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Development of GMP-Compatible Protocols for Thymus-Derived Regulatory T Cell Expansion

MacDonald, Katherine1,2; Hoeppli, Romy3,8; Piret, James2,4; Dijke, Esmé5,6,8; West, Lori5,6,7,8; Levings, Megan3,8

doi: 10.1097/

1Department of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada; 2Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada; 3Department of Surgery, University of British Columbia, Vancouver, BC, Canada; 4Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, BC, Canada; 5Department of Pediatrics, University of Alberta, Edmonton, AB, Canada; 6Alberta Transplant Institute, University of Alberta, Edmonton, AB, Canada; 7Department of Surgery, University of Alberta, Edmonton, AB, Canada; 8Canadian National Transplant Research Program, Edmonton, AB, Canada.

Introduction: Animal and early clinical studies have shown that regulatory T cell (Treg) therapy can prevent graft-versus-host disease (GVHD) following hematopoietic stem cell transplant. Despite these promising results, challenges remain in administering Treg therapy including the difficulty in isolating pure Tregs and expanding them to a therapeutic dose. We have previously found that the thymus, which is routinely removed during pediatric cardiac surgery, is a plentiful source of Tregs. These Tregs can be easily isolated, and can prevent xenoGVHD in mice. However, to use this therapy in humans, standard GMP-compliant methods for isolation and large-scale expansion of thymic Tregs must be developed.

Methods: CD4+CD8-CD25+ thymic Tregs were isolated from pediatric thymuses using GMP-compatible magnetic bead-based separation. Tregs were expanded using artificial antigen-presenting cells or GMP-compatible antibody tetramer complexes that stimulate CD3 and CD28, with or without additional activation of CD2. Cells were cultured with rapamycin and IL-2, and were restimulated after 7 days without rapamycin.

Results: Thymic Tregs expand in vitro when cultured with tetrameric antibody complexes. About 50-fold expansion was achieved using the anti-CD3/CD28/CD2 antibody tetramers, which represents the expansion that will be necessary to reach a clinically relevant dose. A higher expansion rate was obtained using the anti-CD3/CD28/CD2 antibody tetramer complexes than the anti-CD3/CD28. Cells cultured with these antibody tetramers express high levels of FOXP3, similar levels of Treg-characteristic markers as those expanded with artificial antigen-presenting cells, and do not produce inflammatory cytokines.

Conclusion: Thymic Tregs can be isolated and expanded with GMP-compliant methods. Once the protocol is optimized, we can transfer our protocols to a GMP facility for further process development and to generate data required for a clinical trial application. We are the first group to pursue clinical application using allogeneic thymic Tregs, which provide an opportunity for an “off-the-shelf” therapy to treat GVHD.

Canadian National Transplant Research Program.

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