期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 371, 期 4, 页码 934-947出版社
ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2007.05.011
关键词
yeast display; single chain Fv antibody; epidermal growth factor receptor; affinity maturation; immunoliposome
资金
- NCI NIH HHS [P50-CA58207, P50 CA058207] Funding Source: Medline
To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (K-D=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (K-D=264 nM). By combining mutations, higher affinity scFv were generated with K-D ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting. (c) 2007 Elsevier Ltd. All rights reserved.
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