Background: Apolipoprotein B (apoB) is the essential protein component of chylomicrons and very low density lipoprotein that transports lipids in plasma. Results: Domains of apoB interact with model membranes dependent on lipid composition and surface pressure. Conclusion: ApoB domains interact with membranes to initiate lipid recruitment. Significance: The apoB lipid recruitment mechanism provides a target to regulate the secretion of VLDL.

Advances in the treatment of multiple myeloma have been driven by the development of new modalities, including proteasome inhibitors, immunomodulatory agents and monoclonal antibodies. TAK-169 is an engineered toxin body (ETB) comprising a proprietarily engineered, deimmunized form of the ribosome inactivating Shiga-like toxin A-subunit (SLTA) genetically fused to an antibody fragment that specifically targets the CD38 cell surface receptor. Although CD38 is a poorly internalizing receptor, TAK-169 is able to efficiently internalize and directly kill CD38-expressing cells through the enzymatic and irreversible inhibition of protein synthesis. This novel mechanism of action may be relevant in patients who have progressed after or are unlikely to respond to CD38-targeted antibody therapy. A library of unique CD38-targeting antibodies fused to deimmunized SLTA was screened in a variety of efficacy and safety models to identify TAK-169. TAK-169 demonstrated potent cytotoxicity across a range of myeloma cell lines with a range of CD38 expression in vitro as well as in patient-derived samples including with previous exposure to daratumumab. TAK-169 retains activity in whole blood and PBMCs, indicating that the presence of red blood cells is not restrictive to cytotoxicity. Furthermore, TAK-169 retains activity in the presence of excess approved, CD38 targeted therapeutic daratumumab. TAK-169 is efficacious in several myeloma xenograft models, both subcutaneous and disseminated. Complete regressions were observed using both a once-weekly and bi-weekly schedule. In addition, a syngeneic model cell line expressing human CD38 was shown to be responsive to TAK-169 treatment in an immunocompetent mouse model, suggesting the possibility of immune mediated cell death induced by TAK-169. Tolerability studies in non-human primates demonstrate that repeat administration is tolerated at doses expected to be efficacious. Takeda Pharmaceuticals and Molecular Templates jointly discovered TAK-169 and are co-developing the molecule for the treatment of multiple myeloma. TAK-169 is expected to enter clinical studies in 2019. Citation Format: Erin K. Willert, Garrett L. Robinson, Jack P. Higgins, Jensing Liu, Janice Lee, Sakeena Syed, Yuhong Zhang, Dan Tavares, Anya Lublinsky, Nibedita Chattopadhyay, Haiqing Wang, Laura Packer, Pu Shi, Carole Harbison, Sanjay Patel, John Newcomb. TAK-169, an exceptionally potent CD38 targeted engineered toxin body, as a novel direct cell kill approach for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2384.

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Villin-type headpiece domains are ~70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously-folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.

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