Enhanced ADCC and NK Cell Activation of an Anticarcinoma Bispecific Antibody by Genetic Insertion of a Modified IL-15 Cross-linker

Mol Ther. 2016 Aug;24(7):1312-22. doi: 10.1038/mt.2016.88. Epub 2016 May 9.

Abstract

Previously, we constructed a bispecific NK-cell-engager (BiKE) bearing single-chain variable fragments (scFv) against CD16 on NK cells and EpCAM on tumor cells. This BiKE facilitated antigen-specific antibody-dependent cell-mediated cytotoxicity (ADCC) but did not induce NK cell expansion. We incorporated a modified interleukin-15 cross-linker to create a trispecific construct (TriKE) in order to improve activation, proliferation, and survival of NK cells. Synthesis and assembly of hybrid genes encoding the TriKE was accomplished using DNA-shuffling and DNA-ligation techniques. The TriKE was tested for specificity, efficacy, proliferative capability, and cytokine profile using functional assays. The molecular modifications improved yield without compromising binding to EpCAM(+) HT-29 colorectal carcinoma cells. (51)Chromium-release and degranulation assays showed better killing rates with TriKE compared to BiKE. TriKE was more active in a variety of different carcinoma cell lines. TriKE showed the ability to stimulate expansion of CD56(+)CD3(-) NK cells. BiKE and TriKE showed enhanced but not supraphysiologic levels of cytokine secretion. 1615EpCAM TriKE drives enhanced ADCC while significantly improving proliferation, activation, and survival of NK cell effectors. The TriKE provides a selectively delivered self-sustaining signal at the NK/tumor cell synapse. Targeted cytokine stimulation, rather than systemic cytokine administration, may impact toxicity in patients rendering the TriKE a promising new off-the-shelf carcinoma therapy.

Publication types

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

MeSH terms

  • Antibodies, Bispecific / genetics*
  • Antibody-Dependent Cell Cytotoxicity / genetics*
  • Carcinoma / genetics
  • Carcinoma / immunology*
  • Cell Degranulation / immunology
  • Cell Line, Tumor
  • Cell Proliferation
  • Cytokines / biosynthesis
  • Epithelial Cell Adhesion Molecule / genetics
  • Epithelial Cell Adhesion Molecule / metabolism
  • Gene Expression
  • Gene Order
  • Genetic Vectors / genetics
  • Humans
  • Interferon-gamma / genetics
  • Interferon-gamma / metabolism
  • Interleukin-15 / genetics*
  • Killer Cells, Natural / immunology*
  • Killer Cells, Natural / metabolism*
  • Lymphocyte Activation*
  • Mutagenesis, Insertional*
  • Protein Binding
  • Recombinant Fusion Proteins

Substances

  • Antibodies, Bispecific
  • Cytokines
  • Epithelial Cell Adhesion Molecule
  • Interleukin-15
  • Recombinant Fusion Proteins
  • Interferon-gamma