Reducing target binding affinity improves the therapeutic index of anti-MET antibody-drug conjugate in tumor bearing animals.

Many oncology antibody-drug conjugates (ADCs) have failed to demonstrate efficacy in clinic because of dose-limiting toxicity caused by uptake into healthy tissues. We developed an approach that harnesses ADC affinity to broaden the therapeutic index (TI) using two anti-mesenchymal-epithelial transition factor (MET) monoclonal antibodies (mAbs) with high affinity (HAV) or low affinity (LAV) conjugated to monomethyl auristatin E (MMAE). The estimated TI for LAV-ADC was at least 3 times greater than the HAV-ADC. The LAV- and HAV-ADCs showed similar levels of anti-tumor activity in the xenograft model, while the 111In-DTPA studies showed similar amounts of the ADCs in HT29 tumors. Although the LAV-ADC has ~2-fold slower blood clearance than the HAV-ADC, higher liver toxicity was observed with HAV-ADC. While the SPECT/CT 111In- and 124I- DTPA findings showed HAV-ADC has higher accumulation and rapid clearance in normal tissues, intravital microscopy (IVM) studies confirmed HAV mAb accumulates within hepatic sinusoidal endothelial cells while the LAV mAb does not. These results demonstrated that lowering the MET binding affinity provides a larger TI for MET-ADC. Decreasing the affinity of the ADC reduces the target mediated drug disposition (TMDD) to MET expressed in normal tissues while maintaining uptake/delivery to the tumor. This approach can be applied to multiple ADCs to improve the clinical outcomes.

Temporal dynamics of receptor-induced apoptosis in an affinity microdevice.

The temporal dynamics of Fas-induced apoptosis is elucidated. Jurkat cells are captured on the affinity surface of a microdevice coated with anti-CD95, an antibody known to induce apoptosis in cells via the extrinsic (caspase 8) pathway. The timing of apoptosis induction is controlled by the binding of the cells to the surface. Once bound, the cells are continuously stained with the caspase probe, L-bisaspartic acid rhodamine 110 (D(2)R), and the fluorescence of the cells was monitored for 6 h by light microscopy. This approach normalizes the temporal dynamics for each cell, as the binding event is also the start of apoptosis. In addition to providing the number of apoptotic cells over time, the fluorescence of individual cells can be monitored, providing information about the timing of caspase activity in each cell. The rate of caspase cleavage of D(2)R in each cell is also measured and shows good agreement between the cells in a given population. The effects of the caspase inhibitor, z-VAD-FMK, on the timing of caspase activity are also investigated and are shown to dramatically slow the apoptotic process. In the future, other caspase probes could be used to provide additional information about the temporal dynamics of caspase activation. Additional techniques, such as fluorescence correlation spectroscopy, can be coupled to these methods to provide faster temporal response and help to elucidate the heterogeneity of the apoptosis process.

Simultaneous cell capture and induction of apoptosis using an anti-CD95 affinity microdevice.

A microfluidic device is designed and demonstrated for the simultaneous capture and induction of apoptosis in Jurkat cells. In this unique case, the cell capture event initiates a biological process. The device features a single channel made from poly(dimethylsiloxane) sealed to a glass substrate. The channel is coated with a series of reagents used in affinity chromatography separations of cells. In this case, the antibody used to capture the cells is functional anti-CD95 which captures the cells by binding to the Fas receptor on the cell membrane and, at the same time, inducing apoptosis via the caspase 8 pathway. Cells retained on the surface of the channel are known to be induced to undergo apoptosis. Medium is flowed slowly through the channel to maintain cell viability while the cells undergo apoptosis. After 3 h, staining with Annexin V-PE and 7-AAD revealed that 43.5% of the cells bound to the anti-CD95 coated channel are apoptotic, whereas 7.9% of cultured Jurkat cells induced with anti-CD95 for 3 h and stained in the same way were determined to be apoptotic by flow cytometry. The device provides a method of determining when apoptosis is induced, maintaining cell viability for long-term analysis and observing cells in real time as they are exposed to reagents that affect apoptosis. In the future, the device will be an invaluable tool for the study of the temporal dynamics of apoptosis.