35. Establishment of the Dose-Response Relationship Needed for Human Translation of Pulmonary Macrophage Transplantation (PMT) Therapy of Hereditary Pulmonary Alveolar Proteinosis (hPAP)

Hereditary pulmonary alveolar proteinosis (hPAP) is a severe pediatric lung disease caused by mutations in CSF2RA/B (encoding GM-CSF receptor α/β, respectively) without pharmacologic therapy. Disease patho-genesis is mediated by loss of GM-CSF-dependent clearance by alveolar macrophages (AMs) resulting in progressive pulmonary alveolar surfactant accumulation and hypoxemic respiratory failure. We recently reported a conceptually and technically novel, exceptionally efficient gene and cell therapy approach -PMT- as a promising alternative for the existing highly invasive, inefficient whole lung lavage procedure for hPAP. Our preclinical studies revealed that macrophage cell doses from 0.5 to 4 x106/mouse were similarly highly efficacious as therapy of hPAP in Csf2rb-/- mice, an authentic model of human hPAP (Nature, 2014, 514: 450-5). The present study was undertaken to identify a dose-response relationship and to address the hypothesis of an expected ‘trade-off’ between the minimum effective dose and time to treatment effect. We isolated Lineage-, Sca1+, cKit+ hematopoietic stem/progenitors from the bone marrow of wild-type (WT) CD45.1+ mice, differentiated them in vitro into mature macrophages and sorted for highly homogenous CD11cHi, F4/80Hi macrophage population by flow cytometry. We administered cell doses (2.5 x103, 2.5 x104, 2.5 x105, 1 x106 cells/mouse; n=3-4/dose) by PMT to CD45.2+ Csf2rb-/- recipients. At 8 weeks after PMT, therapeutic efficacy was evaluated by measuring the optical density (OD) of bronchoalveolar lavage (BAL turbidity) - an excellent measure of overall PAP disease severity. The CD45.1+ donor macrophages recovered from the BAL were of CD11cHi, F4/80Hi phenotype. BAL turbidity decreased smoothly with increasing PMT cell dose over the entire range but reached significance only at the two highest doses compared to age-matched, untreated Csf2rb-/- controls (OD λ=600 nm = 0.88±0.07 and 0.5±0.08 vs. 2.32±0.18; Pu003c0.001, Pu003c0.0001; respectively). BAL turbidity correlated inversely with the percentage of CD45.1+ donor macrophage engraftment in CD45.2+ Csf2rb-/- recipients (Spearman rank correlation, R2=0.74). Since we have identified an increased ratio of cholesterol (relative to phospholipid) in surfactant as the primary lipid abnormality in hPAP, we also evaluated the relationship of BAL cholesterol level and PMT cell dose. Like turbidity, BAL cholesterol levels decreased smoothly with PMT cell dose over the entire range but reached significance only at the two highest cell doses compared to age-matched, untreated Csf2rb-/- controls (ng/ml BAL = 25±3 and 10±0.3 versus 54±0.3; Pu003c0.01, Pu003c0.001; respectively, R2=0.67). Our pre-clinical results establish a direct relationship between the number of macrophages transplanted and efficacy of PMT therapy in Csf2rb-/- mice and facilitate the design of a clinical trial to test PMT therapy in children with hPAP. Based on these results and the previously demonstrated strong survival advantage of transplanted over endogenous macrophages, we anticipate a ‘trade-off’ between the minimum effective dose and the time to equivalent treatment effect.