Human MAP Tau Based Targeted Cytolytic Fusion Proteins

Abstract

Some of the most promising small molecule toxins used to generate antibody drug conjugates (ADCs) include anti-mitotic agents (e.g., auristatin and its derivatives) which are designed to attack cancerous cells at their most vulnerable state during mitosis. We were interested in identifying a human cystostatic protein eventually showing comparable activities and allowing the generation of corresponding targeted fully human cytolytic fusion proteins. Recently, we identified the human microtubule associated protein tau (MAP tau), which binds specifically to tubulin and modulates the stability of microtubules, thereby blocking mitosis and presumably vesicular transport. By binding and stabilizing polymerized microtubule filaments, MAP tau-based fusion proteins skew microtubule dynamics towards cell cycle arrest and apoptosis. This biological activity makes rapidly proliferating cells (e.g., cancer and inflammatory cells) an excellent target for MAP tau-based targeted treatments. Their superior selectivity for proliferating cells confers additional selectivity towards upregulated tumor-associated antigens at their surface, thereby preventing off-target related toxicity against normal cells bearing tumor-associated antigens at physiologically normal to low levels. In this review, we highlight recent findings on MAP tau-based targeted cytolytic fusion proteins reported in preclinical immunotherapeutic studies.

Keywords: cancer; human cytolytic fusion proteins; immunotherapy; inflammatory diseases; microtubule associated protein tau (MAP).

Figure 1

Mechanistic scenario of events leading to mitotic cell death (MCD) by microtubule targeting drugs. Microtubule stabilizing agents (e.g., paclitaxel, dictyostain, microtubule associated proteins—MAPs) promote the formation of microtubule bundles that cannot disassemble, while microtubule destabilizing agents (e.g., vinca alkaloids, colchicines) prevent the formation of the mitotic spindle by inhibiting tubulin polymerization. Both result in the activation of the spindle assembly checkpoint (SAC), eventually leading to mitotic arrest and apoptosis.

Figure 2

Schematic representation of the expression cassette for the recombinant epidermal growth factor (EGF)–MAP human cytolytic fusion protein. The open reading frame (ORF) was expressed in E. coli BL21(DE3) using protocol for periplasmic stress expression in the presence of compatible solutes and includes an N-terminal pectate lyase B (pelB) leader sequence from Erwinia carotovora which directs the fusion protein to the periplasmic space, a His₁₀ tag to facilitate purification, and an enterokinase cleavage site (ECS) to allow proteolytic separation of the fusion construct from the upstream production sequence. * = C-terminal nuclear localization signal sequence (NLS).