β-Cell regeneration is a key goal of diabetes research. Progression through the cell cycle is associated with retinoblastoma protein (pRb) inactivation via sequential phosphorylation by the “early” cyclins and cyclin-dependent kinases (cdks) (d-cyclins cdk4/6) and the “late” cyclins and cdks (cyclin A/E and cdk1/2). In β-cells, activation of either early or late G1/S cyclins and/or cdks is an efficient approach to induce cycle entry, but it is unknown whether the combined expression of early and late cyclins and cdks might have synergistic or additive effects. Thus, we explored whether a combination of both early and late cyclins and cdks might more effectively drive human β-cell cell cycle entry than either group alone. We also sought to determine whether authentic replication with the expansion of adult human β-cells could be demonstrated. Late cyclins and cdks do not traffic in response to the induction of replication by early cyclins and cdks in human β-cells but are capable of nuclear translocation when overexpressed. Early plus late cyclins and cdks, acting via pRb phosphorylation on distinct residues, complementarily induce greater proliferation in human β-cells than either group alone. Importantly, the combination of early and late cyclins and cdks clearly increased human β-cell numbers in vitro. These findings provide additional insight into human β-cell expansion. They also provide a novel tool for assessing β-cell expansion in vitro.

Type 2 diabetes (T2D) is characterized by insulin resistance and reduced functional β-cell mass. Developmental differences, failure of adaptive expansion and loss of β-cells via β-cell death or de-differentiation have emerged as the possible causes of this reduced β-cell mass. We hypothesized that the proliferative response to mitogens of human β-cells from T2D donors is reduced, and that this might contribute to the development and progression of T2D. Here, we demonstrate that the proliferative response of human β-cells from T2D donors in response to cdk6 and cyclin D3 is indeed dramatically impaired. We show that this is accompanied by increased nuclear abundance of the cell cycle inhibitor, p27kip1. Increasing nuclear abundance of p27kip1 by adenoviral delivery decreases the proliferative response of β-cells from non-diabetic donors, mimicking T2D β-cells. However, while both p27kip1 gene silencing and downregulation by Skp2 overexpression increased similarly the proliferative response of human β-cells, only Skp2 was capable of inducing a significant human β-cell expansion. Skp2 was also able to double the proliferative response of T2D β-cells. These studies define c-Myc as a central Skp2 target for the induction of cell cycle entry, expansion and regeneration of human T2D β-cells.

A biparatopic HER2xHER2 antibody drug conjugate with potent anti-tumor activity. The antibody drug conjugate (ADC) Trastuzumab-emtansine (T-DM1) targets the well-characterized breast cancer oncogene HER2 and is active in tumors that express very high levels of HER2 protein. However, tumors expressing intermediate levels of HER2 remain resistant to T-DM1 therapy, apparently due to insufficient lysosomal trafficking of T-DM1. We generated a series of biparatopic HER2xHER2 antibodies in which each arm recognizes a distinct epitope of HER2. One of our HER2xHER2 antibodies bound to cells with greater avidity than Trastuzumab and formed visible cell surface clusters that were efficiently internalized. While some of the internalized HER2xHER2 antibody trafficked to lysosomes and promoted HER2 degradation, a significant fraction recycled back to the plasma membrane through recycling endosomes. To exploit the increased internalization and recycling of the HER2xHER2 antibody, ADCs were generated by site-specific conjugation of Tubulysin A payload through a protease-cleavable linker. Recent data suggest that cleavage of this linker does not require lysosomal trafficking, but can occur in recycling endosomes as well. HER2xHER2-Tubulysin killed cell lines expressing high (IHC3+) and intermediate (IHC2+) HER2 levels with subnanomolar IC50, while exhibiting very weak killing of cells expressing low (IHC1+) HER2 levels (IC50 greater than 10nM). HER2xHER2-Tubulysin induced complete and durable tumor regression and outperformed T-DM1 in a collection of IHC3+ and 2+ tumor xenograft models (including PDX). Our work shows that a detailed understanding of antibody trafficking can enable the design of more effective ADCs and suggests that HER2xHER2-Tubulysin is a potential clinical candidate for HER2 IHC2+ breast cancer. Citation Format: Andres Perez Bay, Devon Faulkner, Shiwani Tiwari, Carla Castanaro, Xiang Zheng, Arthur Kunz, Thomas Nittoli, Amy Han, William Olson, Gavin Thurston, Christopher Daly, Julian Andreev. A biparatopic HER2xHER2 antibody drug conjugate with potent anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2898.

Trastuzumab-emtansine (T-DM1) targets the well-characterized breast cancer oncogene HER2, and has shown success in the clinic, but tumors expressing intermediate levels of HER2 remain resistant to T-DM1 therapy, apparently due to insufficient lysosomal trafficking of T-DM1. Here, we engineered bispecific HER2-DM1 conjugates that bridge HER2 with the rapidly internalizing receptor APLP2. The high affinity arm of HER2xAPLP2 bispecific antibodies mediates surface binding to HER2+ tumor cells (but not to HER2- normal cells). After binding to HER2, the low affinity APLP2 arm promotes antibody internalization and lysosomal degradation. The strength of the APLP2 arm allows for modulation of the degree of internalization, degradation and killing. In in vitro killing assays, HER2xAPLP2-DM1 were significantly more potent than T-DM1 in cell lines expressing high (IHC3+) and intermediate (IHC 2+) levels of HER2, albeit induced little or no killing in cell lines expressing low HER2 levels (IHC 1+). Monovalent APLP2-ADCs induced little or no killing in all cell lines tested. Selected HER2xAPLP2-DM1 conjugates had acceptable PK profile in APLP2 humanized mice while significantly outperforming T-DM1 in inhibiting growth of IHC2+ tumor xenografts. These data pave the way to generate clinical candidate ADCs that will improve on T-DM1. In addition, our study opens up a possibility to exploit internalizing proteins such as APLP2 in combination with bispecific antibodies to enhance efficacy of ADCs. Citation Format: Andres Perez Bay, Anuj Kalsy, Shiwani Tiwari, Bo Luan, Art Kunz, Zhaoyuan Chen, Li Zhang, Terra Potocky, Thomas Nittoli, Gavin Thurston, Christopher Daly, Julian Andreev. Bispecific HER2 ADC: Making more potent HER2 ADC by improving target internalization [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 233.