A screening assay to identify agents that enhance T-cell recognition of human melanomas

Assay Drug Dev Technol. 2012 Apr;10(2):187-201. doi: 10.1089/adt.2011.0379. Epub 2011 Nov 15.


Although a series of melanoma differentiation antigens for immunotherapeutic targeting has been described, heterogeneous expression of antigens such as Melan-A/MART-1 and gp100 results from a loss of antigenic expression in many late stage tumors. Antigen loss can represent a means for tumor escape from immune recognition, and a barrier to immunotherapy. However, since antigen-negative tumor phenotypes frequently result from reversible gene regulatory events, antigen enhancement represents a potential therapeutic opportunity. Accordingly, we have developed a cell-based assay to screen for compounds with the ability to enhance T-cell recognition of melanoma cells. This assay is dependent on augmentation of MelanA/MART-1 antigen presentation by a melanoma cell line (MU89). T-cell recognition is detected as interleukin-2 production by a Jurkat T cell transduced to express a T-cell receptor specific for an HLA-A2 restricted epitope of the Melan-A/MART-1 protein. This cellular assay was used to perform a pilot screen by using 480 compounds of known biological activity. From the initial proof-of-principle primary screen, eight compounds were identified as positive hits. A panel of secondary screens, including orthogonal assays, was used to validate the primary hits and eliminate false positives, and also to measure the comparative efficacy of the identified compounds. This cell-based assay, thus, yields consistent results applicable to the screening of larger libraries of compounds that can potentially reveal novel molecules which allow better recognition of treated tumors by T cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibody Specificity
  • Antigens, Neoplasm / biosynthesis
  • Cell Line, Tumor
  • Cloning, Molecular
  • Cytokines / metabolism
  • Drug Screening Assays, Antitumor / methods*
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry
  • Genes, Reporter / genetics
  • Genetic Vectors
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunotherapy
  • Jurkat Cells
  • Lentivirus / genetics
  • MART-1 Antigen / immunology
  • Melanoma / immunology*
  • Melanoma / therapy
  • Receptors, Antigen, T-Cell / drug effects
  • T-Lymphocytes / immunology*
  • Tumor Necrosis Factor Receptor Superfamily, Member 7 / analysis


  • Antigens, Neoplasm
  • Cytokines
  • MART-1 Antigen
  • Receptors, Antigen, T-Cell
  • Tumor Necrosis Factor Receptor Superfamily, Member 7
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins