Abstract 4156: Molecular and phenotypic characterization of breast and colorectal cancer cells selected for resistance to antibody-dependent cell-mediated cytotoxicity (ADCC)
Authors: Biswas Tanuka , Fritzemeier Rebecca , Mark Adam , Meißner Tobias , Young Brandon , Jones Brian-Leyland , Pegram Mark 
Published: July 2017

Monoclonal antibody (mAb) therapy is limited by clinical resistance. For example, alterations in PI3K/Akt /Ras-MAPK signaling, over activation/expression of alternate receptor kinases (c-Met/ IGF-1R), or proteolysis of extracellular domains harboring target epitopes are postulated mechanisms of clinical resistance to trastuzumab. ADCC is a major mechanism of action for mAbs. Most studies have focused on cell models of resistance through in vitro selection, in presence of mAbs, in the absence of immune effector cells. We developed a unique model of immune selection, where target cells (HER2 +ve breast cancer cells SKBR3, BT474; and EGFR +ve colon cancer cells HT29, DLD1) were subjected to ADCC (&gt;90% cell death) with saturating concentrations of trastuzumab or cetuximab (100 µg/ml), respectively, and human peripheral blood mononuclear cells (PBMCs). Selection (in triplicate) was continued for 10 consecutive passages and surviving cells were allowed to grow to confluence over a period of 8-10 weeks. Mock-treated parent cells, IgG1 isotype control, mAb-alone and PBMCs only were utilized as controls. Compared to parent controls, immune-selected DLD1 and SKBR3 cells demonstrated statistically significant increase in proliferation (WST viability assays, P&lt;0.01), and significantly higher colony forming efficiency (soft agar assays, P&lt;0.001); immune-selected HT29 and SKBR3 cells showed significantly higher motility (trans-well migration assays, P&lt;0.05, and P&lt;0.001 respectively). In vitro ADCC assays (calcein labeling) suggested significant reduction (max 20%, p&lt;0.005) in cell lysis in all the immune-selected cell lines compared to parent. Immune-selected HT29 cells demonstrated a 5-fold reduction in gene copy number of EGFR by fluorescence in situ hybridization (FISH, p&lt;0.0001), 2-fold reduction in mRNA levels (qPCR, p&lt; 0.01) and 1.8-fold reduction in protein levels of EGFR (densitometry scans, western blot analyses, p&lt;0.01). Transcriptome-wide next-generation RNA sequencing (Illumina NextSeq 500, 2 x 75 bp paired-end, with over 100 million paired-end reads/sample), coupled with bioinformatic analyses (Reactome pathway database) revealed gene expression changes in ADCC-selected cells. These involve changes in key immune signaling pathways such as class I/II MHC antigen processing, cell chemotaxis, inflammatory and immune regulatory pathways among others. Our data indicate immune-selection by effector cells contributes to ADCC resistance in vitro. Further investigation will determine whether mAb combination with an agonist antibody against CD137 (co-stimulatory NK cell receptor) or Fc-engineered MAbs (e.g. afucosylated) will sensitize the resistant cells. It is hoped that this investigation will better inform mAb therapy by elucidating potentially targetable pathways/ markers that emerge from immune-selection with therapeutic mAbs.

Citation Format: Tanuka Biswas, Rebecca Fritzemeier, Adam Mark, Tobias Meißner, Brandon Young, Brian-Leyland Jones, Mark Pegram. Molecular and phenotypic characterization of breast and colorectal cancer cells selected for resistance to antibody-dependent cell-mediated cytotoxicity (ADCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4156. doi:10.1158/1538-7445.AM2017-4156