Manufacturing and validation of Good Manufacturing Practice-compliant regulatory dendritic cells for infusion into organ transplant recipients

Abstract

Background aims: Regulatory (or "tolerogenic") dendritic cells (DCregs) are a highly promising, innovative cell therapy for the induction or restoration of antigen-specific tolerance in immune-mediated inflammatory disorders. These conditions include organ allograft rejection, graft-versus-host disease following bone marrow transplantation and various autoimmune disorders. DCregs generated for adoptive transfer have potential to reduce patients' dependence on non-specific immunosuppressive drugs that can induce serious side effects and enhance the risk of infection and certain types of cancer. Here, our aim was to provide a detailed account of our experience manufacturing and validating comparatively large numbers of Good Manufacturing Practice-grade DCregs for systemic (intravenous) infusion into 28 organ (liver) transplant recipients and to discuss factors that influence the satisfaction of release criteria and attainment of target cell numbers.

Results: DCregs were generated in granulocyte-macrophage colony stimulating factor and interleukin (IL)-4 from elutriated monocyte fractions isolated from non-mobilized leukapheresis products of consenting healthy adult prospective liver transplant donors. Vitamin D3 was added on day 0 and 4 and IL-10 on day 4 during the 7-day culture period. Release and post-release criteria included cell viability, purity, phenotype, sterility and functional assessment. The overall conversion rate of monocytes to DCregs was 28 ± 8.2%, with 94 ± 5.1% product viability. The mean cell surface T-cell co-inhibitory to co-stimulatory molecule (programmed death ligand-1:CD86) mean fluorescence intensity ratio was 3.9 ± 2.2, and the mean ratio of anti-inflammatory:pro-inflammatory cytokine product (IL-10:IL-12p70) secreted upon CD40 ligation was 60 ± 63 (median = 40). The mean total number of DCregs generated from a single leukapheresis product (n = 25 donors) and from two leukapheresis products (n = 3 donors) was 489 ± 223 × 10⁶ (n = 28). The mean total number of DCregs infused was 5.9 ± 2.8 × 10⁶ per kg body weight. DCreg numbers within a target cell range of 2.5-10 × 10⁶/kg were achieved for 25 of 27 (92.6%) of products generated.

Conclusions: High-purity DCregs meeting a range of quality criteria were readily generated from circulating blood monocytes under Good Manufacturing Practice conditions to meet target cell numbers for infusion into prospective organ transplant recipients.

Keywords: Cell therapy; Clinical trials; GMP manufacturing; Regulatory dendritic cells; Release criteria.