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. Author manuscript; available in PMC: 2024 Nov 14.
Published in final edited form as: Immunity. 2023 Oct 30;56(11):2570–2583.e6. doi: 10.1016/j.immuni.2023.09.013

Figure 1. KRASG12D mutation-specific dIgA1, but not IgG4, neutralizes KRASG12D inside tumor cells and expels the oncodriver outside tumor cells.

Figure 1.

(A) Immunoblots showing that recombinant anti-KRASG12D-IgA1 and anti-KRASG12D-IgG4 antibodies specifically recognize this mutation in KRAS, but not G12C or G13D mutant KRAS, or WT KRAS (n=3). (B) Confocal immunofluorescence microscopy images showing that Alexa Fluor 647-conjugated anti-KRASG12D-IgA1, but not with anti-KRASG12D-IgG4, penetrates KRASG12D-mutant A427 and SK-LU-1 NSCLC cells, while KRASG12D-specific, but not control irrelevant dIgA, co-immunoprecipitated with KRASG12D; Scale bar, 25 μm. (C) OVCAR3 cells were transduced with KRASG12D or WT KRAs fused to PamCherry (Red). Confocal microscopy shows that treatment with Alexa Fluor 488-conjugated anti-KRASG12D-IgA1 (middle panel), but not with anti-KRASG12D-IgG4 (right panel), disarrays KRASG12D intracellular distribution, without effects on KRASWT cells (left panel). (n=3). Scale bar, 50 μm (D) The secretory component of human PIGR was immunoprecipitated from media from KRASG12D-PamCherry-transduced OVCAR3 cells, treated with anti-KRASG12D-IgA1, IgG4, irrelevant IgA or PBS vehicle. Eluted immunocomplexes were ran by SDS-PAGE, stained with Ponceau Red, and immunoblotted for KRASG12D, IgA, and pIgR (n=3). I Immunoblots showing KRASG12D levels in anti-KRASG12D-IgA1/IgG4 or irrelevant IgA-treated KRASG12D-OVCAR3 cell lysates. (F) Supernatants from KRASG12d-PAmCherry-transduced OVCAR3 cells, treated with anti-KRASG12D-IgA1, IgG4, irrelevant IgA or PBS vehicle, were subjected to liquid chromatography with tandem mass spectrometry (LC–MS/MS). Heatmaps showing intensities of mCherry (left panel) and pIgR (right panel) peptides detected (n=3). (G) Left, Transcytosis experiment adding biotinylated anti-KRASG12D-IgA1/IgG4 or irrelevant IgA to the upper chamber of a transwell system, where physical access of antibodies to the basal chamber is prevented by WT or KRASG12D-OVCAR3 cells grown to confluence in transwell inserts. Right, dot blots showing presence of KRASG12D (top panel) or beads (bottom panel) in the streptavidin immunoprecipitates of the basal and upper chamber contents.