Targeting Lewis Y-Positive Multiple Myeloma and Acute Myeloid Leukemia with Gene-Modified T Cells Demonstrating Memory Phenotype

Abstract

AML and MM are sensitive to immune control as evidenced by T cell mediated allogeneic graft versus leukemia/myeloma effect. Adoptive immunotherapy with gene modified T cells has shown clinical activity in some solid tumors and B cell non Hodgkin lymphomas. The carbohydrate antigen Lewis Y (LeY) is a tumor associated antigen expressed on numerous epithelial cancers. Our aim was to generate gene-modified clinical-grade T cells directed against LeY positive hematological malignancies. Moreover, we aimed to produce cells that possessed T cell memory, essential for in vivo T cell persistence and long-term control of tumor cell targets. MM and AML cell lines were found to express differing levels of the LeY antigen ranging from negative (median fluorescence intensity (MFI) equal to mature lymphocytes (lymph) as internal control) to strongly positive (up to10×MFI lymph). Furthermore, 25/46 (54%) and 15/29 (52)% of primary MM and AML bone marrow samples were LeY-positive (≥5×MFI lymph), respectively. Lewis Y expression did not correlate with patient age, gender, clinical risk status, cytogenetic abnormalities, extent of previous chemotherapy, degree of bone marrow infiltrate, disease subtype, cytopenias in peripheral blood (MM and AML), LDH, WBC > or ≤ 30×109/L (AML), beta2 microglobulin, albumin or pretreatment with bortezomib, thalidomide or lenalidomide (MM). We manufactured a novel retroviral vector construct enabling efficient transduction of PBMC-derived T cells with resultant high expression (up to 65%) of a single-chain anti-LeY chimeric T cell receptor comprising T cell activation via CD3zeta and CD28 signaling domains. Under GMP conditions, we achieved >100-fold expansion of T cells using a 12 day culture protocol. Anti-LeY T cells lysed LeY-positive tumor cells in vitro while sparing LeY-negative control tumor cells and LeY expressing neutrophils (moderate LeY expression, >3×MFI lymph). Similar transduction rates were achieved in CD4 and CD8 T cell subsets. Twelve-day culture T cells showed low expression levels of CD45RA and CCR7, moderate levels of the co-stimulatory molecules CD27 and CD28, and active proliferation in response to IL-2 and IL-15, suggesting an effector memory phenotype. On re-exposure to LeY expressing tumor cells, anti-LeY T cells displayed active proliferation and IFN-gamma production. In vivo efficacy was demonstrated in three independent experiments of a MM xenograft mouse model showing improved disease free survival of mice receiving anti LeY T cells compared to control mice treated with untransduced T cells. Transplantation of syngeneic murine anti LeY T cells into sublethally irradiated Balb/C mice subsequently monitored for up to two years was found to be safe without generation of lymphoproliferative disorders arising from the anti LeY T cells and no impact on OS compared to irradiated control mice not receiving adoptive T cell transfer. Consequently, we have developed a first in human phase I trial of autologous anti LeY T cells for patients with LeY-positive MM or AML. LeY is a promising and immunologically relevant target for T cell immunotherapy and our product is likely to lead to persistence of anti-LeY T cells in patients, an outcome which will be specifically addressed in our upcoming study.