Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation

Abstract

<jats:p>Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.</jats:p>

Structural and functional characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 function via a novel non-competitive mechanism of action

“…The structural and epitope interaction rationale behind the antibody sequence evolution seen during the affinity maturation process is described and discussed in detail in a related paper. 25 The ARG2 binding affinity of 173 pM for C0021158 equates to an approximately 50-fold improvement in affinity relative to the Representative data further characterizing clones C0020185 (Δ), C0020186 (▲) and C0020187 (□) originally identified in the parallel in vitro ARG2 biochemical and inhibition high throughput screens. Each clone binds (a) recombinant biotinylated trimeric human ARG2, but not (b) recombinant biotinylated trimeric human ARG1 and (c) effectively neutralizes recombinant trimeric human ARG2 in a scFv concentration-dependent manner.…”

“…This was later confirmed by comparison of crystal structures solved for the parent (C0020187) and selected affinity matured (C001158 and C001181) inhibitory antibodies bound to ARG2. 25 In addition, no amino acid changes were seen in either V L CDR1 or V H CDR3, which suggests that Figure 1. High throughput screening of ARG2 phage display round two outputs identified large numbers of human ARG2-specific binders, but few ARG2 inhibitors.…”

“…No binding of C0021158 to human ARG1 was detected using bio-layer interferometry. 25 Commensurate with an increase in affinity, C0021158 displayed significant improvements in in vitro potency, fully inhibiting recombinant human ARG2, with an IC 50 of 18.5 ± 5.1 nM as an IgG ( Figure 5). There is also a significant change in the steepness of the inhibitory profile seen for C0021158 compared to its parent C0020187 ( Figure 5), with C0020187 appearing to show negative cooperativity in inhibition, implying three non-equivalent binding sites on the ARG2 trimer.…”

“…C0020187 was affinity optimized using a comprehensive affinity maturation campaign that targeted, in a nonbiased manner, the parental scFv's entire sequence, as described in detail by Chan et al 25 The main thrust of this work sought to exploit and recombine advantageous mutations across all six of C0020187's hypervariable complementarity-determining regions (CDRs). Importantly, lead optimized scFvs continued to be screened for ARG2 specificity and inhibition at each stage of the affinity optimization process, helping to ensure that C0020187's inhibitory epitope was retained.…”

“…ScFv clones were reformatted to human IgG1 and the corresponding Fab fragment to enable functional screening in a T-cell proliferation assay and to determine antibody affinity using bio-layer interferometry, respectively. Using this approach, several significantly more tightly binding antibodies were identified, including C0021158, C0021181, C0021177, and C0021144 (K D values of 173 pM, 288 pM, 173 pM and 338 pM, respectively 25 ). The lead antibody C0021158 differs from the parent antibody C0020187 by 1 residue in V H FW1 (Kabat residues: S30R), 5 residues in V H CDR1 (Kabat residues: S31Y; Y32E; A33V; M34A; and S35A), 5 in V H CDR2 (Kabat residues: S53P; G54I; G55P; S56K; T57G), 2 in V L CDR2 (Kabat residues: P55T, S56A), 1 residue in V L FW3 (Kabat residue: I58V) and 4 in V L CDR3 (Kabat residues: S93E, S94L, L95T, A95bN) ( Figure 4).…”