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Development of breast cancer immunotherapy using MUC1 retargeted T lymphocytes
Breast Cancer Research volume 8, Article number: P38 (2006)
The MUC1 mucin represents an excellent target for breast cancer immunotherapy since it is overexpressed and under-glycosylated in 90% of cases. To exploit this, we are developing a genetic approach to retarget T-cell specificity to MUC1, using chimeric antigen receptor (CAR) technology.
A panel of MUC1-specific CAR have been generated using scFv derived from the SM3 and HMFG2 hybridomas. All CAR were generated by overlap extension PCR and incorporate a fused signalling domain comprising CD28 and CD3ζ. Stable CAR expression was achieved in up to 75% of human T cells using the SFG oncoretroviral expression vector, following activation using PHA or CD3+28 beads.
Our first-generation MUC1-specific CAR, termed S28z, contained an SM3 scFv fused to a CD28 hinge. Surprisingly, however, S28z grafted T cells were poorly activated by a MUC1 + IgG fusion protein or MUC1 expressing T47D breast cancer cells. By contrast, S28z enabled T-cell activation when the MUC1 epitope was presented as a crosslinked peptide. Together, these findings suggested that steric hindrance and/or poor access to the epitope are limiting factors in CAR-based targeting of MUC1. To overcome this, a flexible monomeric hinge derived from IgD was introduced, thereby creating SD28z. Despite reduced stability, the SD28z CAR enabled T cells to proliferate in response to MUC1 glycoforms found in breast cancer. Stability of SD28z was further improved by inclusion of IgG1 Fc sequences in the extracellular domain (giving SDF28z). SDF28z exhibited greater functional activity, enabling T cells to kill T47D tumour cells. In a second approach to optimize function, a scFv was cloned from the MUC1-specific HMFG2 hybridoma. HMFG2 binds breast tumour cells with greater intensity than SM3. In keeping with this, all HMFG2-derived CAR exhibited greater functional activity than their SM3 counterparts. In the MUC1-specific CAR that exhibits greatest activity (HDF28z), an HMFG2 scFv has been fused to the IgD hinge and IgG1 Fc (HDF28z). HDF28z grafted human T cells exhibit potent cytolytic activity against MUC1 expressing breast cancer cells, associated with cytokine production and subsequent T-cell clonal expansion.
Following extensive protein engineering, we have developed a stable and highly potent CAR to retarget human T cells to the ubiquitous tumour antigen MUC1.
This work is supported by a Health Foundation/Royal College of Pathologists Senior Clinician Scientist Research Fellowship and a Project Grant awarded by Breast Cancer Campaign.
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Wilkie, S., Brenner, M., Taylor-Papadimitriou, J. et al. Development of breast cancer immunotherapy using MUC1 retargeted T lymphocytes. Breast Cancer Res 8 (Suppl 2), P38 (2006). https://doi.org/10.1186/bcr1593