Journal: Journal for Immunotherapy of Cancer
Article Title: Fc-null anti-PD-1 monoclonal antibodies deliver optimal checkpoint blockade in diverse immune environments
doi: 10.1136/jitc-2021-003735
Figure Lengend Snippet: Similar Fc requirements for anti-PD-1 mAb therapy in cold tumors are not accompanied by improved long-term survival. C57BL/6 mice received 5×10 5 9464D cells s.c. on day 0. (A–C) When tumors became 5×5 mm mice received weekly doses of 200 µg (i.p) anti-PD-1 isotypes or irrelevant mAbs. Tumor growth was monitored and mice culled when mean tumor area exceeded 225 mm 2 . Data are presented as tumor area (mm 2 ) for each individual mouse (A) or the mean of the group (B). (C) Kaplan-Meier curves showing percentage survival to humane end point on days after tumor inoculation. Experiment performed once, N=5 mice per group. Log-rank (Mantel-Cox) Test, *p<0.05. (D–K) When tumors became 7×7 mm, mice received three doses of 200 µg (i.p) anti-PD-1 mIgG1, mIgG2a, mIgG1-N297A or irrelevant mAbs on days 1, 5 and 8. Mice were sacrificed on day 9 and spleen and tumor analyzed by flow cytometry. (D, E) Frequency of CD45+ immune infiltrates (D) and T lymphocyte populations (expressed as % of CD45+ cells) (E). F) CD8:Treg ratios expressed as fold change compared with control mice. (G, H) Expression of PD-1 on T lymphocyte populations as % (G) or MFI (H). I–J) Expression of PD-L1 on tumor cells (I) or myeloid infiltrating subpopulations (J) presented as MFI. (K) Heat map indicating relative expression of FcγRs in treatment groups compared with controls. Colors represent the mean ratio of a group, where 1=no change; 1upregulation relative to controls. Experiment performed twice, n=10 mice per group. Bars represent mean±SD, *p<0.05, **p<0.01, ***p<0.001 (One-way ANOVA). ANOVA, analysis of variance; i.p, intraperitoneally; mAb, monoclonal antibodie; MFI, mean fluorescence intensity; ns, not significant; PD-1, programmed cell-death; s.c., subcutaneously.
Article Snippet: Purified CD8 T cells were plated in the presence of 1 µg/mL plate-bound anti-CD3 (clone 145–2 C11; in-house) plus 5 µg/mL mPD-L1-Fc or irrelevant Fc-recombinant proteins (R&D Systems).
Techniques: Flow Cytometry, Expressing, Fluorescence
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![Engineered mouse anti-PD-1 mAbs retain equivalent in vitro binding properties and function. (A) Mouse PD-1-transfected HEK293F cells were incubated with the indicated anti-PD-1 mAb isotypes at a range of concentrations prior to staining with a PE- or APC-labeled secondary antibodies. Data show mean fluorescence intensity (MFI) as a percentage of maximum. (B) Cells were incubated with anti-PD-1 mAb as in (A) in the presence of 1 µg/mL AF488-conjugated rat anti-PD-1 mAb. Data are presented as MFI of the rat anti-PD-1 relative to the concentration of competitive mouse mAb. Data in (A) and (B) show one representative experiment of two. Bars represent mean±SEM of triplicates. (C) Surface plasmon resonance analysis illustrating anti-PD-1 mAb binding to mouse PD-1 and blockade of <t>PD-L1/2.</t> Mouse anti-PD-1 mAbs (100 µg/mL) were passed over His-tagged PD-1 captured with an anti-His mAb. Recombinant PD-L1 or PD-L2-Fc were passed over (25 µg/mL) to demonstrate PD-1 binding or blockade. (D) Purified CD8 T cells from C57BL/6 mice were incubated with 1 µg/mL plate-bound anti-CD3, 5 µg/mL plate-bound PD-L1-Fc or irrelevant controls, and 5 µg/mL soluble mouse anti-PD-1 mAbs or irrelevant isotypes. Proliferation was assessed by [3H]-thymidine incorporation. Data show combined means from two independent experiments. Bars represent mean±SD. (E) Activated CFSE-labeled murine splenic T cells were opsonized with anti-PD-1 isotypes (filled bars) or irrelevant controls (open bars) prior to coculture with BMDMs. Experiment performed twice in triplicates. Bars show mean±SD. Student T-test, **p<0.01. BMDM, bone marrow-derived macrophages; CFSE, carboxyfluorescein succinimidyl ester; mAb, monoclonal antibodies; PD-1, programmed cell-death.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3441/pmc08753441/pmc08753441__jitc-2021-003735f01.jpg)
