Chimeric antigen receptor (CAR)-T cell therapy has shown spectacular objective clinical responses in the treatment of haematological malignancies. However, early trials employing CARs specific for solid tumour antigens such as carcinoembryonic antigen (CEA) have been far less successful. Preclinical studies suggest that this is partly due to several immunosuppressive mechanisms within solid tumours. Therefore, the aim of the project is to improve CAR-T cell therapy targeting CEA by increasing the potency of CAR-T cells and superseding the immunosuppressive tumour microenvironment. To achieve this, CAR-T cells which secrete the pro-inflammatory cytokine IL-12 or disrupt TGF-Î² or PD-1 signalling through the secretion of antagonistic scFvs were developed. Mouse anti-CEA CAR-T cells were successfully engineered to constitutively or inducibly express IL-12. Compared to non-IL-12-secreting CAR-T cells in vitro, IL-12-secreting CAR-T cells significantly improved CEA-specific cytotoxicity and increased IFN-Î³ production. Whilst in vivo results showed that a single dose of anti-CEA CD28-CD3Î¶ CAR-T cells constitutively secreting IL-12 mediated complete regression of subcutaneous CEA+ tumour in some mice, this observation needs to be reproduced. Furthermore, anti-CEA CD28-CD3Î¶ CAR-T cells inducibly secreting IL-12 did not achieve similar results. Combination of CAR-T cells constitutively or inducibly secreting IL-12 with lymphodepletion pre-conditioning via 5Gy total body irradiation (TBI) resulted in lethal toxicity, which was highly associated with IL-12. Mouse T cells were also effectively transduced to express an anti-CEA CD28-CD3Î¶ CAR construct and constitutively secrete Î±TGF-Î² or Î±PD-1 scFv with functional binding and blocking capacity. The secretion of scFv did not enhance the CEA-specific cytotoxicity and cytokine production of CAR-T cells upon co-culture in vitro. With regards to the in vivo function, scFv-secreting CAR-T cells could not efficiently eradicate subcutaneous CEA+ tumour, although delayed tumour growth was observed in the therapy of CAR-T cells secreting Î±PD-1 scFv. Given no significant difference in the infiltration of CD8+ T cells in tumour sites between mock T cell therapy and CAR-T cell therapy demonstrated by IHC analysis, the therapeutic effects of anti-CEA CAR-T cells were possibly limited by the level of T cell infiltration or CAR-T cell retention. Overall, this thesis has shown that immune modulation on anti-CEA CAR-T cells is a feasible immunotherapeutic strategy for solid tumours. The therapy of anti-CEA CAR-T cells constitutively secreting IL-12 appears to be efficacious in tumour regression in vivo, whilst it needs to be further validated. More efforts are also required to determine whether the inducible secretion of IL-12 or the constitutive secretion of Î±TGF-Î² or Î±PD-1 scFv results in improved anti-tumour responses of anti-CEA CAR-T cells.
|Date of Award||31 Dec 2020|
- The University of Manchester
|Supervisor||John Bridgeman (Supervisor), Anne Armstrong (Supervisor), Eleanor Cheadle (Supervisor) & Gray Kueberuwa (Supervisor)|