Development of breast cancer immunotherapy using MUC1 retargeted T lymphocytes

Background

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.

Methods

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.

Results

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.

Conclusion

Following extensive protein engineering, we have developed a stable and highly potent CAR to retarget human T cells to the ubiquitous tumour antigen MUC1.