Pulmonary Physiology: Pulmonary Physiology |

Establishment of a Novel Nanobody Specific for Anti-human Pulmonary Surfactant Protein A and Its Targeting Capacity FREE TO VIEW

Xian He; Shanmei Wang; Zhao-Fang Yin; Nan Li; Liusheng Wang; Yang Hu; Huiping Li
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Shanghai Pulmonary Hospital, Shanghai, China

Copyright 2016, American College of Chest Physicians. All Rights Reserved.

Chest. 2016;149(4_S):A486. doi:10.1016/j.chest.2016.02.506
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SESSION TITLE: Pulmonary Physiology

SESSION TYPE: Original Investigation Poster

PRESENTED ON: Saturday, April 16, 2016 at 11:45 AM - 12:45 PM

PURPOSE: To develop an novel anti-human pulmonary surfactant protein A (SP-A) nanobody to provide an ideal ligand for development of lung-targeted new drugs in clinical use

METHODS: 1) Anacquire a tertiary biopanning nanobody library by using the solid-phase biopanning technique; 2) human lung tissue was formulated into tissue homogenates whose weight and activity of SP-A were validated by Western blot analysis and enzyme-linked immunosorbent assay; 3) the clonal tertiary biopanning nanobody library was primarily detected by phage-ELISA to select the high affinity clonal strains; 4) the obtained clonal strains were constructed, inducibly expressed, and purified by genetic recombination; 5) the targeting capacity, binding specificity, and stability of the anti-hSP-A nanobody were evaluated by laser scanning confocal microscopy, immunohistochemistry, in vivo imaging of nude mice, and so on; 6) the safety of the anti-hSP-A nanobody was determined either through acute toxicity or chronic toxicity test

RESULTS: 1) Establishment a 3.8 ×106 human pulmonary nanobody phage display library. 2) Extraction and validation of human pulmonary SP-A ( (hSP-A) whose molecules weighing approximately 35 kDa, 70 kDa, and 120 kDa. 3) a total of 15 gene sequences of the nanobody that specifically bound to the hSP-A, and soluble prokaryotic expression of Nb4 (∼19 kDa) was performed. 4) Nb4 bound specifically to the extracted hSP-A, as indicated by the Western blot and ELISA results. 5) The extrapulmonary targeting capacity of Nb4 was assessed by immunofluorescence of human lung A549 cells while lung-specific capacity in the immunohistochemistry of tissues from normal human lungs as well as in the in vivo imaging of nude mice. Stability revealed that the optimal pH was 7.0-7.6, and the most favorable temperature was 37 °C. 6) mild liver and kidney injuries were documented 3 months after the chronic toxicity test. Pathologically, mild pulmonary infiltrates were present in both acute and chronic toxicity tests indicating favorable safety

CONCLUSIONS: we successfully constructed an anti-human pulmonary SP-A nanobody that specifically targets the highly expressed SP-A in the lungs to provide an ideal ligand for the development of effective and low-toxic lung-targeted drugs

CLINICAL IMPLICATIONS: we selected SP-A as the target spot and successfully constructed anti-human-SP-A Nanobody (Nb4) for the first time, then confirmed its human lung tissue-specific targeting combination. This novel anti-hSP-A Nanobody provides an ideal targeting ligand for preparing drugs for pulmonary diseases

DISCLOSURE: The following authors have nothing to disclose: Xian He, Shanmei Wang, Zhao-Fang Yin, Nan Li, Liusheng Wang, Yang Hu, Huiping Li

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