Investigating determinants of sensitivity and resistance to T cell redirecting antibodies in colorectal cancer through patient derived organoid models

Abstract

Most colorectal cancers (CRCs) do not respond to treatment with immune-checkpoint-inhibitors due to modest mutation loads, low cytotoxic T cell infiltrates, and immunosuppressive microenvironments. The CEA-TCB bispecific antibody is a novel therapeutic agent that can help overcome the unfavourable immune landscape in CRC. CEA-TCB binds CD3 on T cells and targets them to the carcinoembryonic antigen (CEA) which is overexpressed on the cell surface of many CRC cells. Recent clinical trial data has shown that despite pre-selecting patients with CEA positive tumours, only 11% treated with CEA-TCB monotherapy responded to the treatment. Mechanisms of resistance to T cell redirecting antibodies remain poorly understood, indicating a need for pre-clinical model systems that can be used to dissect the determinants of CEA-TCB response and resistance. It has been demonstrated that patient-derived organoids (PDO) may more accurately represent patient tumours than established cell lines. The aim of my thesis was to use PDOs to investigate factors determining sensitivity and resistance to CEA-TCB therapy. After characterising CEA expression of eight PDOs generated from multi-drug resistant metastatic CRCs, three PDOs with persistently high CEA expression and good CEA-TCB response were selected to develop a co-culture technology of PDOs and allogeneic T cells which can be used to screen for microenvironmental factors that influence CEA-TCB activity. This model allowed me to evaluate the effect of different immunosuppressive cytokines and immune and stromal cell types commonly present in the CRC tumour microenvironment on CEA-TCB response. This screen revealed that TGFb, commonly overexpressed in CRCs, confers resistance to CEA-TCB by suppression of T cell cytolytic and proliferative abilities. I was able to reverse immunosuppressive effects of TGFb receptor inhibitor galunisertib, IL-2, and tumour targeted CEA-4-1BBL and stroma targeted FAP-IL2v bispecific T cell co-stimulatory agents, thus informing rational combination therapies for clinical testing. The 3D configuration of this co-culture model was used to assess T cell infiltration into the PDOs and evaluate the impact of different PDO sizes and morphologies on CEA-TCB mediated killing of tumour cells. Distinct PDO morphologies as well as large orgnaoid size did not impair T cell infiltration as CD8 T cells were able to penetrate into the centre of large organoids growing in different architectural patterns and showed strong killing activity. the versatile in vitro organoid and T-cell co-culture screening platform I developed can be used for identification of resistance mechanisms and rational combination therapies.