Assessing the Potency of T Cell-Redirecting Therapeutics Using In Vitro Cancer Cell Killing Assays

Abstract

The development of in vitro cell-based assays that can best recapitulate as near as possible to a human in vivo setting is urgently needed, as animal models are unsuitable for the preclinical assessment of the growing number of fully human cancer therapeutics. In the field of immune-oncology (I/O), interest is focused on efforts to better understand and manipulate the immune system to either overcome tolerance or promote tumor cell killing. However, T cell responses are frequently assessed using in vitro cell-based assays that have traditionally focused on simple endpoint measurements, such as cytokine release or cell proliferation, which do not represent a true readout of potency (i.e., the ability of T cells to induce apoptosis of cancer cells). In addition, traditional T cell killing assays, including Cr⁵¹ or lactate dehydrogenase (LDH) release assays, usually have short-term end points, and use reagents or technology that can present technical challenges. Furthermore, from the drug discovery perspective, these assays are not well suited to high throughput 96 or 384-well screening assay formats.

In this chapter, we describe in detail reliable methods to quantify anticancer cell activity mediated by T cell killing in extended duration and kinetic assays using real-time measurement of apoptosis or cell lysis. We offer a comparison of the relative merits of two platforms, IncuCyte (a Sartorius brand) and xCELLigence (ACEA Biosciences), highlighting critical areas of assay optimization and discuss technical considerations. We have found these methods to be robust and believe them suitable for testing a broad spectrum of I/O therapeutic modalities either alone or in combination with additional agents. Their broad applicability will also be of value to researchers undertaking basic research as well as drug discovery in I/O or other therapeutic areas associated with measuring T cell responses.