A rapid in vitro methodology for simultaneous target discovery and antibody generation against functional cell subpopulations

Abstract

Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin β6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin β6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.

The Rational Development of CD133-Targeting Immunotherapies for Glioblastoma

“…Immunodetection of CD133 has presented a major limitation toward therapeutic development, as currently available antibodies are limited in their ability to detect CD133 splice variants and aberrantly post-translationally modified CD133. We used iterative cellbased panning, which combines the use of matched HEK293 and HEK293-CD133-overexpressing cells (Mak et al, 2011), a phagedisplayed synthetic antibody fragment (Fab) library (Nixon et al, 2019;Persson et al, 2013), and DNA sequencing, to identify Fabs targeting human CD133, encoded by the PROM1 gene (Figure 1A). This effort yielded a Fab called RW03 (i.e., RW03-Fab), which demonstrated highly specific binding to the CD133-overexpressing cells with minimal binding to the parental HEK293-CD133-non-expressing cells.…”

Probing Surface Membrane Receptors Using Engineered Bacteriophage Bioconjugates

“…Fluorescence-activated cell sorting (FACS) is the most popular and widely used method for staining of mammalian surface receptors . Several recent articles described approaches combining FACS and bacteriophage panning to generate antibodies to the plasma membrane or extracellular proteins displayed on the yeast surface, transfected mammalian cells, or cancer cells . It is worth highlighting identification of antigen-specific B-cells.…”

A T cell redirection platform for co-targeting dual antigens on solid tumors

“…25 Notably, the trastuzumab framework has proven to be highly robust for supporting many diverse paratopes, and we and others have used this framework to construct naïve phage-displayed libraries that have yielded thousands of Fabs recognizing hundreds of diverse antigens. 29,42,[56][57][58][59] Consequently, virtually any stable Fab derived from natural IgGs or synthetic libraries could be converted into the DATE format to provide a vast toolkit for exploring novel TAAs for T-cell recruitment against diverse tumors. To further enhance specificity in a modular manner, we developed d-DATEs by adding a second tumor-targeting moiety in the form of an autonomous VH domain, built on a modified version of the trastuzumab framework.…”

“… 41 HEK293T cells stably expressing CD133-Green Fluorescent Protein (GFP) have been described previously. 42 Patient tumor-derived pancreatic ductal adenocarcinoma cell lines GP5A, GP13A, GP14A, and GP16A were a generous gift from Dr. David Hedley, Toronto, Canada, and described previously. 43 Cell lines were maintained as follows: KP4 cell lines in RPMI (ThermoFisher Cat# A1049101) supplemented with 10% FBS, HCT116 cells in McCoy’s 5A (ThermoFisher Cat# 16600–108) supplemented with 10% FBS, RCC243 cells in IMDM (ThermoFisher Cat# 12440061) supplemented with 10% FBS, HEK293T cells in DMEM (ThermoFisher Cat# 11995073), GP5A, GP13A, GP14A in DMEM/F12 1:1 (ThermoFisher Cat# 11320033) supplemented with 10% FBS, GP16A in DMEM/F12 1:1 (ThermoFisher Cat# 11320033) supplemented with 2.5% FBS, GP9A in RPMI (Cat# 11875119) supplemented with 10% FBS.…”