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. 2022 Jan 19;10(1):e003488. doi: 10.1136/jitc-2021-003488

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© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

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Generation and characterization of novel Treg-depleting αCTLA-4 mAbs and oncolytic VVs expressing Treg-depleting αCTLA-4 and GM-CSF. (A) Heatmap shows function-first isolated antibody clones (vertical lines) binding to T cells from CT26 tumor-bearing and naïve BALB/c mice. (B) Antibody-mediated survival (left panel) and TIL modulation (right panel) in CT26 tumor-bearing BALB/c mice. Animals with established tumors received four injections (10 mg/kg) of antibodies with indicated Treg-associated specificity or control mIgG2a antibody (n=5–15). (C) CTLA-4-specific mAbs induce ADCC of in vitro-activated CD4⁺ T cell. Lysed target T cells were identified by FACS. Figure shows mean±SD (n=4–8); **p<0.01 by Student’s t-test. (D) Anti-CTLA-4 (IgG1) mAbs mediate Treg depletion in vivo in PBMC-humanized mice. Clone 4-E03 shows enhanced depletion of human Treg cells (left panel) but not CD8⁺ T cells (right panel) compared with ipilimumab. Each dot represents one mouse. Graph shows mean data from two experiments. *p<0.05 by one-way analysis of variance. Right: Level of 4-E03-induced cell depletion plotted in relation to CTLA-4 expression as determined by flow cytometry. (E) 4-E03 hIgG1 and ipilimumab binding to human, mouse, and cynomolgus CTLA-4 and CD28 by ELISA. (F) 4-E03 IgG1 binding to in vitro-activated CTLA-4-expressing human T cells was preblocked with rhCTLA-4-Fc protein (blue line) (G) 4-E03 and 2-C06 block CD80 and CD86 binding to CTLA-4 by ELISA. (H) Functional ligand blockade in vitro. Graphs show interleukin-2 in supernatants following treatment of in vitro activated human PBMCs with αCTLA-4. A representative donor is shown (n=6). (I) Schematic illustration of the VV vectors used to encode heavy (at J2R locus) and light chains of the αCTLA-4 antibody and GM-CSF (at the I4L locus). (J) Replication kinetics in LoVo cells and (K) oncolytic activity on MIA PaCa-2 cells of VV_GM-αhCTLA-4 (BT-001). TG6002 (recombinant J2R and I4L deleted VV) was added as control. (L) Functional assessment of αCTLA-4 mAb 4-E03 produced by BT-001-infected MIA PaCa-2 cells in vivo (Treg depletion) as in figure 1D. ADCC, antibody-dependent cell cytotoxicity; FACS, fluorescent-activated cell sorting; ICOS, inducible costimulatory molecule; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; RLU, relative light unit; TIL, tumor-infiltrating lymphocyte; VV, vaccinia virus.