Improving antibody drug development using bionanotechnology

Abstract

Monoclonal antibodies are being used to treat a remarkable breadth of human disorders. Nevertheless, there are several key challenges at the earliest stages of antibody drug development that need to be addressed using simple and widely accessible methods, especially related to generating antibodies against membrane proteins and identifying antibody candidates with drug-like biophysical properties (high solubility and low viscosity). Here we highlight key bionanotechnologies for preparing functional and stable membrane proteins in diverse types of lipoparticles that are being used to improve antibody discovery and engineering efforts. We also highlight key bionanotechnologies for high-throughput and ultra-dilute screening of antibody biophysical properties during antibody discovery and optimization that are being used for identifying antibodies with superior combinations of in vitro (formulation) and in vivo (half-life) properties.

Figure 1.. Overview of emerging bionanotechnologies that are improving antibody drug discovery and early-stage developability analysis.

One of the key challenges in antibody drug development is the generation of antibodies specific for membrane proteins given the difficulty in preparing soluble and functional versions of membrane proteins that can be used for immunization and in vitro antibody selections. Advances in generating diverse types of biological and synthetic lipoparticles displaying functional membrane proteins is simplifying the discovery of antibodies against a myriad of membrane proteins as well as diverse panels of epitopes and cross-specifies reactive epitopes. A second key challenge in antibody drug development is the assessment of antibody biophysical properties such as self-association and non-specific binding at the earliest stages of antibody drug discovery. This is particularly important because the complementarity-determining regions of antibodies, which govern antibody affinity and specificity, also mediate antibody self-association and non-specific binding. Advances in biophysical screening methods using bionanotechnologies is enabling the unusually large-scale screening of antibody self-association and non-specific binding in addition to affinity and specificity to identify antibodies with global superior properties.

Figure 2.. Discovery of conformation-specific antibodies against the membrane protein GLUT4 using lipoparticles.

(A) Presentation of the native conformations of GLUT4 (inward-open and outward-open) for immunization and B-cell isolation was achieved using lipoparticle (also known as virus-like particle) technology. Phage panning with GLUT4 lipoparticles led to the isolation of several high affinity antibodies. (B-C) Two selected GLUT4 antibodies (mAb 1 and mAb 2) display conformational specificity for either the (B) inward-open (mAb 1) or (C) outward-open (mAb 2) conformations. The figure is adapted from a previous publication [18].

Figure 3.. Isolation of antibodies against the membrane-pass membrane protein, VSD4-NavAb, using nanodiscs.

(A) Mice were immunized using the membrane protein (VSD4-NavAb) prepared in proteoliposomes, hybridomas were generated, and then hybridomas were single-cell FACS sorted using biotinylated nanodiscs presenting VSD4-NavAb. (B) Single-cell sorting of hybridoma cells using nanodiscs resulted in the selection of large numbers (>400) of antibodies with high binding activity, as judged by an ELISA assay with immobilized nanodiscs. (C) Selected VSD4-NavAb antibodies generally display similar or higher levels of binding to nanodiscs than proteoliposomes. The figure is adapted from a previous publication [29].

Figure 4.. Charge-stabilized affinity-capture nanoparticle spectroscopy (CS-SINS) enables ultra-dilute screening of antibody self-association for identifying candidates with low viscosity and opalescence in concentrated antibody formulations.

(A) Gold nanoparticles coated with anti-human Fc capture antibodies aggregate at weakly acid pHs (e.g., pH ~5–6.5) and low ionic strengths (e.g., 10 mM histidine or acetate) because the zeta potential of the conjugates is low and crosses zero net charge around pH ~5.5. (B) Gold nanoparticles co-adsorbed with anti-human Fc capture antibodies and positively-charged polymers (polylysine) fail to aggregate at weakly acidic pH values and low ionic strengths because of the increased charge of the conjugates. (C and D) CS-SINS measurements of >0.35, which are measured at a mAb concentration of 0.01 mg/mL, display low risk for high viscosity (>30 cP) or high opalescence (>12 NTU) when formulated at 150 mg/mL. In (C) and (D), the solution conditions were pH 6 and 10 mM histidine. In (D), well-behaved mAbs refer to those that display both viscosity values <30 cP and opalescence values <12 NTU. The figure is adapted from a previous publication [39].