| Autor: |
Austin M; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK.; Antibody Discovery & Protein Engineering, BioPharmaceuticals R&D, AstraZeneca , Cambridge, UK., Burschowsky D; Leicester Institute of Structural and Chemical Biology and the Department of Molecular and Cell Biology, University of Leicester , Leicester, UK., Chan DTY; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Jenkinson L; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Haynes S; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Diamandakis A; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Seewooruthun C; Leicester Institute of Structural and Chemical Biology and the Department of Molecular and Cell Biology, University of Leicester , Leicester, UK., Addyman A; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Fiedler S; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Ryman S; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Whitehouse J; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Slater LH; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Hadjinicolaou AV; MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford , Oxford, UK., Gileadi U; MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford , Oxford, UK., Gowans E; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Shibata Y; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Barnard M; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Kaserer T; Cancer Research UK, Cancer Therapeutics Unit, The Institute of Cancer Research , London, UK., Sharma P; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Luheshi NM; Early Oncology Discovery, Oncology R&D, AstraZeneca , Cambridge, UK., Wilkinson RW; Early Oncology Discovery, Oncology R&D, AstraZeneca , Cambridge, UK., Vaughan TJ; Antibody Discovery & Protein Engineering, BioPharmaceuticals R&D, AstraZeneca , Cambridge, UK., Holt SV; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK., Cerundolo V; MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford , Oxford, UK., Carr MD; Leicester Institute of Structural and Chemical Biology and the Department of Molecular and Cell Biology, University of Leicester , Leicester, UK., Groves MAT; Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK.; Antibody Discovery & Protein Engineering, BioPharmaceuticals R&D, AstraZeneca , Cambridge, UK. |
| Abstrakt: |
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 p K 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. |
