期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 298, 期 4, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.jbc.2022.101798
关键词
-
资金
- Cancer Prevention and Research Institute of Texas grant [RP180690]
- Welch Foundation [F-1767]
By engineering the human IgG1 Fc domain to enhance pH-selective binding to the receptor Fc gamma RIIIa, the study has successfully achieved antibody engineering with enhanced tumor specificity.
Despite the exquisite specificity and high affinity of antibody based cancer therapies, treatment side effects can occur since the tumor-associated antigens targeted are also present on healthy cells. However, the low pH of the tumor microenvironment provides an opportunity to develop conditionally active antibodies with enhanced tumor specificity. Here, we engineered the human IgG1 Fc domain to enhance pH-selective binding to the receptor Fc gamma RIIIa and subsequent antibody-dependent cellular cytotoxicity (ADCC). We displayed the Fc domain on the surface of mammalian cells and generated a site-directed library by altering Fc residues at the Fc-Fc gamma RIIIa interface to support interactions with positively charged histidine residues. We then used a competitive staining and flow cytometric selection strategy to isolate Fc variants exhibiting reduced Fc gamma RIIIa affinities at neutral pH, but physiological affinities at the tumor-typical pH 6.5. We demonstrate that antibodies composed of Fab arms binding the breast cell epithelial marker Her2 and the lead Fc variant, termed acid-Fc, exhibited an ,~& nbsp;2-fold pH-selectivity for Fc gamma RIIIa binding based on the ratio of equilibrium dissociation constants K-d,K-7.4/K-d,K-6.5, due to a faster dissociation rate at pH 7.4. Finally, in vitro ADCC assays with human Fc gamma RIIIa-positive natural killer and Her2-positive target cells demonstrated similar activities for anti-Her2 antibodies bearing the wild-type or acid-Fc at pH 6.5, but nearly 20-fold reduced ADCC for acidFc at pH 7.4, based on EC50 ratios. This work shows the promise of mammalian cell display for Fc engineering and the feasibility of pH-selective Fc activation to provide a second dimension of selective tumor cell targeting.
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