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.

精氨酸酶2（ARG2）是一种双核锰金属酶，可催化L-精氨酸的水解。ARG2在特定肿瘤微环境中表达失调会产生免疫抑制位，有效地使肿瘤对宿主的免疫系统“不可见”。增加的ARG2表达导致局部L-精氨酸水平的同时消耗，进而导致抑制抗肿瘤T细胞介导的免疫反应。在这里，我们描述了高亲和力抗体（C0021158）的分离和表征，该抗体可完全抑制ARG2的酶功能，有效地在体外恢复T细胞增殖。酶动力学研究证实，C0021158表现出非竞争性作用机制，独立于L-精氨酸浓度抑制ARG2。为了在结构水平上阐明C0021158的抑制机理，解决了Fab与三聚体ARG2形成复合物的共晶体结构。因此，将C0021158的表位定位在距酶的底物结合裂口一定距离的区域，这表明正在采用变构机制。结合C0021158之后，ARG2的不同区域会发生重大构象变化。值得注意的是，表面暴露环的主链结构被完全重新排列，从而导致在Fab-ARG2界面处形成新的短螺旋结构。此外，ARG2表位的这种大规模结构重塑转化为酶活性位点内的更细微变化。39位的精氨酸残基向内重新定向，在空间上阻碍L-精氨酸的结合。预计Arg39也会改变p 160位关键催化组氨酸残基的K _A进一步减弱ARG2的酶功能。在计算机分子对接模拟中，预测结合抗体时L-精氨酸无法有效结合，这一预测得到了使用L-精氨酸模拟物的等温量热实验的支持。具体而言，通过应用C0021158（可能与标准化疗方案或替代免疫疗法结合）在肿瘤微环境中靶向ARG2代表了靶向免疫性冷肿瘤的潜在新策略。