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

Abstract

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pK A of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.

Characterisation of the immune microenvironment of primary breast cancer and brain metastasis reveals depleted T-cell response associated to ARG2 expression

“…Our data agree with other studies showing the prognostic potential of ARG2, 22 , 43 , 44 , 45 while a recent study showed that ARG2 promotes melanoma progression and metastasis through STAT3 signalling, also involved in BM. 46 , 47 Since arginase has been identified as a potential biomarker of disease progression, investigating the therapeutic efficacy of arginase inhibitors as monotherapy or in combination with PD-1/PD-L1 inhibitors has been of great research interest 38 , 40 , 48 , 49 , 50 , 51 , 52 and a number of clinical trials are ongoing ( Supplementary Table S2 , available at https://doi.org/10.1016/j.esmoop.2022.100636 ) in advanced solid tumours and glioblastoma. Finally, we observed that there was a significant difference between the transcript levels of ARG2 and cytotoxic cells and T cells in both BC and BCBM samples ( Figure 4 ) indicative of a depleted T-cell response.…”

Affinity maturation: highlights in the application of in vitro strategies for the directed evolution of antibodies

“…In this context, high-affinity binding and a potent inhibitory effect are essential properties for the therapeutic, which is ideal for an antibody approach. An antibody candidate was first isolated from AstraZeneca's naïve phage display libraries [ 6 ], which showed specific binding to human ARG2 and inhibitory activity in an enzymatic assay in vitro [ 7 ]. This antibody was then taken through a comprehensive affinity maturation process, in which each of the six complementarity-determining regions (CDRs) were targeted for diversification and taken through selections in parallel.…”

“…Sequence and structural comparison of the parental and affinity-matured antibodies revealed that the antibody has gone through some very extensive changes during the affinity maturation process [ 4 ]. Substantial mutations across several regions of the antibody were translated into a large epitope shift that facilitated increases in the interface area and shape complementarity to the antigen, whilst preserving the key contacts of a hydrophobic cleft that is essential for its inhibitory mechanism [ 7 ]. Essentially, these changes have enabled the antibody to re-orient itself into a position allowing for superior binding without changing the observed mechanism of action, and in doing so seemed to have overcome some initial limitations of the parent.…”