Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

Abstract

The ability of monoclonal antibodies (mAbs) to target specific antigens with high precision has led to an increasing demand to generate them for therapeutic use in many disease areas. Historically, the discovery of therapeutic mAbs has relied upon the immunization of mammals and various in vitro display technologies. While the routine immunization of rodents yields clones that are stable in serum and have been selected against vast arrays of endogenous, non-target self-antigens, it is often difficult to obtain species cross-reactive mAbs owing to the generally high sequence similarity shared across human antigens and their mammalian orthologs. In vitro display technologies bypass this limitation, but lack an in vivo screening mechanism, and thus may potentially generate mAbs with undesirable binding specificity and stability issues. Chicken immunization is emerging as an attractive mAb discovery method because it combines the benefits of both in vivo and in vitro display methods. Since chickens are phylogenetically separated from mammals, their proteins share less sequence homology with those of humans, so human proteins are often immunogenic and can readily elicit rodent cross-reactive clones, which are necessary for in vivo proof of mechanism studies. Here, we compare the binding characteristics of mAbs isolated from chicken immunization, mouse immunization, and phage display of human antibody libraries. Our results show that chicken-derived mAbs not only recapitulate the kinetic diversity of mAbs sourced from other methods, but appear to offer an expanded repertoire of epitopes. Further, chicken-derived mAbs can bind their native serum antigen with very high affinity, highlighting their therapeutic potential.

Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another

“…To illustrate these displacements, we use mAbs produced from two independent sources, namely mAb C21 generated from the immunization of chickens and mAb 28H6 (also known as M27) and mAb 14C7 (also known as M4) generated from the immunization of mice [15]. A one-shot kinetic analysis of PGRN over immobilized C21, 28H6, and 14C7 yielded apparent K D values of < 2, ~20, and ~ 600 pM (Fig 5A and Table 1).…”

“…A one-shot kinetic analysis of PGRN over immobilized C21, 28H6, and 14C7 yielded apparent K D values of < 2, ~20, and ~ 600 pM (Fig 5A and Table 1). Using a chimeric swap epitope mapping strategy [15], the epitopes of all three mAbs were co-localized to the same subdomain of PGRN, namely granulin E [18], and when tested for cross-blocking in a classical sandwich assay format, C21 and 28H6 blocked one another and both potently displaced 14C7. Upon reversing the assay orientation, 14C7 blocked both C21 and 28H6, and appeared to partially displace them but only when 14C7 was used at high analyte concentrations (1 μM).…”

“…High-throughput epitope binning experiments on PCSK9 were performed in a classical sandwich assay format using an MX96 SPR imager in a 96-ligand array configuration, as described previously [15], but using more sophisticated software for the data analysis (ECTO module 2.0 version 33, Wasatch Microfluidics) that help to identify (and discard) non-ideal or problematic behaviors from ligands (coupled mAbs) and analytes (solution mAbs). Representative sensorgrams obtained for multiple binning cycles on a single spot are shown as overlay plots (S2, S3 and S6 Figs).…”

“…Serum experiments were performed as described previously [15, 16] using a Dylight-labeled anti-PGRN mAb 27D5 (M14) as secondary detection, chosen because its epitope does not overlap with that of mAb 14C7 (M4) or mAb 28H6 (M27).…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…The immunological mechanisms underlying B-cell immunodominance are poorly understood, and patterns of immunodominance probably are not completely conserved across species. 29 …”