Fig 1.
Accelerated workflow for the discovery of antagonistic A2AR antibodies.
(A) Immune and synthetic libraries were sourced and built from antibody diversities obtained from hA2AR-immunized Abveris DiversimAb/DivergimAb mice and humanized ATX-GK mice, as described in the materials and methods. (B) Pipeline for the discovery of antagonistic A2AR antibodies from immune and synthetic libraries. After phage panning, hits were sequenced, reformatted to IgG, purified, and triaged in a series of binding and functional assays.
Table 1.
Summary of libraries and triage results for lead identification.
Fig 2.
TB206-001 is a high-affinity, specific, and cross-reactive binder of A2AR.
(A) TB206-001 binds to hA2AR-overexpressing HEK293 cells with high apparent affinity (EC50 = 5.76 nM). (B) TB206-001 cross-reacts with mA2AR but not hA1R, hA2BR, and hA3R. p<0.05. (C) TB206-001 cross-reacts with cynomolgus PBMCs.
Fig 3.
In vitro cAMP cell-based functional assay and primary immune cell activation assay.
(A) Detection of cAMP in HEK293 cells that overexpress hA2AR. TB206-001 dose-dependently increased the RFU ratio (665/615 nm), indicating an antagonistic effect on NECA-stimulated cAMP production. (B) TB206-001 antagonized NECA-stimulated IFN-γ release in T cell-activated (CD3/CD28-simulated) PBMCs. The small molecule A2AR antagonist ZM-241385 served as a positive control. (C) Effect of NECA ligand concentration on TB206-001 antagonism of NECA-stimulated IFN-γ release. (D, E) Isolated T cells activation is assessed by cell proliferation and up-regulation of activation marker CD25. The small molecule A2AR antagonist AB928 served as a positive control. (F, G) Isolated NK cell activation is detected by IFN-γ release and activation marker CD107. IL15 stimulation (10 ng/mL) serve as a positive control for NK cell activation.
Fig 4.
TB206-001 suppresses the growth of COLO 205 tumors in HuCD34-NCG mice.
(A) Dosing and clinical monitoring schedule. Dosing was initiated when the average tumor volume of the cohort reached ~100 mm3. Mice were monitored three times a week for changes in tumor size and other clinical signs until termination 24 days after dosing initiation. (n = 6) (B) TB206-001-IgG1, pembrolizumab, and the small molecule AZD4635 suppressed the growth of COLO 205 tumors in HuCD34NCG mice. (C) TB206-001-IgG4 binds to hA2AR-overexpressing HEK293 cells (EC50 = 87.5 nM). (D) TB206-001-IgG4 promotes IFN-γ release from NECA-stimulated, T cell-activated PBMCs. (E) TB206-001-IgG4 and TB206-001-IgG1 suppressed the growth of COLO 205 tumors in HuCD34NCG mice with similar potency. *p ≤ 0.05 vs. isotype; **p ≤ 0.01 vs. isotype.