Comparative Analysis of Clinical-Scale IFN-γ-Positive T-Cell Enrichment Using Partially and Fully Integrated Platforms

Christoph Priesner; Ruth Esser; Sabine Tischer; Michael Marburger; Krasimira Aleksandrova; Britta Maecker-Kolhoff; Hans-Gert Heuft; Lilia Goudeva; Rainer Blasczyk; Lubomir Arseniev; Ulrike Köhl; Britta Eiz-Vesper; Stephan Klöß

doi:10.3389/fimmu.2016.00393

Comparative Analysis of Clinical-Scale IFN-γ-Positive T-Cell Enrichment Using Partially and Fully Integrated Platforms

Front Immunol. 2016 Sep 30:7:393. doi: 10.3389/fimmu.2016.00393. eCollection 2016.

Affiliations

¹ Institute of Cellular Therapeutics, Hannover Medical School, Niedersachsen, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany.
² Institute for Transfusion Medicine, Hannover Medical School , Niedersachsen , Germany.
³ Institute of Cellular Therapeutics, Hannover Medical School , Niedersachsen , Germany.
⁴ Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany; Department of Pediatric Hematology and Oncology, Hannover Medical School, Niedersachsen, Germany.
⁵ Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany; Institute for Transfusion Medicine, Hannover Medical School, Niedersachsen, Germany.

Abstract

Background and aims: The infusion of enriched CMV-specific donor T-cells appears to be a suitable alternative for the treatment of drug-resistant CMV reactivation or de novo infection after both solid organ and hematopoietic stem cell transplantation. Antiviral lymphocytes can be selected from apheresis products using the CliniMACS Cytokine-Capture-System^® either with the well-established CliniMACS^® Plus (Plus) device or with its more versatile successor CliniMACS Prodigy^® (Prodigy).

Methods: Manufacturing of CMV-specific T-cells was carried out with the Prodigy and Plus in parallel starting with 0.8-1 × 10⁹ leukocytes collected by lymphapheresis (n = 3) and using the MACS GMP PepTivator^® HCMVpp65 for antigenic restimulation. Target and non-target cells were quantified by a newly developed single-platform assessment and gating strategy using positive (CD3/CD4/CD8/CD45/IFN-γ), negative (CD14/CD19/CD56), and dead cell (7-AAD) discriminators.

Results: Both devices produced largely similar results for target cell viabilities: 37.2-52.2% (Prodigy) vs. 51.1-62.1% (Plus) CD45⁺/7-AAD^- cells. Absolute numbers of isolated target cells were 0.1-3.8 × 10⁶ viable IFN-γ⁺ CD3⁺ T-cells. The corresponding proportions of IFN-γ⁺ CD3⁺ T-cells ranged between 19.2 and 95.1% among total CD3⁺ T-cells and represented recoveries of 41.9-87.6%. Within two parallel processes, predominantly IFN-γ⁺ CD3⁺CD8⁺ cytotoxic T-cells were enriched compared to one process that yielded a higher amount of IFN-γ⁺ CD3⁺CD4⁺ helper T lymphocytes. T-cell purity was higher for the Prodigies products that displayed a lower content of contaminating IFN-γ^- T-cells (3.6-20.8%) compared to the Plus products (19.9-80.0%).

Conclusion: The manufacturing process on the Prodigy saved both process and hands-on time due to its higher process integration and ability for unattended operation. Although the usage of both instruments yielded comparable results, the lower content of residual IFN-γ^- T-cells in the target fractions produced with the Prodigy may allow for a higher dosage of CMV-specific donor T-cells without increasing the risk for graft-versus-host disease.

Keywords: CliniMACS Cytokine-Capture-System; closed GMP-compliant systems; immunoaffinity cell selection; single platform and multicolor flow cytometry; virus-specific T-cells.