Abstract
Abstract 930
The orphan tyrosine kinase receptor ROR1 was previously identified as a highly expressed gene by expression profiling of B cell chronic lymphocytic leukemia (B-CLL), [Klein et al. J Exp Med 2001], and has subsequently been shown to be expressed on mantle cell lymphoma (MCL) and a subset of B cell acute lymphoblastic leukemias (B-ALL). ROR1 encodes a 105 kDa protein that contains Ig-like, cysteine rich, kringle, tyrosine kinase and proline rich domains and is expressed during embryonic development but is absent on normal adult tissues including non-malignant B cells. The function of ROR1 in normal and malignant cells is not known, although secreted Wnt proteins have been proposed as candidate ligands. Analysis of ROR1 protein expression using specific polyclonal antibodies revealed uniform, stable, and restricted cell surface expression on B-CLL, suggesting this molecule is a candidate for targeted immunotherapy of B cell malignancies [Baskar et al. Clin Cancer Res 2008]. We constructed a lentiviral vector that encodes a chimeric antigen receptor (CAR) consisting of single chain variable (scFV) fragments of the heavy and light chains of a murine monoclonal antibody specific for ROR1, linked to an IgG4 Fc domain, the T cell receptor CD3 zeta chain and a CD28 costimulatory domain. The specificity and function of the ROR1 CAR was compared with a similarly designed CAR specific for the CD20 molecule, which is expressed on both malignant and normal B cells, and is being targeted with gene-modified T cells in clinical trials. Primary human CD8+ T cells were transduced with the ROR1 and CD20-specific CARs respectively, and T cells expressing high levels of the receptors were sort-purified using an anti-Fc antibody. T cells that expressed either the ROR1-specific CAR or the CD20-specific CAR efficiently lysed primary B-CLL samples (5/5) obtained from patients with advanced disease, and also lysed a MCL cell line (JeKo-1), and a ROR1+ B-ALL cell line (BALL-1). ROR1-specific T cells did not recognize the myeloid leukemia cell line K562, but efficiently lysed K562 cells that had been transfected to express ROR1, confirming the specific recognition of ROR1 on target cells. Consistent with the expression pattern of the target molecules, T cells that expressed the CD20-specific CAR also efficiently lysed normal primary and EBV-transformed B cells, but T cells that expressed the ROR1-specific CAR did not recognize nonmalignant or EBV-transformed B cells. Activation of normal B cells by engagement of the B cell receptor or activation through CD40 induced B cell proliferation and upregulation of the CD80 and CD86 costimulatory molecules, but did not result in ROR1 surface expression by flow cytometry or recognition by T cells that expressed the ROR1-specific CAR. These results suggest that targeting ROR1 with gene-modified T cells may have advantages over targeting B cell-lineage restricted molecules such as CD19 and CD20 that are expressed on normal mature B cells. Studies to determine whether ROR1 is expressed during a stage of normal B cell development are in progress. ROR1 is highly conserved in non-human primates and this model may be suitable to determine potential toxicities of adoptive immunotherapy with ROR1-specific CAR expressing T cells.
Disclosures:
No relevant conflicts of interest to declare.
CD8+ T Cells Expressing an HLA-DR1 Chimeric Antigen Receptor Target Autoimmune CD4+ T Cells in an Antigen-Specific Manner and Inhibit the Development of Autoimmune Arthritis
