PF446 EVALUATING EX VIVO EXPANSION OF FUNCTIONALLY COMPETENT MULTI-SPECIFIC T LYMPHOCYTES TO MINOR HISTOCOMPATIBILITY ANTIGEN FOR THE TREATMENT OF HEMATOLOGICAL MALIGNANCIES

Abstract

Background: Allogeneic hematopoietic stem cell transplantation represents the sole curative treatment for high risk and relapsing leukemia. The presence of donor T cells in the graft mediates the powerful graft-versus-leukemia (GvL) effect. Recognition of minor histocompatibility antigen (MiHA) by T cells forms the basis of this GvL effect. MiHAs are small major histocompatibility antigen-restricted peptides encoded by germline polymorphism. Their preferential tissue restriction makes them an excellent target for adoptive T cell therapy for hematological malignancies. However, this form of anti-cancer activity is highly toxic due to the induction of detrimental graft-versus-host-disease mediated by donor T cells that recognize MiHAs expressed on non-hematopoietic cells. Thus, there is a scope for improving this form of adoptive T cell therapy by pre-activating and expanding donor-derived-T lymphocytes against specific MiHAs that are predominantly expressed on hematopoietic cells and may lead to improved cancer immunotherapy for hematologic cancers. Aims: The aim of this study was to evaluate the ex vivo expansion protocol of functionally competent MiHA specific CD8+ T cells. As an alternative to clinical studies where monocyte-derived dendritic cells (Mo-DC) are pulsed with single MiHA peptide, we propose an approach where Mo-DC pulsed with several MiHA peptides are used to activate and expand MiHA specific CD8+ T cells. We hypothesized that priming CD8+ T cells with multiple MiHAs may result in a multi-specificity product consisting of an oligoclonal T cell population reactive to multiple MiHAs and/or a dominant T cell response to one of the MiHAs. Further, we aim to understand potentially differential MiHA immunogenicity by quantifying MiHA presentation by dendritic cells using labeled-MiHAs. Methods: Mature Mo-DC were pulsed with single or multiple MiHA peptides, irradiated and co-cultured with autologous peripheral blood mononuclear cells for 21 days. Expansion of MiHA specific T cells was evaluated using dextramer staining. The functionality of antigen-specific T cells was assessed by interferon-γ (IFN-γ) and tumor necrosis factor-α production, CD107a degranulation, and IFN-γ ELISpot. In parallel, labeled-MiHA presentation by Mo-DC was quantified using flow cytometry. Results: In this study, we concluded on the feasibility of using Mo-DC pulsed with multiple peptides to expand MiHA specific T cells. Importantly, in several experiments, we also observed CD8+ T cells reactive to more than one MiHA demonstrating multiple antigen-specific responses within the same culture. However, variability was observed in the magnitude of this response. This variability might rely, in part, on the MiHA presentation by Mo-DC; Mo-DCs pulsed with four or more peptides resulted in higher cell mortality and had lower expression of the costimulatory molecule CD80, thus suggesting that pulsing Mo-DCs with four or more MiHAs may impact the antigen-presenting capacity of these cells. Interestingly, we demonstrated, using labeled-MiHAs that the peptides were presented by Mo-DC at variable levels. Summary/Conclusion: Our results demonstrate that we could generate a multi-specific T cell response using Mo-DC pulsed with multiple MiHA peptides. Ongoing experiments will shed light on the variability seen in the antigen presentation by Mo-DC derived from different donors and peptides used. Understanding the fundamental biology of MiHA specific T cells for cancer immunotherapy will allow us to develop a personalized approach for the treatment of high-risk leukemia patients.