Abstract
Synthetic antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal Abs against human antigens. Yet, they typically depend on purified proteins, which exclude integral membrane proteins that require the lipid bilayers to support their native structure and function. Here, we present an Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins on native cells in complex environment. As proof of concept, we targeted three transmembrane proteins linked to cancer, tetraspanin CD151, carbonic anhydrase 9, and integrin-α11. First, we performed in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders. Then, we designed next-generation sequencing procedures to explore Ab diversities and abundances. Finally, we developed motif-based scoring and sequencing error-filtering algorithms for the comprehensive interrogation of next-generation sequencing pools to identify Abs with high diversities and specificities, even at extremely low abundances, which are very difficult to identify using manual sampling or sequence abundances.
Highlights
Summary of identified clones showing the NGS unique sequence counts, abundance (% total), and the enrichment in the positive pool versus the negative pool, the obtained p value using the motif-based method and the corresponding effect size (Cohen’s index), the Ab concentrations that result in half-maximal affinities (EC50) for CD151, and the sequence of amino acids in the diversified CDR loop regions, as defined by the IMGT nomenclature[77]
The round 4 phage selection output consisted of two pools, a positive and a negative pool
We screened all 96 clones by cellular phage ELISA37, where phage signals were measured for binding to HEK293TCD151+ cells and compared to control HEK293T-CD151− cells
Summary
The CD151 knockdown (HEK293T-CD151−) and CD151 overexpressing (HEK293T-CD151+) cell lines were gifts from the Dr Rottapel lab at University of Toronto, Princess Margaret Cancer Centre. The overexpressing integrin-α11 (C2C12-α11+) cells were previously described[36], and the CA9 overexpressing (HEKT-CA9+) cells were a gift from Northern Biologics Inc. and generated as previously described[78], which stably introduced CA9 ORF cDNA (OriGene Technologies) into the cell line genome. The HEK293T and C2C12 cell backgrounds were cultured in Dulbecco’s Modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). The human fibrosarcoma H1080 cell line (ATCC; CCL-121) was cultured in Eagle’s Minimum Essential Medium (EMEM) with 10% FBS. All cells were cultured at 37 °C in a humid incubator with 5% CO2
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