期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 519, 期 2, 页码 267-273出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2019.08.160
关键词
PD-1; Nanobody; P. pastoris; Human serum albumin
资金
- National Natural Science Foundation of China [81773620, 81573332, 31872746]
- National Key Basic Research Program of China [2015CB931800]
- Shanghai Science and Technology Funds [18431902800]
PD-1/PD-L1 pathway blocking with antibodies offers a vital and efficient therapeutic strategy to restore T cell-associated antitumor immunity and treats a variety of cancers in clinic. Nanobodies (Nbs) give several advantages over conventional monoclonal antibodies such as size, solubility, stability and costs. Additionally, P. pastoris is a suitable host for Nb production. Herein, we aim to produce and evaluate anti-PD-1 Nb derived from the P. pastoris. Our findings indicated that we successfully established the Nbs phage-displayed library against PD-1 with qualified library capacity and insert ratio. Anti-PD-1 Nb Nb97 was screened through PE-ELISA and flow cytometry. To extend half-life of Nb97, we contracted pPICZaA-Nb97-Nb97-HSA recombination vector, which was then transformed into the system of P. pastoris X-33. The yield of purified Nb97-Nb97-Human serum albumin (HSA) fused protein (MY2935) reached to 2.3 g/L after 147 h of fermentation. Meanwhile, the blocking effect of MY2935 is similar to that of MY2626 (humanized Nb97-Fc), and MY2935 showed better performance on stimulating the immune function through PD-1 reporter assay. Hence, P. pastoris X-33 expressing and secreting functional anti-PD-1 Nb-HSA fusion protein might be a system of high yield and low cost. (C) 2019 Published by Elsevier Inc.
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