Towards Novel Improved Anti-Kallikrein Agents: Extending the Circulatory Half-Life of C1INH and Identifying Novel Variants of AAT M358R with Enhanced Anti-Kallikrein Selectivity

Abstract

Hereditary Angioedema (HAE) is a rare disorder characterized by recurrent and severe swelling caused by excessive activation of bradykinin due to unregulated plasma kallikrein (Pka). Current therapies, including plasma derived or recombinant C1-esterase inhibitor (C1INH), aim to suppress Pka activity but are limited by short circulatory half-lives, necessitating frequent dosing. Additionally, while the engineered serpin Alpha1-antitrypsin M358R (AAT M358R) can inhibit Pka, it lacks sufficient selectivity, raising concerns about off-target effects on other proteases. This thesis addresses these challenges through three complementary approaches. First, I engineered C1INH-mouse serum albumin (MSA) fusion proteins to prolong in vivo half-life. I found that N-terminal MSA fusions preserved C1INH function and significantly extended circulatory persistence in mice, with orientation-dependent effects on SDS stability and activity. Second, using T7 phage display, I screened AAT M358R libraries mutated at key positions within the reactive center loop (RCL). Third, I performed rational loop-swapping experiments between AAT M358R and C1INH to explore whether segments of the C1INH RCL could improve Pka selectivity. Both the second and third projects identified novel sequence motifs that enhanced reactivity toward Pka, uncovering previously uncharacterized RCL combinations with potential therapeutic relevance. Together, these studies contribute new insights into therapeutic serpin engineering. The half-life extension work highlights the potential of albumin fusions to improve pharmacokinetics without compromising activity. The AAT M358R variant screening uncovers previously uncharacterized RCL sequences that promote Pka selectivity, expanding the known sequence space for functional serpin design. Overall, this thesis advances the development of improved biologics for HAE and provides a framework for engineering more selective, long-acting protease inhibitors for future therapeutic applications.