Date of Graduation
5-2014
Document Type
Dissertation (PhD)
Program Affiliation
Immunology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Laurence Cooper, M.D., Ph.D.
Committee Member
Oliver Bogler, Ph.D.
Committee Member
Bradley McIntyre, Ph.D.
Committee Member
Jeffrey Molldrem, M.D.
Committee Member
Kimberly Schluns, Ph.D.
Abstract
T cells can be redirected to target tumor-associated antigen (TAA) by genetic modification to express a chimeric antigen receptor (CAR), which fuses the specificity derived from an antibody to T-cell activation domains to result in lysis of TAA-expressing cells. Due to the potential for on-target, off-tissue toxicity, CAR+ T-cell therapy is currently limited to unique or lineage-restricted TAAs. Glioblastoma, a grade IV brain malignancy, overexpresses epidermal growth factor receptor (EGFR) in 40-50% of patients. EGFR also has widespread normal tissue expression. To target EGFR on glioblastoma while reducing the potential for normal tissue toxicity, EGFR-specific CAR generated from cetuximab, Cetux-CAR, was transiently expressed in T cells by RNA-modification. RNA-modified CAR+ T cells demonstrated similar cytotoxicity against EGFR+ cells, including normal renal cells, as DNA-modified CAR+ T cells. However, RNA-modified T cells lost CAR expression over time, concomitant with loss of functional specificity to EGFR. Transient expression of CAR limits potential for off-tissue toxicity at the expense of anti-tumor activity, and does not protect normal tissue from immediate toxicity. Recognizing that EGFR is overexpressed at a higher density on glioblastoma relative to normal tissue, we generated an EGFR-specific CAR from nimotuzumab, an EGFR-specific antibody with reduced binding to low density EGFR. While Cetux-CAR+ T cells produced cytokine and mediated lysis independent of EGFR density, function of Nimo-CAR+ T cells directly correlated with the EGFR density of targets, with reduced activity in response to low density EGFR, but equivalent activity in response to high density EGFR relative to Cetux-CAR+ T cells. Cetux-CAR+ T cells and Nimo-CAR+ T cells demonstrated equivalent control of intracranial glioma xenograft with intermediate EGFR density, but only Cetux-CAR+ T cells controlled xenografts with low EGFR density. In sum, transient expression of CAR has the potential to reduce long-term toxicity to normal tissue, but at the expense of anti-tumor activity. Rational design of CAR based on an antibody with reduced binding to low density EGFR generated EGFR-specific CAR able to tune T-cell function to antigen density resulting in discrimination of high EGFR density on malignant cells from low EGFR density on normal tissue.
Keywords
genetically modified T cells, cancer immunotherapy, affinity, EGFR, glioma