title: Evaluation of Targets for Maytansinoid ADC Therapy Using a Novel Radiochemical Assay.
abstract: OBJECTIVE
  Many antibody-drug conjugates (ADCs) become active only after antigen-mediated internalization and release of the cytotoxic agent via antibody degradation. Quantifying these processes can provide critical information on the suitability of a particular receptor target or antibody for ADC therapy by providing insight into the amount of cytotoxic agent released. We describe a simple and inexpensive radiolabel assay to monitor this process in cultured cancer cells.
  
  
  METHODS
  Monoclonal antibodies were trace-labeled at their lysine residues by treatment with the N-hydroxysuccinimide ester of [(3)H]propionic acid. Human cancer cell cultures were treated with the labeled antibody at concentrations sufficient to saturate the targeted antigen. After washing to remove unbound antibody, cells were incubated and analyzed for antigen expression, conjugate degradation and catabolite formation. Results were compared with data obtained from similar assays run with radiolabeled antibody-[(3)H]maytansinoid conjugates ([(3)H]AMCs). To exemplify the method, studies were conducted with a panel of [(3)H]propionamide-antibodies to evaluate processing efficiency in EGFR-expressing SCCHN cell lines, and in NHL cell lines expressing the B-cell targets CD19, CD20, CD22 and CD37.
  
  
  RESULTS
  Use of the [(3)H]propionamide-antibody assay yielded cell-mediated processing results similar to those obtained with corresponding maytansinoid ADCs. Further exploration allowed comparison of expression levels, antigen-dependent degradation, and catabolite formation across a panel of EGFR-expressing SCCHN cell lines, and for multiple targets in various B-cell cancer indications.
  
  
  CONCLUSIONS
  The [(3)H]propionamide-antibody assay described here is a sensitive, facile method which enables rapid and robust assessment of relative antibody processing amounts for target, antibody, and cell line evaluation.
authors: ImmunoGen, Inc., Waltham, Massachusetts, USA. Kate.Lai@immunogen.com.
authors: Katharine C
authors: KC
authors: Lai
authors: ImmunoGen, Inc., Waltham, Massachusetts, USA.
authors: Jutta
authors: J
authors: Deckert
authors: ImmunoGen, Inc., Waltham, Massachusetts, USA.
authors: Yulius Y
authors: YY
authors: Setiady
authors: ImmunoGen, Inc., Waltham, Massachusetts, USA.
authors: Prerak
authors: P
authors: Shah
authors: ImmunoGen, Inc., Waltham, Massachusetts, USA.
authors: Lintao
authors: L
authors: Wang
authors: ImmunoGen, Inc., Waltham, Massachusetts, USA.
authors: Ravi
authors: R
authors: Chari
authors: ImmunoGen, Inc., Waltham, Massachusetts, USA.
authors: John M
authors: JM
authors: Lambert
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000494:pharmacology*; D008453:Maytansine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D058990:Molecular Targeted Therapy; D011869:Radioligand Assay; D014316:Tritium
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D018796:Immunoconjugates; D014316:Tritium; D008453:Maytansine
keywords: ADC; antibody-drug conjugate; antigen-mediated delivery; maytansine
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