Breaking barriers in antibody discovery: harnessing divergent species for accessing difficult and conserved drug targets

Abstract

To exploit highly conserved and difficult drug targets, including multipass membrane proteins, monoclonal antibody discovery efforts increasingly rely on the advantages offered by divergent species such as rabbits, camelids, and chickens. Here, we provide an overview of antibody discovery technologies, analyze gaps in therapeutic antibodies that stem from the historic use of mice, and examine opportunities to exploit previously inaccessible targets through discovery now possible in alternate species. We summarize the clinical development of antibodies raised from divergent species, discussing how these animals enable robust immune responses against highly conserved binding sites and yield antibodies capable of penetrating functional pockets via long HCDR3 regions. We also discuss the value of pan-reactive molecules often produced by these hosts, and how these antibodies can be tested in accessible animal models, offering a faster path to clinical development.

Keywords: Camelid antibodies; chicken antibodies; conserved epitopes; divergent species; membrane protein antibodies; pan-reactive antibody; paratope; rabbit antibodies; therapeutic antibody discovery.

Figure 1.

Sequence conservation of drug targets. a) Human drug targets show high conservation with mouse orthologs. A collection of 663 small and large molecule human drug targets curated in the ECOdrug database⁷ were analyzed with respect to their sequence identity in mice. 48% of targets showed sequence identity of >90%, and 24% of targets showed sequence identity of >95% compared to their mouse orthologs. Seven targets were not predicted to have a murine ortholog. b) Phylogenetic tree of divergent species used as hosts for immunization, shown with respect to evolutionary distance from humans.^8,9

Figure 2.

Technologies enabling MAbs from divergent species. a) The development of antibody discovery technologies that provide an alternative to hybridomas have enabled the discovery and approval of therapeutic MAbs from divergent species, including camelid, rabbit, and chicken. b) Increasing use of divergent species for therapeutic MAb discovery and development. Analysis of the TABS therapeutic antibody database²³ between 2005 and 2020 demonstrates the growing pipeline of MAbs from divergent species in clinical and preclinical development.

Figure 3.

HCDR3 length and paratope shape. Antibodies co-crystalized with target proteins corroborate the influence of HCDR3 length with paratope shape. In these examples, MAbs with smaller HCDR3 regions have concave (4 amino acid) and flat (11 amino acid) paratopes (left and middle panels). However, erenumab (right panel) with an HCDR3 length of 21 amino acids forms a protruding convex paratope that reaches into a pocket of the CGRP receptor. Structures were obtained from the Protein Data Bank (nivolumab 5WT9, cetuximab 5SX5, and erenumab 6UMG). Cetuximab was derived from mouse immunization, while nivolumab and erenumab were derived from transgenic mice that produce human MAbs.

Figure 4.

Few currently marketed therapeutic antibodies have long HCDR3. We analyzed the HCDR3 sequences of 137 FDA approved and late clinical stage MAbs.⁵ The average HCDR3 length of these MAbs was 10 amino acids. Only 16% of these MAbs have an HCDR3 length of 14 amino acids or more, the predicted threshold to form a protruding topology. The relationship between HCDR3 length and paratope shape noted here is based on the study by Ramsland et al.³²

Figure 5.

Human drug targets are less conserved in chickens compared with mice. A collection of 663 human drug targets curated in the ECOdrug database were analyzed with respect to their amino sequence homology. Almost half (48%) of the human drug targets have the highest degree of conservation (>90%) when compared with mice, but only 15% of targets are conserved at this high level with chickens.

Figure 6.

Sequence divergence of SLC2A4, a conserved 12-TM transporter. SLC2A4 is a complex immunogen with small extracellular loops and 95% sequence identity between mouse and human. The extracellular loops of human SLC2A4 have only 9 amino acid differences relative to mouse, but 20 amino acid differences (and only 65% identity) with the closest chicken paralog, SLC2A1. The use of a chicken host enabled the discovery of antibodies with diverse epitopes.¹¹