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.

Keywords: Binning; Immunology; chicken immune repertoire; epitope; monoclonal antibody.

Figure 1.

Affinity comparison of mAbs derived from chicken immunizations and other sources for 2 unrelated model antigens, PCSK9 and PGRN. (A) Biacore binding curves and global fits of select anti-PCSK9 mAbs from chicken immunization showing a diverse set of kinetic profiles. The colored curves represent the measured binding responses of hPCSK9 when injected at concentrations of 5 nM (red) and 50 nM (green), with the global fit overlaid in black. (B) Isoaffinity plot comparing anti-PCSK9 mAbs generated from chicken immunization (olive green) with those from human phage display libraries (blue). (C) Isoaffinity plot comparing anti-PGRN mAbs generated from immunizations in chicken (olive green) and mouse (purple). The red dotted line indicates the k_d limit of 5.70 × 10⁻⁵ (1/s) that was placed on interactions which showed < 5 % signal decay within the allowed dissociation phase of 15 min, also known as the “5 % rule” (see Methods).

Figure 2.

Solution affinity determination of 2 high affinity clones obtained from chicken immunization toward their respective serum antigens. (A) Schematic representation of the KinExA assay set-up. The mAb of interest is titrated into human serum and the equilibrated mixtures are injected over beads absorption-coated with a competing mAb to capture the free antigen. The bead-captured antigen is then detected using a Dylight-labeled sandwiching mAb. (B) Global analysis of mAb C34 binding serum PCSK9. (C) Global analysis of mAb C25 binding serum PGRN. In each case, the reported apparent K_D value is the best fit and 95 % confidence interval of the fit.

Figure 3.

Chicken-human merged binning results for PCSK9. (A) Heat map showing binning assignments for 39 mAbs generated from chicken immunization (olive green) and 24 mAbs generated by human phage display (blue). SPR-derived K_D values toward human PCSK9 are reported (conditionally formatted using a color gradient), along with their Octet-based cross-reaction toward human (h), mouse (m) and rat (r) PCSK9 (n/d = not determined). (B) Dendrogram showing antibody sequence lineages of the chicken mAbs alongside the binning heat map for these clones (drawn from panel A, transposed, and resorted). See Table S1.

Figure 4.

Chicken-mouse merged binning results for PGRN. (A) Heat map showing binning assignments for 32 mAbs generated from chicken immunization (olive green) and 20 mAbs generated from mouse immunization (purple). SPR-derived K_D values toward human PGRN are reported, along with their cross-reaction toward mouse (m) PGRN, and their GEP subdomain assignment (just for the human-specific clones; n/d = not determined). (B) Blocking network plot, colored by mAb library, with GEP assignments indicated (determined empirically, or inferred). (C) Dendrogram of the antibody sequence lineages for the chicken mAbs alongside the binning heat map for these clones (drawn from panel A, transposed, and resorted). (D) Blocking network plot for the chicken clones, colored by bin, with GEP assignments indicated. See Table S2.

Figure 4.

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