Molecular biorecognition of coiled-coiled motifs in the construction of hybrid hydrogels and drug-free macromolecular therapeutics

Molecular biorecognition is at the heart of all biological processes and has come to the center stage in designing new biologics. Coiled-coils result from molecular biorecognition of multiple protein α-helices. We have designed a series of coiled-coil motifs that self-assemble into supercoils, which in turn function as physical cross-linkers in the construction of hybrid biomaterials. This dissertation describes our endeavors following the hypothesis below: the selfassembly of coiled-coil forming peptides into coiled-coils can be used as a cross-linking mechanism in the construction of hybrid hydrogels and the development of a drug-free macromolecular therapeutic. In the first part, a macromonomer free radical copolymerization strategy was developed and HPMA graft copolymers containing coiled-coil motifs of different chain lengths were synthesized. Results indicated that the primary structure of these motifs greatly influenced gel formation. At least four heptads were needed to mediate effective gelation. The gelation process was highly dependent on the environmental temperature and copolymer concentration. In the second part, a drug-free macromolecular therapeutic was designed to take full advantage of the facts that CD20 is one of the most reliable biomarkers for B-cell non- Hodgkin's lymphoma (NHL), and cross-linking of CD20 antigens induces apoptosis of B cells. The drug-free therapeutic was composed of two components: Fab' fragment of 1F5 iv anti-CD20 antibody conjugated with one coiled-coil motif (CCE) and polyHPMA copolymer grafted with multiple copies of the complementary coiled-coil motif (CCK). In vitro studies showed the conjugates could colocalize on Raji cell surfaces and a clinically relevant magnitude of apoptosis was achieved. A systemic NHL murine model was used to evaluate in vivo efficacy. Significant delay of hind-limb paralysis onset was observed after treatment. In groups receiving multiple-dose treatment, the surviving mice were disease-free and no presence of Raji B cell in bone marrow could be detected after 100 days. In summary, the possibility of integrating molecular biorecognition of short nonnatural coiled-coil motifs into new smart biomaterials is presented. This specific biorecognition can be used with or without adjuvant pharmacology to mediate a biofunctional process in combating diseases.