591. Development of Anti-JC Virus “Designer T Cells” for Progressive Multifocal Leukoencephalopathy (PML) Immunotherapy
Authors: Wen Yang, Esther Beaudoin, Renaud A. Du Pasquier, Igor J. Koralnik, Richard P. Junghans
Journal: Molecular Therapy (2006)

Progressive multifocal leukoencephalopathy (PML) is a deadly brain disease caused by the polyomavirus JC (JCV), mainly arised in subjects with immunodeficiency. We and other have shown that some patients who are able to mount a cellular immune response against JCV survive PML, those who have undetectable JCV specific T cells, as seen by the absence of T cell receptors (TCRs) recognizing JCV peptides, have a rapidly fatal outcome. "Designer T cells" armed with anti-JCV TCRs by gene modification would be a genuine therapy for PML, by providing PML patients the missing T cells with anti-viral specificity. The aim of this study is to develop such "designer T cells". Two immunodominant CTL epitopes derived from JCV VP1 protein (termed p36 and p100 respectively) in HLA- A0201+ subjects, were previously identified by one of our labs. In this study, T cells lines specific to these antigenic peptides and enriched for certain TCR Vb usage were first generated, from an HLA-A0201+PML survival by repeated in vitro antigen stimulation followed by cell-sorting for peptide/HLA-A0201 positive cells and in combination with specific anti-TCR Vbeta antibodies. Two cell lines, one specific to p36 and enriched for Vb5+ T cells and another specific to p100 and selected for Vb2+, were then chosen for TCR cloning. Two distinct dominant a chains (Va6 and Va12) and a unique beta chain (Vb5.1) were cloned from the p36-specific cell line, while only one alpha (Va8.6) and one beta (Vb2) chain were dominantly from the p100-specific line. The Vb usage of the two cloned dominant beta chains (Vb5.1 and Vb2) are consistent with specificity of the anti-Vb antibodies used for the enrichment of the T cell lines. Based on the 5 cloned TCR alpha and beta chains, DNA constructs encoding chimeric immune receptors (CIRs) were created that the extracellular domains of TCR alpha or beta chains were fused to the transmembrane and cytoplasmid portions of a CD3zeta (VaCaCD3z or VbCbCD3z). Recombinant retroviruses encoding each of the 5 CIRs were then constructed. PG13 cells were co-transduced with each pair of alpha and beta chains of CIR format with corresponding recombinant retroviruses (two pairs for p36 due to two alpha chains and one for p100). The surface expression of the CIRs was confirmed by FACS in association with specific anti-Vb antibodies. The PG13 cells transduced with the pair of CIRs derived from p100 specific T cells lines (Va8.6 and Vb2) were stained specifically with the p100/ HLA-A0201 tetramer as shown by FACS analysis; however, but not surprisingly, only cells tranduced with one of the two possible combination of the alpha and beta chains, Va12+Vb5.1, were specifically bond by the p36/HLA-A0201 tetramer. Tranduced cells expressing either of the two functional pairs of anti-JCV CIRs were not stained by unrelated p36 or p100/HLA-A0201 tetramer, indicating the functional paired CIRs remain their antigen specificity. Further functional tests of the CIRs with human T cells are currently undergoing.