T cell immunotherapy is a promising and relatively new field of medicine focused on tailoring a person’s own immune cells to fight disease more effectively. The field has enjoyed early success in the area of cancer immunotherapy, most notably the development of chimeric antigen receptor (CAR) T cells designed to express antigens that specifically target cancer cells. Regulatory T cells, or Tregs, are the foundation of another promising type of T cell immunotherapy. Tregs mediate immune tolerance, helping to suppress overactive, potentially harmful immune responses in the body. Early clinical studies have shown Tregs to have efficacy against autoimmune disease, and in suppressing immune rejection during organ or stem cell transplant. Unfortunately, Tregs circulate at a low frequency compared to other immune cells, so expanding these cells for use in cell therapy is time and resource intensive.
Improving Rapamycin Bioavailability
The San Diego based researchers were inspired to find a way to make it easier to expand Tregs in culture. Their research was based on rapamycin (Rapa), an anti-inflammatory compound that suppresses proliferation of the more common effector T cells but has little effect on Tregs. The challenge, however, is that Rapa has poor solubility and a short half-life in the bloodstream. To make it easier for Rapa to exert its biological effects, the scientists would need to modify the compound to increase its bioavailability and prolong its effect on effector cell suppression in culture.
One of the most common ways of increasing drug solubility is to make a complex between the drug of interest and beta-cyclodextrin (bCD), a hydrophilic glucose-based molecule that can confer solubility, helping drugs to cross biological membranes. The scientists consequently designed a formulation of Rapa complexed with bCD derivatives and set out to characterize the effectiveness of the new cyclodextrin-Rapa complexes (CRCs) at enhancing Treg expansion.
Preliminary characterization showed that bCD complexed rapamycin was approximately 154 -fold more soluble than rapamycin alone and allowed for Rapa bioactivity to be maintained for at least 30 days under biologically relevant conditions.
Characterization of Treg Expansion
The research team began their study by obtaining human immune cell starting material from Excellos, a full-service CDMO company which at that time was operating as part of the San Diego Blood Bank. The company also supplies freshly isolated or cryopreserved immune cells, including rare T cells subsets.
Briefly, primary human PBMCs were isolated from healthy human donor blood by density centrifugation and then cryopreserved. Pan T cells were later isolated from the thawed PBMCs using magnetic bead separation and then cultured in standard T cell media. The scientists carried out equivalent studies using mouse primary T cells in parallel to the human cell studies.
After a day in culture, human or murine IL-2 was added as a standard method of promoting Treg proliferation, along with any CRC preparation being tested. In addition to IL-2, either a null control, Rapa alone, or CRCs were added to the culture media at a 10, 100 or 1000 nM concentration. 7 days later, cells from each test condition were evaluated by flow cytometry for the presence of distinct T cell subtypes.
The researchers found that CRC preferentially enhanced the fraction of Treg (CD4+CD25+FoxP3+) type cells up to 6-fold in murine cell cultures and 2-fold in human cell cultures. At the same time, the addition of CRCs suppressed the overall expansion of T effector cells in a dose-dependent manner by up to 5-fold in both species. Combining CRCs with growth factor TGF-β1 synergistically boosted the expansion of Treg cells. Addition of CRCs to the culture media did not result in any observable toxic effects.
The research team concluded that CRCs are an effective method of preferentially enriching for Tregs in culture and could consequently help promote the successful development of future T cell immunotherapies.
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- McBride, David A., et al. Characterization of regulatory T cell expansion for manufacturing cellular immunotherapies. Biomaterials Science | (2020) 8:4186 | https://doi.org/10.1039/d0bm00622j
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