Ex Vivo Expansion of Donor-Derived Anti-Tumor T Cells for CLL-Specific Adoptive Immunotherapy

Abstract 978

Although chronic lymphocytic leukemia (CLL) is sensitive to graft-versus-leukemia (GVL) effects, strategies to enhance donor-derived tumor immunity are needed to prevent relapse and improve outcomes after allogeneic hematopoietic stem cell transplantation (HSCT). Post-transplant infusion of mature donor T cells specific for recipient CLL cells could provide an effective treatment approach personalized to the individual tumor. However, unmanipulated CLL cells are weak antigen presenting cells (APCs), expressing low levels of costimulatory molecules, and therefore only poorly stimulate the expansion of tumor-reactive T cells.

To overcome this barrier, we evaluated a novel formulation of human recombinant CD40L, a molecule known to enhance the immunostimulatory capacity of normal and malignant B cells. This formulation of CD40L (designated CD40L-Tri) was designed with the extracellular domain of CD40L connected by a long flexible linker to a leucine zipper for trimerization and an octahistidine motif for purification. We compared the immunostimulatory activity of CD40L-Tri with a murine fibroblast cell line that was engineered to express human CD40L (tCD40L/NIH3T3). In 3 of 3 cases, CD40L-Tri (at 0.5, 1, and 2 mg/ml) significantly expanded normal CD19+ B cells over 14 days by an average fold change of 21.5, 27.0 and 29.5, respectively (all p<0.05). We further observed that exposure of normal CD19+ B cells to CD40L-Tri (at 2mg/ml, n=3) resulted in significantly increased expression of the costimulatory molecules CD80, CD83 and CD86 at 48 hours by an average fold change of 28.7, 24, and 169.9, respectively(all p<0.05), which was comparable to the average fold change of tCD40L/NIH3T3 (24.7, 21.8, and 144.9, respectively, all p<0.05). In three separate experiments, thymidine incorporation assays revealed that exposure of normal B cells to CD40L-Tri consistently generated APCs with high capacity to stimulate allogeneic CD4+ and CD8+ T cells, at comparable levels to tCD40L/NIH3T3 cells. Consistent with these results, CD40L-Tri-activated B cells could be used to present pulsed peptide to specifically expand T cells specific for the influenza peptide M1 from a normal HLA-A2+ volunteer. Together, these studies confirmed that CD40L-Tri has potent immune-stimulatory effects on B cells, and can be utilized to expand human T cells without the risk of expansion of xenoantigen-specific T cells incurred through the use of conventional tCD40L/NIH3T3 cells.

To evaluate the ability of CD40L-Tri to facilitate the expansion of CLL-specific T cells, we examined T cell responses against 4 cryopreserved CLL tumors for which cryopreserved HLA-matched normal donor cells were available. Donor CD8+ T cells were subjected to four weekly in vitro stimulations with CD40L-Tri-activated recipient CLL-B cells in the presence of IL-7, IL-12, and IL-15. In 3 of 4 cases, donor CD8+ T cells with specificity against recipient tumor were expanded. These CTL were not reactive with recipient-derived fibroblasts or PHA blasts, suggesting that T cell reactivity was tumor- rather than allo-specific. CD8+ CLL-reactive T cells secreted IFNg by Elispot and were cytotoxic to recipient CLL cells. Reactivity was blocked in the presence of MHC class I blocking antibody (W6/32). Cloning of CLL-reactive T cells from 2 patients resulted in the isolation of up to 8 tumor-reactive T cell clones each, that were confirmed to have tumor-specific and MHC class I-restricted reactivity. These T cell clones were also reactive against other CLL cells that shared at least one HLA molecule with original donor/recipient, suggesting that these CTL clones recognize common CLL antigens.

Our results demonstrate that the combination of CD40L-Tri-activation of CLL cells and supportive cytokines can reliably generate CLL-specific T cells from HLA-matched donors. Our ongoing studies focus on the identification of CLL antigens recognized by these T cells, which may serve to identify useful immunogens for CLL immunotherapy. These studies suggest a potentially effective strategy for adoptive T cell therapy to enhance GVL after allogeneic HSCT in patients with CLL and possibly other mature B cell malignancies.