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Abstract
Background While Chimeric Antigen Receptor (CAR) technology has shown promises, FDA commercially approved applications are not yet approved for solid tumors. This could be related to a traditional centralized cold-chain manufacturing process, with multiple freeze/thaw cycles and transport, that ultimately produces substandard effector cells at a much higher cost, while increasing patient wait time (figure 1). If cell manipulation can be reduced to a minimum, it is likely that cells processed in this fashion will be maximally fit and best suitable therapeutical modalities. Here we show that a full ‘warm chain’ manufacturing model at the POC, where immune cell fractions are immediately processed upon collection, can improve immune cells critical quality attributes (CQA) for cancer cell therapies applications.
Methods Samples from healthy volunteers and cancer patients are collected by phlebotomy or leukapheresis and processed for peripheral blood mononuclear cells (PBMCs), CD8+ lymphocytes and Natural Killer (K) cell fractions isolation. Samples are processed immediately (warm chain), after 4 hours, and after 24 hours or frozen (cold-chain). Cell recovery, viability, identity, CD8+ phenotype, NK cell phenotype, functional cytotoxicity (e.g.,degranulation) are determined by flow cytometry methods, and IFNγ release was measured by ELISA.
Results Samples processed after 4 hours yielded lower recovery and viability overall compared to immediate processing which was best. CD8+ lymphocytes were significantly impacted after 4 hours, but not NK cells. Sample freezing significantly impacted recovery and viability overall, as well as for NK and CD8+ subtypes. While CD8+ exhaustion was not significantly impacted, NK cells delayed in processing or frozen showed a significant decrease in CD16 and NkP46 expression. Likewise, CD8+ degranulation and IFNγ release were not affected by a delay in processing, but NK cells were significantly affected. Freezing affected both cell types with a decrease in both degranulation capabilities and IFNγ production.
Conclusions Immune samples collected at the POC, processed immediately in a warm chain environment produce higher yields at slightly higher viability, especially for CD8+ cytotoxic lymphocytes. NK cells, prime candidates for off-the-shelf, allogeneic cell therapies, showed a significant decrease in antibody-dependent cytotoxic marker CD16. This study highlights the problem of focusing on cell function (through genetic modifications or specific immune engagers) while neglecting cell functional fitness: even the best modalities may not be able overcome a lack of cell functional fitness. New warm chain models with manufacturing production facilities, such as a distributed decentralized POC model, are needed to augment cancer cell therapies capabilities and accessibility.
Ethics Approval Clinical trial NCT00571389 is sponsored by BioCytics, Inc. and approved by Western Institutional Review Board-Copernicus Group IRB. All participants provided written informed consent before participating in any study procedures.
Logistics and pitfalls of cell and gene therapy manufacturing process
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