Abstract 2123: Antibody-dependent cell-mediated cytotoxicity (ADCC) tolerance to monoclonal antibody therapeutics in human breast and colon cancer cell lines
Authors: Biswas Tanuka , Fritzemeier Rebecca , Mark Adam , Meißner Tobias , Young Brandon M. , Pegram Mark D. 
Published: July 2016

Abstract
Resistance to monoclonal antibody (mAb) therapy limits clinical utility. ADCC is a significant mechanism of action of mAb-elicited tumor response. Postulated mechanisms of mAb resistance include, alteration in cell signaling pathways (PI3K/Akt/Ras-MAPK signaling), over activation/expression of alternate receptor kinases (c-Met/ IGF-1R), or proteolysis of extracellular domains harboring target epitopes. However, most studies on mAb resistance have utilized cancer cells conditioned with mAbs alone, in absence of immune effector cells. We developed an in vitro selection model wherein human HER2 positive breast cancer cell lines (BT474, SKBR3) and EGFR-expressing colorectal cancer cell lines (HT29, DLD1) were subjected to acute ADCC (&gt;90% cell death) with high effector-target (100:1) ratio repeatedly with trastuzumab and cetuximab, respectively, using human peripheral blood mononuclear cells (PBMCs) as effectors. Surviving cells were allowed to recover to confluence over 8-10 weeks for 10 total rounds of ADCC selection. Internal controls included mock-treated parental, isotype-IgG1, effector cells only, and mAb alone. In vitro ADCC assays based on calcein fluorescent labeling of live target cells, demonstrated significant reduction (20%, p&lt;0.005) in cell lysis in immune-selected cell lines compared to parental controls. ADCC-conditioned HT29 cells demonstrated 5-fold reduction in EGFR gene copy number by fluorescence in situ hybridization (FISH, p&lt;0.0001), 2-fold reduction in mRNA level (qPCR, p&lt; 0.01) and 1.8-fold reduction in protein level (immunoblot densitometry, p&lt;0.01). In BT474 breast cells, ADCC-conditioned cells acquired elongated fibroblast-like morphology. Transcriptome-wide next-generation RNA sequencing (Illumina NextSeq 500, 2 × 150 bp paired-end, with over 100 million paired-end reads/sample), coupled with bioinformatic analyses (Reactome pathway database) revealed gene expression changes in immune-selected cells. These involve changes in key immune signaling pathways such as NK-cell and T-cell maturation, class I MHC antigen processing, and immune regulatory pathways, among others. Further validation through q-PCR analyses and loss/ gain of expression is ongoing. Future investigation will also determine whether mAb combination with agonist CD137 mAb (co-stimulatory NK receptor) or Fc-engineered mAbs (e.g. afucosylated) will re-sensitize resistant cells. Our data indicate immune-selection by effector cells contributes to ADCC resistance in vitro. Such investigation may help to elucidate potentially targetable pathways/ markers that emerge from immune-selection with mAbs and effector cells.
Citation Format: Tanuka Biswas, Rebecca Fritzemeier, Adam Mark, Tobias Meißner, Brandon M. Young, Mark D. Pegram. Antibody-dependent cell-mediated cytotoxicity (ADCC) tolerance to monoclonal antibody therapeutics in human breast and colon cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2123.