In Vitro Stimulation with WT1-Peptide Mix Elicits Functional Polyclonal CTL From Adult Donors and Cord Blood
Authors: Krishnadas Deepa Kolaseri , Stamer Mindy , Dunham Kim , Bao Lei , Lucas Kenneth 
Published: October 2019

Abstract
Abstract 2096
Elevated expression of Wilms’ tumor antigen (WT1) in most leukemias and limited expression on normal tissues makes WT1 a potential target for cancer immunotherapy. Fighting leukemia using in vitro generated WT1-CTL has been under active study. Previous studies have focused mainly on cloning WT1-CTL or generating CTL that are limited to a single epitope. We have recently generated WT1-A*0201 peptide-specific CTL from peripheral blood lymphocytes (PBL) of healthy adult donors by enriching and expanding antigen-specific CD137+ cells. Using this method we demonstrate the generation of polyclonal WT1-CTL from healthy adult donors and from cord blood by stimulating with an overlapping pool of peptides derived from full length WT1. The WT1-peptide mix (WT1-pMix) is a mix of 110 peptides consisting 15-mers overlapped by 11 amino acids. The WT1-pMix stimulated PBL from adult donors were selected based on CD137+ expression. After three stimulations, 0.3% of the cells were positive for CD137. These cells were expanded with anti-CD3 and IL-2 resulting in 100–200 fold expansion. Therefore, from a typical apheresis product yielding approximately 4–10 × 109 cells, approximately 1.2 – 3 × 109 WT1-specific cells could be generated for immunotherapy. The expanded CTL displayed antigen-specific IFN-γ production in four out of five adult donors studied. They also killed antigen-specific target cells and WT1 expressing leukemia lines in a MHC restricted manner. Occasionally, these CTL did not produce IFN-γ although they demonstrated killing. WT1-specific cytotoxicity directed against partially matched leukemia lines (Molt-4 and 697) sharing MHC-class I was partially blocked with mAb against MHC-class I but not against MHC-class II and an unmatched leukemia line (BA25) remained close to negative control. Similar to functionally active WT1-CTL from healthy adult donors, polyclonal CTL were generated from cord blood using WT1-pMix with a potential specificity for several HLA backgrounds. These CTL are CD4 and CD8 positive. Three out of five donors studied produced IFN-γ and lysed autologous B blast (BB) pulsed with WT1-pMix and partially HLA matched WT1 expressing leukemia lines. Since recipients of unrelated donor cord blood transplants have no options for donor derived cellular immunotherapy in the event of leukemic relapse, WT1-specific CTL generated from cord blood mononuclear cells could be used to prevent or treat leukemic relapse in these patients. This method utilizes unmodified PBL or cord blood lymphocytes to generate clinically relevant number of CTL in 5–6 weeks following initial stimulation, potentially avoiding the need for CD8+ selection. The generation of WT1-specific CTL using an overlapping WT1-pMix enhances our ability to treat patients from several HLA backgrounds. These findings provide the foundation for further pre-clinical studies in which these cells could be isolated from donor T lymphocytes, and then infused back into patients for treatment or prevention of leukemic relapse in high risk stem cell transplant (SCT) patients.

Disclosures:
No relevant conflicts of interest to declare.