MUC1 is a highly attractive target for immunotherapy of breast cancer owing to its overexpression, altered glycosylation and loss of polarity in over 90% of tumours. To exploit this, we are developing genetic approaches to retarget T-cell specificity to MUC1 using chimeric antigen receptor (CAR) technology.
A panel of CARs have been generated using scFv derived from the SM3 and HMFG2 hybridomas. Using the SFG oncoretroviral expression vector, gene transfer was achieved in up to 75% of human T cells.
Two parameters proved crucial in engineering an optimized CAR ectodomain. First, we found that MUC1-mediated activation of engineered human T cells is subject to steric hindrance. This was observed using anchored but not soluble MUC1 and was independent of MUC1 glycosylation status. To circumvent this, we increased the flexibility and reach of CAR binding arms using the elongated hinge found in IgD. Second, CAR function was highly dependent upon strong binding capacity across a broad range of tumour-associated MUC1 glycoforms, including MUC1 Tn, T and sialylated derivatives. This was realized using an scFv cloned from the HMFG2 hybridoma. To optimize CAR signalling, tripartite endodomains were constructed that contain modules derived from TNF receptor family members in addition to CD28 and CD3ζ. Ultimately, this iterative design process yielded a potent MUC1-specific CAR termed HOX that contains a fused CD28/OX40/CD3ζ endodomain. HOX-expressing T cells proliferate vigorously in vitro upon repeated encounter with soluble or membrane-associated MUC1, mediate production of proinflammatory cytokines (IFNγ and IL-17) and elicit brisk antigen-dependent killing of MUC1+ tumour cells. To test function in vivo, a human breast cancer xenograft model has been established using MDA MB 435 tumour cells engineered to coexpress MUC1 and firefly luciferase. When introduced into SCID/Beige mice by intraperitoneal injection, rapid tumour growth occurs that can be monitored longitudinally and noninvasively by bioluminescent imaging. Mice bearing established tumour have been treated intraperitoneally with a single dose of human T cells grafted with HOX, two control CARs (DOX: lacking the HMFG2 scFv; HDFTr: lacking a functional endodomain) or medium alone. We observed that treatment with HOX-expressing T cells resulted in a significant delay in tumour growth, as measured by bioluminescent imaging, compared with control mice (Figure 1). In addition, HOX-grafted T cells confer a significant survival advantage upon mice bearing MCF7 breast cancer xenografts.
Despite its role in tumorigenesis and immune evasion, we show that the near-ubiquitous breast cancer antigen MUC1 can be targeted using CAR grafted T cells.
Supported by a Health Foundation/Royal College of Pathologists Senior Clinician Scientist Research Fellowship and a Project Grant awarded by Breast Cancer Campaign.
Breast Cancer Biology Group, King's College London, Guy's Hospital, London, UK
Centre for Cancer Imaging, Institute of Cancer and the CR-UK Clinical Centre, Barts and The London, Queen Mary's School of Medicine and Dentistry, Department of Nuclear Medicine, St Bartholomew's Hospital, London, UK
Department of Immunobiology, King's College London, Guy's Hospital, London, UK
Department of Allergy and Clinical Immunology, King's College Hospital, London, UK