Fig 1: Clonally expanded B cells produce antibodies against extracellular regions of transmembrane receptor antigens that are locally overexpressed in the tumor tissues(A) Non-denatured lysates of #1 and #2 HGSOC primary cell lines and healthy hepatocyte cells were immunoprecipitated with #1 TLS-kappa, or #1 TLS-lambda, or #2 TLS-kappa antibodies (n = 3; using the same germline IGHV03/IGHJ06-01∗02 VH) and the precipitates were subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS). Heatmap showing exclusive detection of GPR85 in #1 TLS-kappa-immunoprecipitates. IgA and J chain are shown as controls.(B) Heatmap of all peptides across the length of GPR85. Scale in (A and B) represents log2-transformed intensities of peptides detected in LC-MS/MS.(C) Immunoblot showing #1 TLS-kappa antibody, but not #1 TLS-lambda or #2 TLS-lambda produced a distinct band at the same position of recombinant GPR85. β-actin was used as loading control. Experiments were repeated three times.(D) Left, histograms showing FACS analysis of WT or GPR85-ablated #1 HGSOC primary cells upon incubation with #1 TLS-kappa, or #1 TLS-lambda, or #2 TLS-kappa antibodies. Experiments were repeated three times. Right, FACS analysis comparing #1 TLS-kappa Ab vs. commercial anti-GPR85 (rabbit polyclonal Abcam ab140783).(E) Top, schema showing positions of the immunogenic peptide sequences within the extracellular region of GPR85. Middle, dot plots of Imagestream analysis showing binding of GPR85 extracellular region peptide-1, but not peptide-2, with #1 TLS-kappa antibody. Middle, histogram showing maximum intensity for antibody: GPR85 extracellular peptide interaction when #1 TLS-kappa antibody was incubated with peptide-1. Bottom, representative Imagestream multi-spectral images showing bright field (BF), antibodies-PE (yellow), peptide-biotin-streptavidin-APC (Red), and the merge showing the interaction or co-localization of #1 TLS-kappa antibody with the N-biotinylated-peptide-1. Experiments were repeated two times.(F) Dot blots showing the specificity of #1 TLS-kappa antibody for the GPR85 extracellular region peptide-1, along with cancer and non-cancer cell lysates. Recombinant GPR85 was used as positive control.Non-specific IgA (irrIgA) was used as an antibody negative control. Experiments were repeated three times.