Improvement of Methodologies for Engineering and Use of Chimeric Immune Receptors in Adoptive Immunotherapy of Cancer

Abstract

The adoptive transfer of CIR expressing T cells is a promising new strategy in cancer immunotherapy. However, to improve the methodology, a safer and more robust method for the delivery of CIR genes into primary T cells is required. The current clinical protocol for CIR adoptive transfer involves retroviral delivery systems, which raise safety issues concerning long-term expression and the risk of insertional mutagenesis. Therefore, in this study a non-viral, electroporation-based nucleofection was assessed for the transfer of CIR genes into primary T cells. Preliminary experiments involving nucleofection of Jurkat T cells resulted in very high CIR expression (70%-90%), maintained for at least 5 days with very high survival (80%-90%). CIR expression was also found very high in stimulated human T cells with 80% cells expressing CIR, compared to 30% in non-stimulated T cells. The viability of stimulated human T cells was observed to initially fall to ~30% but then was followed by rapid recovery, whereas the viability of nucleofected non-stimulated human T cells was stably maintained at ~60%. Murine T cells were found to be more difficult to nucleofection with highest expression levels of 30% and cell survival less than 40%. Nucleofection was not found to be detrimental to T cell function as CIR modified T cells retained the ability to undergo CIR directed activation and upregulated expression of CD69 following antigen stimulation. However, in the preliminary in vivo experiment using a CEA tumour mouse model, these CIR nucleofected cells did not show any obvious anti-tumour effect. To further improve CIR function and make the CIR therapy applicable to a wide range of malignancies, a wide repertoire of scFv's specific to tumour antigen is required. The current method for the identification of therapeutic scFv's is usually phage display. Although phage display has proven to be successful in many cases, its main limitation is the fact that it uses a prokaryotic based system. Many scFv's identified using phage display encounter translational problems when expressed in eukaryotic systems. Therefore, this study also sought to develop an alternative screening method in which scFv's are identified in a eukaryotic system, and in the context of CIR structure. Analysis of several model systems identified the Jurkat T cell line and antigen-dependent CD69 upregulation as an appropriate model for the selection of antigen-specific scFv's. Proof of principle experiments based on this system that involved mini-libraries consisting of two scFv's: MFE, a scFv specific for CEA and anti-CD19, which does not bind to CEA, showed up to 4-log enrichment of MFE scFv following two rounds of selection. The feasibility and efficiency of the developed screening protocol was further proved by screening of a CEA specific scFv library, in which 5-fold enrichment of MFE was achieved in a single selection round. We also generated a CA19-9 specific scFv library and performed a preliminary screen in which no CA19-9 specific scFv's were identified due to the low diversity of the scFv repertoire in the scFv library.

Date of Award	1 Aug 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Robert Hawkins (Supervisor), David Gilham (Supervisor) & Dominic Rothwell (Supervisor)