Generating Anti-CD276 scFv Antibodies from Yeast Display Library

A yeast display naïve, single-chain variable fragment (scFv) human antibody library was used to generate the anti-human CD276 scFvs as described previously (35 (link)). The library was constructed using a collection of human antibody gene repertoires, including the genes used for the construction of a phage display Fab library and those from more than 50 additional individuals and contained a total of 1e10 scFvs. In vitro selection of the yeast display library involved three rounds of sequential panning on biotinylated, purified recombinant CD276(ED)-AP (alkaline phosphatase) fusion proteins. For this, 10 μg of biotinylated hCD276(ED)-AP was incubated with approximately 5e10 cells from the initial naïve antibody library in 50 mL PBSA (PBS containing 0.1% BSA) for 2 hours, washed with PBSA, and captured with streptavidin-conjugated microbeads from Miltenyi Biotec using the AutoMACS system. The sorted cells were amplified and the panning was repeated once with the human hCD276 (ED)-AP and once with the mouse mCD276(ED)-AP protein to enrich for cross-reactive binders. After characterizing several scFvs for binding specificity, cross-species reactivity, a panel of five binders (1 (link)–5 (link)) were sequenced and used for CAR construction.

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Majzner R.G., Theruvath J.L., Nellan A., Heitzeneder S., Cui Y., Mount C.W., Rietberg S.P., Linde M.H., Xu P., Rota C., Sotillo E., Labanieh L., Lee D.W., Orentas R.J., Dimitrov D.S., Zhu Z., St Croix B., Delaidelli A., Sekunova A., Bonvini E., Mitra S.S., Quezado M.M., Majeti R., Monje M., Sorensen P.H., Maris J.M, & Mackall C.L. (2019). CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(8), 2560-2574.

Publication 2019

streptavidin-conjugated microbeads AutoMACS system

A gene Alkaline phosphatase Antibody Cells Cross reactivity Genes Human Library Microbeads Mouse Pbsa Phage display Protein Single chain variable fragment antibody Streptavidin Yeast

Corresponding Organization :

Other organizations : Stanford University, University of Colorado Anschutz Medical Campus, University of Colorado Denver, California Institute for Regenerative Medicine, University of Virginia, University of Washington, University of Pittsburgh, University of British Columbia, BC Cancer Agency, MacroGenics (United States), Center for Cancer Research, Children's Hospital of Philadelphia, University of Pennsylvania

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Variable analysis

independent variables

Yeast display naïve, single-chain variable fragment (scFv) human antibody library

dependent variables

Anti-human CD276 scFvs generated from the yeast display library

control variables

Biotinylated, purified recombinant CD276(ED)-AP (alkaline phosphatase) fusion proteins used for panning the yeast display library
Streptavidin-conjugated microbeads used for capturing the cells after incubation with the biotinylated CD276(ED)-AP fusion proteins

controls

Positive control: Human hCD276(ED)-AP protein used for panning the yeast display library
Negative control: Mouse mCD276(ED)-AP protein used for panning the yeast display library to enrich for cross-reactive binders

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