A super-resolution platform for in situ molecular analysis of receptor co-localization mediated by co-stimulatory trispecific antibodies

Trispecific T cell-engager (TriTCE) antibodies are an emerging class of therapeutics designed to enhance the body’s ability to fight cancer by targeting three distinct sites. Bispecific TCE antibodies, which target two binding sites, have been developed and clinically approved for treating various types of cancer. These antibodies typically work by binding to both a tumor antigen and the T cell receptor (CD3), thus activating T cells and redirecting cytotoxicity towards cancer cells. However, their effectiveness relies on having a high baseline of T cell infiltration in the tumor microenvironment. This poses a challenge for treating solid tumors that are poorly infiltrated by T cells and rapidly growing, as the bispecific antibodies may not adequately inhibit their growth.

To overcome this limitation, co-stimulatory TriTCE antibodies have been developed. These antibodies not only target a tumor antigen and CD3, but also stimulate a second co-stimulatory T cell signal, known as CD28, which has been shown to enhance T cell activation. This trispecific design has demonstrated enhanced cytokine production and sustained T cell proliferation, leading to improved cytotoxicity against tumor cells. However, the precise mechanism by which TriTCE antibodies bring together the tumor cell antigen, CD3, and CD28 to enhance co-stimulation remains poorly understood.

Super-resolution microscopy offers a powerful tool to visualize and understand protein interactions within cells. Fluorescent imaging enables the direct visualization of the three key targets: the tumor cell antigen, CD3, and CD28. However, traditional fluorescence imaging does not have the resolution to visualize these interactions. With the ability to achieve up to 20nm resolution, super-resolution microscopy can directly image and distinguish the organization of the immune synapse by co-engagement of the tumor cell antigen, CD3, and CD28. Super-resolution microscopy will enable us to observe how TriTCEs organize their targets in the intact cell.

Our project aims to utilize super-resolution microscopy to gain insights into the mechanism of action of TriTCEs. The intern will play a crucial role in integrating super-resolution microscopy with research on TriTCEs. The intern will learn to apply super-resolution microscopy for therapeutic applications and learn to work in an industrial setting. The intern will utilize STED, dSTORM super resolution microscopes, as well as conventional fluorescence microscopy. The intern will optimize labeling parameters, such as antibody concentrations and microscope settings, to identify optimal imaging conditions. Additionally, the intern will present qualitative visualizations and quantitative measures for the interaction of TriTCEs and develop new analysis paradigms for assessing and quantifying receptor-target interactions.

Through this work, we aim to better understand how these antibodies organize their targets, ultimately contributing to our understanding of how co-stimulatory TriTCEs function. Implementing super resolution imaging technology will lead to the development of an analysis platform to provide in situ molecular analysis of the efficacy of current and future trispecific antibody therapeutics under development at Zymeworks Inc.