It has been recently shown that CD4+ and CD8+ T lymphocytes are critically involved in the collateral brain injury and neurological deficit associated with experimental stroke. Naturally occurring CD4+CD25+Foxp3+ (nTreg) cells derived from thymus play a key role in modulating the function of effector T cells and antigen- presenting cells that maintain self tolerance and immunological homeostasis. In the experimental models of stroke, deletion of nTreg cells profoundly increased delayed brain damage and deteriorated functional outcome in mice. Conversely, administration of nTreg cells resulted in a marked reduction of brain infarct and improvement of neurological functions while attenuating blood-brain barrier disruption with therapeutic window of at least 24 h. Remarkably, nTreg cell treatment corrected poststroke lymphopenia and decreased bacterial loads in the blood during recovery. Hence, nTreg cells represent a potentially safe and powerful approach for stroke therapy. Given the daunting manufacturing complexity, regulatory and economic obstacles to the establishment of human nTreg cells as a widespread and viable pharmaceutical platform, we have been focusing on developing pharmacological approach to stimulate nTreg cells. Interleukin-2 (IL-2) is the key cytokine for the generation, survival, and function of nTreg cells to control immune responses and prevent autoimmune disease by binding to a high affinity receptor consisting of three subunits, IL2Ra (CD25), IL2Rb (CD122) and gc (CD132). Recent clinical trial shows that treatment with low-dose IL2 increased nTreg cells and was associated with reversal of glucorticoid-refractory chronic graft-versus host disease in patients who had undergone allogeneic hematopoietic stem cell transplantation. Similar treatment also led to substantial clinical improvement in both cryoglobulinemia and vasculitis in patients with hepatiris C virus. In both trials, the repeated dose treatment for 4-8 weeks was safe without any serious complications or infections. Meanwhile, high-dose IL2 (100 fold higher dose) also activates naturally killer cells, na?ve CD4+ T cells, memory and memory-phenotype CD8+ T cells expressing high levels of the intermediate affinity receptor without IL2Ra (10-100 fold lower affinity), which has been used to treat renal carcinoma and melanoma patients with narrow therapeutic window and limited efficacy (partially due to increased nTreg cells. Current IL2 therapy (Aldesleukin, 15kD) is an unglycosylated recombinant IL2 from E coli. Rapid renal elimination results in a very short in vivo half-life of IL-2 in the range of minutes due to its smll molecular size. This has been a major limitation for IL-2-based strategies, thus requiring the use of high and multiple doses of IL-2 and causing severe toxic side effects, termed vascular leak syndrome. We have designed and produced APT602, a fusion protein of human serum albumin (HSA) and IL-2 in mammalian cells. The large size of fusion protein (85kD) improves potency and minimizes renal clearance, extending plasma half-life by 25 fold. Moreover, albumin is distributed between vascular and extravascular compartments through endothelial junctions and active transcytotic transport, which increases uptake in spleen and lymph nodes and exposure of APT602 to lymphatic tissues compared with IL2. Hence, low-dose APT602 will enable more effective and steady stimulation of nTreg cells with abundant high-affinity ??? receptor while minimizing activation of effector immune cells with intermediate affinity ?? receptors. In addition this therapeutic agent does not require the toxic detergent SDS to solubilize aggregates needed for Aldesleukin. In randomized and blinded studies with the clinically relevant models of thrombo-embolic stroke in rats, treatment with low-dose APT602 6h after stroke significantly decreased infarct volume by 54% and improved neurological deficit score by 31% 7 days after stroke (Prior Work). These data provide strong justification for continuing the preclinical validation of APT602 as innovative monotherapy for stroke.
Specific Aims i nclude: 1) To produce APT602 in a human cell line. 2) To determine the optimal dose and therapeutic time window in the thrombo-embolic stroke model of adult (3-4 months old) male rats.
APT602 is a safe and powerful inducer of natural regulatory T cells that attenuates adverse immune responses associated with experimental stroke. We propose to determine whether low-dose APT602 will be safe and beneficial with at least 12h therapeutic time window in a clinically relevant model of thrombo-embolic stroke in rats.
