Analysis of antibody self-interaction by bio-layer interferometry as tool to support lead candidate selection during preformulation and developability assessments.

Developability assessment of therapeutic mAb candidates before entering CMC development mitigates the risk of later failure because of manufacturing and stability issues. For mAbs derived from library based screenings, such evaluation starts with the first panning and ends with the selection of a lead candidate. This candidate should show, amongst others, high affine target binding and beneficial conformational as well as chemical stability. In addition, colloidal stability, reflected by the self-interaction propensity, should be superior in order to reduce aggregate formation and unacceptably high viscosity at elevated protein concentrations. Here, we present a study demonstrating the application of self-interaction bio-layer interferometry (SI-BLI) in a developability assessment, including the evaluation of preformulations. We reveal that the formulation rankings based on SI-BLI, DLS and viscosity measurements correlate. SI-BLI provides a deeper understanding of influencing factors on mAb self-interaction such as ionic strength or cation species. The attractive mAb self-interaction propensity was significantly more suppressed by Mg2+ compared to Na+. SI-BLI can be performed in high throughput with minimal material and sample preparation needs. Therefore, it can be applied in early stages of developability assessment going beyond the use of a platform formulation and a small number of analysis, to screen more parameters before proceeding with candidate selection and further extensive development.

Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.