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* The Breast Cancer Biology Group and
Department of Immunobiology, King’s College London School of Medicine,
Department of Clinical Immunology and Allergy, King’s College Hospital National Health Service Foundation Trust; and
Centre for Cancer Imaging, Institute of Cancer and the Cancer Research U.K. Clinical Centre, Barts and The London, Queen Mary’s School of Medicine and Dentistry, Department of Nuclear Medicine, St. Bartholomew’s Hospital, London, United Kingdom
MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glycosylation, and loss of polarity in >80% of human cancers. To exploit this, we have constructed a panel of chimeric Ag receptors (CAR) that bind selectively to tumor-associated MUC1. Two parameters proved crucial in optimizing the CAR ectodomain. First, we observed that the binding of CAR-grafted T cells to anchored MUC1 is subject to steric hindrance, independent of glycosylation status. This was overcome by insertion of the flexible and elongated hinge found in immunoglobulins of the IgD isotype. Second, CAR function was highly dependent upon strong binding capacity across a broad range of tumor-associated MUC1 glycoforms. This was realized by using an Ab-derived single-chain variable fragment (scFv) cloned from the HMFG2 hybridoma. To optimize CAR signaling, tripartite endodomains were constructed. Ultimately, this iterative design process yielded a potent receptor termed HOX that contains a fused CD28/OX40/CD3
endodomain. HOX-expressing T cells proliferate vigorously upon repeated encounter with soluble or membrane-associated MUC1, mediate production of proinflammatory cytokines (IFN-
and IL-17), and elicit brisk killing of MUC1+ tumor cells. To test function in vivo, a tumor xenograft model was derived using MDA-MB-435 cells engineered to coexpress MUC1 and luciferase. Mice bearing an established tumor were treated i.p. with a single dose of engineered T cells. Compared with control mice, this treatment resulted in a significant delay in tumor growth as measured by serial bioluminescence imaging. Together, these data demonstrate for the first time that the near-ubiquitous MUC1 tumor Ag can be targeted using CAR-grafted T cells.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by a Royal College of Pathologists/ Health Foundation Senior Clinician Scientist Research Fellowship (to J.M.), Breast Cancer Campaign Project Grant 2003:552 (to J.M.), and a Cancer Research U.K. Programme Grant (to S.J.M.).
2 Address correspondence and reprint requests to Dr. John Maher, Breast Cancer Biology Group, Division of Cancer Studies, Third Floor Thomas Guy House, King’s College London School of Medicine, Guy’s Hospital, St. Thomas Street, London SE1 9RT, U.K. E-mail address: john.maher{at}kcl.ac.uk
3 Abbreviations used in this paper: VNTR, variable number tandem repeat; BLI, bioluminescence imaging; CAR, chimeric antigen receptor; CM, conditioned medium; ffLUC, firefly luciferase; mIgG, mouse IgG; 435-MUC-LUC, MUC1+ffLUC+ MDA-MB-435 cell; scFv, single-chain variable Ab fragment; sTn, sialyl Tn.
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