Development and Optimization of a GMP-Compliant Manufacturing Process for a Personalized Tumor Lysate Dendritic Cell Vaccine

Abstract

With the emergence of immune checkpoint inhibitors and adoptive T-cell therapies, there is a considerable interest in using personalized autologous dendritic cell (DC) vaccines in combination with T cell-targeting immunotherapies to potentially maximize the therapeutic impact of DC vaccines. Here, we describe the development and optimization of a Good Manufacturing Practice (GMP)-compliant manufacturing process based on tumor lysate as a tumor antigen source for the production of an oxidized tumor cell lysate loaded DC (OC-DC) vaccine. The manufacturing process required one day for lysate preparation and six days for OC-DC vaccine production. Tumor lysate production was standardized based on an optimal tumor digestion protocol and the immunogenicity was improved through oxidation using hypochloric acid prior to freeze-thaw cycles resulting in the oxidized tumor cell lysate (OC-L). Next, monocytes were selected using the CliniMACS prodigy closed system and were placed in culture in cell factories in the presence of IL-4 and GM-CSF. Immature DCs were loaded with OC-L and matured using MPLA-IFNγ. After assessing the functionality of the OC-DC cells (IL12p70 secretion and COSTIM assay), the OC-DC vaccine was cryopreserved in multiple doses for single use. Finally, the stability of the formulated doses was tested and validated. We believe this GMP-compliant DC vaccine manufacturing process will facilitate access of patients to personalized DC vaccines, and allow for multi-center clinical trials.

Recent Advances in Good Manufacturing Practice-Grade Generation of Dendritic Cells

“…One of these comprises IL-1β, TNFα, IFNα, IFNγ, and poly(I:C), focusing on TH1 maturation of MoDCs [84]. Another frequently used PGE 2 -free cocktail consists of IFNγ and LPS DOI: 10.1159/000512451 or its alternative monophosphoryl lipid A [47,89]. Similar to the single use of IFNγ, this combinations leads to the release of TH1-inducing factors such as CCL3, CCL5, IL-1, IL-6, and IL-12 and is able to induce the attraction of various immune cells [84,89].…”

“…Another option of a closed system relies on the use of magnetic enrichment of target cells, implementing positive selection or depletion of unwanted cells from leukocyte apheresis products. Here, positive selection of monocytes using α-CD14 monoclonal antibodies was shown to produce up to 96–97% purity and recovery rates of approximately 89% [36, 46, 47]. Due to the lack of a cytoplasmic domain required for TLR4 activation, CD14-targeting antibodies should not be able to activate monocytes, yet a small residual risk remains that positive selection might affect the finished products’ functionality [32, 48, 49].…”

“…In recent years, several new GMP-compliant cultivation methods have been introduced, all with their own pros and cons, concerning costs, required personnel, and product size. These include cell factory flasks [47], bioreactors (wave bioreactor [54], Quantum Terumo [55, 56]), and culture bags in a closed cultivation system (CliniMACS Prodigy [57]). The introduction of closed cell culture bags and the understanding that vessel materials and geometry have a significant impact on the efficacy of the cell therapeutic products reflect the complexity scientists and clinicians face in protocol development.…”

“…In a different approach, tumor lysates were tested to increase the availability of immunogenic antigens, comprising lysates from tumor cell lines (e.g., KATO-III [65, 93]) or autologous patient tumor lysates [46, 47, 94]. Using cell lines provides the advantage of preparing readily available lysates in advance; however, again antigen loss of the patient’s tumor cannot be addressed.…”

“…Using tumor lysates from autologous tumor samples is advantageous in many ways due to the absence of HLA restriction, reduced cost, and time necessary for preparation as well as the presence of numerous neoantigens within the lysate. A recent publication by Boudousquié et al [47] optimized the immunogenicity of tumor lysate by repeatedly freeze-thawing hypochloric acid-treated autologous tumor lysates. Here, both CD4+ and CD8+ T cells were brought to proliferation through coculture with oxidized lysate-pulsed MoDCs.…”