Pharmacokinetics of natural and engineered secreted factors delivered by mesenchymal stromal cells

Abstract

Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use. Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone, would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes of MSC transplantation and potentially other transient cell therapeutics.

Figure 1. Combined pharmacokinetic monitoring of MSCs and MSC-secreted IL-6.

(A) Bioluminescent images of C57Bl/6 mice over a period of three days after IV cell administration of one million luciferase-engineered human MSCs. (B) Photon flux of bioluminescent signal over time after IV cell administration. Durable BLI signals were detected up to 24 hours in mice that were injected IV with MSCs. (C) Serum ELISA measurements of human IL-6 released by IV cell transplants over time. Time points for serum and imaging analyses were 0.5, 8, 24, and 72 hours after cell injection. Pooled mouse serum was serially analyzed as batches of N = 5.

Figure 2. Enhanced delivery of IL-6 by MSC transplants compared to MSC conditioned medium.

(A) Serum profiles of human IL-6 after IV administration of concentrated conditioned medium into C57Bl/6 mice. The plot was normalized to the dose of conditioned medium that was contributed by 1×10⁶ cells. Pharmacokinetic parameters (B) Cmax, (C) AUC, (D) Tmax, (E) Half-life, and (F) Elimination constant were calculated for IL-6 exposure by cell transplants compared to CM administration. Significant differences between cell transplants compared to CM whereby higher levels of IL-6 and longer artificial duration was observed in plasma after cell transplantation. Time points for serum analyses were 0.5, 8, and 24 hours after cell or media injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.

Figure 3. Golgi-dependent secretion mechanism of MSC-derived IL-6 in vivo.

Brefeldin A pre-treatment of MSCs was used to evaluate blockade of IL-6 release in vitro and in vivo. (A) MTT assay of MSCs treated at different concentrations of brefeldin. A non-toxic dose of 5 ug/ml was used for functional studies. (B) Human IL-6 levels in vitro after brefeldin pre-treatment. Significant reduction in 24 hour release of IL-6 was observed across all doses. (C) Alteration in serum IL-6 delivery by MSCs pretreated with a Golgi-apparatus inhibitor, Brefeldin A. MSCs were incubated with 5 µg/ml of BFA for one day and then injected into C57Bl/6 mice and compared to untreated MSCs in terms of serum IL-6 delivery. Brefeldin treatment of MSCs led to diminished release of human IL-6 in vitro and in vivo. (D) Area-under-curve analysis of human IL-6 after MSC pre-treatment with brefeldin A and transplantation. Exposure to IL-6 was significantly reduced by inhibition of the Golgi apparatus. Time points for serum analyses were 0.5, 8, and 24 hours after cell injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.

Figure 4. The immune system limits the bioavailability of MSC-derived IL-6.

Pharmacokinetic profile of sera IL-6 after IV cell administration. Approximately 1×10⁶ MSCs were injected into C57Bl/6, Foxn1−/−, or NSG mice by IV injection. At different time points after cell injection, mice were sampled for blood plasma and serum human IL-6 levels were measured by ELISA. (B) AUC analysis of IL-6 exposure as a function of mouse strain. IV administration was significantly affected by mouse strain, particularly in severely immunodeficient mice, which had the highest exposure of IL-6 for a given cell mass. Data represent mean ± standard derivation of duplicate or triplicate experiments. Time points for serum analyses were 0.5, 8, and 24 hours after cell injection. Mice were serially analyzed as batches of N = 5 per study. * denotes P>0.01.

Figure 5. Blood Monitoring of Engineered Human MSCs with the Secreted Gaussia Luciferase Reporter.

A lentivirus vector expressing GLuc and GFP was transduced into human MSCs at a confluence of 70% and multiplicity of infection of 4∶1 in complex with 8 ug/ml of polybrene. (A) GFP micrograph and (B) flow cytometry showing high expression level and therefore transduction efficiency of construct. (C) The activity of GLuc was successfully measured in MSCs conditioned medium using a luminometer. (D) Five different engineered cell lines were infused into NOD-SCID mice and serum was individually collected at 0.5, 8, 24, 72, and 168 hours after cell injection in batches of N = 5 per study. MSCs constitutively expressing GLuc were detected in many cases over a week in duration.