Improved Tumor Penetration and Single-Cell Targeting of Antibody–Drug Conjugates Increases Anticancer Efficacy and Host Survival

Abstract

Current antibody-drug conjugates (ADC) have made advances in engineering the antibody, linker, conjugation site, small-molecule payload, and drug-to-antibody ratio (DAR). However, the relationship between heterogeneous intratumoral distribution and efficacy of ADCs is poorly understood. Here, we compared trastuzumab and ado-trastuzumab emtansine (T-DM1) to study the impact of ADC tumor distribution on efficacy. In a mouse xenograft model insensitive to trastuzumab, coadministration of trastuzumab with a fixed dose of T-DM1 at 3:1 and 8:1 ratios dramatically improved ADC tumor penetration and resulted in twice the improvement in median survival compared with T-DM1 alone. In this setting, the effective DAR was lowered, decreasing the amount of payload delivered to each targeted cell but increasing the number of cells that received payload. This result is counterintuitive because trastuzumab acts as an antagonist and has no single-agent efficacy, yet improves the effectiveness of T-DM1 Novel dual-channel fluorescence ratios quantified single-cell ADC uptake and metabolism and confirmed that the cellular dose of T-DM1 alone exceeded the minimum required for efficacy in this model. In addition, this technique characterized cellular pharmacokinetics with heterogeneous delivery after 1 day, degradation and payload release by 2 days, and cell killing and tumor shrinkage 2 to 3 days later. This work demonstrates that the intratumoral distribution of ADC, independent of payload dose or plasma clearance, plays a major role in ADC efficacy. This study shows how lowering the drug-to-antibody ratio during treatment can improve the intratumoral distribution of a antibody-drug conjugate, with implications for improving the efficacy of this class of cancer drugs. .

Single‐Cell Intravital Microscopy of Trastuzumab Quantifies Heterogeneous in vivo Kinetics

“…Further, it is possible to evaluate HER2‐dependent Tzm uptake by phagocytes with in vivo competition experiments using no‐fluorescent Tzm. However, we note that such competition experiments could be complicated to interpret, as prior reports have counter‐intuitively found that co‐administration of multiple types of HER2‐targeted mAbs can lead to enhanced tumor penetration, for instance by mitigating the impact of the binding site barrier .…”

Understanding the In Vivo Fate of Advanced Materials by Imaging

“…[310] A strategy to overcome this barrier, which is based on saturating the antigens around the tumor vessels, has recently been developed to improve the penetration of ado-trastuzumab emtansine. [311] Lastly, otherwise identical NPs or biologics can have distinct biodistribution in different types of tumors due to the difference in the microenvironment. In fact, certain types of tumors, such as pancreatic tumors, have microenvironment that is so unfavorable to drug delivery (lack of blood vessels and ultradense ECM) that even small molecule therapeutics fail to accumulate.…”

Practical Guide for Quantification of In Vivo Degradation Rates for Therapeutic Proteins with Single-Cell Resolution Using Fluorescence Ratio Imaging

“…Although these agents act at the molecular scale, it is necessary to quantify both the microscopic (sub-cellular and cellular) and macroscopic (tissue and organ) distribution in order to bridge the relationship between pharmacokinetics (PK) and pharmacodynamics (PD) [5][6][7]. For example, in the case of antibody-drug conjugates (ADCs), efficacy can be enhanced by understanding the internalization/degradation and payload release at the subcellular scale, the average number and variability of payload molecules required to achieve cell death in vivo at the cellular scale [8], the number of cells in the tumor receiving a therapeutic dose at the tissue scale, and the healthy tissue exposure and resulting toxicity at the whole organ level [9]. The distribution of biologics across multiple length scales is readily measured using near-infrared (NIR) labeling to track biologics; however, to quantify protein degradation, a second NIR label is needed.…”