Background

Most colorectal cancers (CRCs) do not respond to checkpoint-inhibitor immunotherapy due to modest mutation loads and immunosuppressive microenvironments. The CEA-TCB bispecific antibody can help overcome these hurdles for more effective immunotherapy. CEA-TCB binds CD3 on T-cells and targets them to the carcinoembryonic antigen (CEA) which is overexpressed on the surface of many CRC cells. Recent clinical trial data showed that not all patients with CEA positive CRCs responded to this treatment. We developed immunocompetent CRC in vitro models to  assess the contribution of CD4 and CD8 T-cell subtypes to CEA-TCB efficacy and the potential role of CRC microenvironmental immunosuppressive factors for resistance.

Method

We developed co-culture systems incorporating patient derived CRC organoids (PDOs) and allogeneic CD8 or CD4 T-cells. We subsequently added cell types and microenvironmental factors which are commonly found in the CRC microenvironment and considered immunosuppressive. PDO killing and immune cell flow cytometry were main readouts. 

Results

CD4 and CD8 T-cells showed similar growth inhibition of PDOs when CEA-TCB treatment was added. However, CEA-TCB treated CD8 T-cells expressed higher granzyme levels than CD4 T-cells whereas treatment with IFNγ-neutralising antibodies revealed a greater dependency of CD4 T-cells on IFNγ as a mechanism of PDO growth control (57% reduction of CD4 T-cell mediated growth inhibition vs 15% reduction for CD8 T-cells). Inhibiting Fas-FasL interactions had no effect on CEA-TCB efficacy with CD4 or CD8 T-cells. Ongoing testing of IL-10, TGFβ, M2 macrophages, regulatory T-cells and hypoxia (1% O2), all microenvironmental factors that have been described as immunosuppressive in the CRC microenvironment, showed that TGFβ most effectively impaired tumour control. When treated with TGFβ in the presence of CD8 T- cells 57% of PDO growth was restored compared to 51% with CD4 T-cells.

Conclusion

Our data shows that CD4 T-cells can acquire granzyme expression and use IFNγ as a main mechanism of CEA-TCB mediated tumour control. TGFβ was the most potent immunosuppressive factor for CEA-TCB efficacy among the tested conditions and cell types. We will now investigate combination therapies to counter immunosuppressive TGFβ effects on CD8 and CD4 T-cells.

Impact statement

Our results will inform future combination therapies with the bispecific antibody CEA-TCB in CRC.