genetic machinery, but this method requires cell purification and can change other off-target cell behaviors. Here, we instead show a method to photoconjugate protein fragments onto live cell receptors and show that covalent conjugation of these proteins results in their extended expression on the cell surface, thereby avoiding proteolysis.<br/>First, we synthesized anti-EGFR affibodies, which when functionalized with benzophenone-maleimide can specifically conjugate to Epidermal Growth Factor Receptors (EGFR) under UV irradiation. Of the twelve cysteine mutants designed through site-specific mutagenesis, only one showed successful crosslinking to EGFR. This modified affibody demonstrated enhanced retention in EGFR-expressing 2D breast cancer cells and 3D tumor spheroids for up to 24 hours, when compared to non-crosslinkable affibodies.<br/>Next, to monitor the structural fidelity of proteins attached to the EGFR, a fusion protein was designed that contains the anti-EGFR affibody and enzyme cytosine deaminase (CD), capable of converting the prodrug 5-Flucytosine (5FC) to an anticancer metabolite 5-Fluracil (5FU). The enzyme-affibody fusions were conjugated to a pH-sensitive dye (pHAb) and a pH-insenstive dye (AlexaFluor 488). The dye-conjugated fusions were incubated with EGFR positive breast cancer cells and their fluorescence was monitored using flow cytometry. The results revealed that the fusion proteins were endocytosed after binding to EGFR and the photocrosslinked proteins were ejected out of endosome, without degradation, and were therefore retained on the cell membrane. On the other hand, the non-crosslinked control proteins underwent proteolysis, evidenced by a reduction in both the pHAb and AF488 fluorescence after 24 h. These results were then confirmed by confocal microscopy. Finally, the fusions and the prodrug 5FC were incubated with EGFR-expressing MDA MB 468 cells, and it was observed that the fusions were capable in converting the prodrug into 5FU, even 48 hours post conjugation, without any apparent loss in activity. Therefore, this work not only demonstrate the therapeutic efficiencies of such moieties in cancer cell killing but also shows that their activity can be preserved by covalent conjugation to cell membrane proteins.

Photoconjugating affibodies to their receptor prevents their proteolytic degradation and preserves their activity: Strategy for gene-free cell modification

2019 Fall National Meeting

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