Highly sensitive detection of antibody nonspecific interactions using flow cytometry

Abstract

The rapidly evolving nature of antibody drug development has resulted in technologies that generate vast numbers (hundreds to thousands) of lead antibody candidates during early discovery. These candidates must be rapidly pared down to identify the most drug-like candidates for in-depth analysis of their safety and efficacy, which can only be performed on a limited number of antibodies due to time and resource requirements. One key biophysical property of successful antibody therapeutics is high specificity, defined as low levels of nonspecific binding or polyspecificity. Although there has been some progress in developing assays for detecting antibody polyspecificity, most of these assays are limited by poor sensitivity or assay formats that require proprietary antibody surface display methods, and some of these assays use complex and poorly defined polyspecificity reagents. Here we report the PolySpecificity Particle (PSP) assay, a sensitive flow cytometry assay for evaluating antibody nonspecific interactions that overcomes previous limitations and can be used for evaluating diverse types of IgGs, multispecific antibodies and Fc-fusion proteins. Our approach uses micron-sized magnetic beads coated with Protein A to capture antibodies at extremely dilute concentrations (<0.02 mg/mL). Flow cytometry analysis of polyspecificity reagent binding to these conjugates results in sensitive detection of differences in nonspecific interactions for clinical-stage antibodies. Our PSP assay strongly discriminates between antibodies with different levels of polyspecificity using previously reported polyspecificity reagents that are either well-defined proteins or highly complex protein mixtures. Moreover, we also find that a unique reagent, namely ovalbumin, results in the best assay sensitivity and specificity. Importantly, our assay is much more sensitive than standard assays such as ELISAs. We expect that our simple, sensitive, and highthroughput PSP assay will accelerate the development of safe and effective antibody therapeutics.

Co-optimization of therapeutic antibody affinity and specificity using machine learning models that generalize to novel mutational space

“…Next, we displayed the library on the surface of yeast as single-chain Fab fragments and sorted the library via magnetic-activated cell sorting (MACS, rounds 1–2) against the antigen (HGFR) to remove fragmented or non-displaying antibodies. The MACS-sorted libraries were then sorted by fluorescence-activated cell sorting (FACS, round 3) for high levels of antigen binding and high and low levels of non-specific binding to two polyspecificity reagents (ovalbumin 39 and soluble membrane proteins isolated from CHO cells 15 , 40 , 41 ; Supplementary Fig. 2 ).…”

“…For FACS selections, HGFR-Fc was used as purchased following reconstitution. Soluble membrane proteins (SMPs) were prepared as previously described 39 , 40 , and SMPs and ovalbumin (OVA; Sigma A5503) were biotinylated with Sulfo-NHS-LC-Biotin (Pierce, P121335).…”

“…Variable heavy (V H ) domains of selected in-library clones were isolated from yeast display plasmids or ordered as geneblocks (IDT). The V H domains were cloned into pTT5 mammalian expression plasmids containing a common IgG1 heavy and light chain (kappa) framework, as described previously 39 . Briefly, the PCR-amplified fragments and expression vectors were digested with the desired restriction enzymes (EcoRI-HF and NheI-HF for V H ; EcoRI-HF and BsiWI-HF for V L ; New England Biolabs).…”

“…Affinity analysis was performed as reported previously 39 . Briefly, Protein A Dynabeads (Invitrogen, 10002D) were washed three times and diluted to 54 μg/mL in PBSB.…”

Reliable energy-based antibody humanization and stabilization

“…Unlike the results from conventional homology-based humanization methods, CUMAb generates several unique humanization alternatives that exhibit stability and binding properties that are on a par with those of the animal antibody. Since these alternative designs encode dozens of surface mutations from one another, they are likely to exhibit differences in critical developability properties, such as nonspecificity and solution properties at high antibody concentrations 26,27 .…”