Bi-Specific Killer Cell Engager Enhances NK Cell Activity against Interleukin-13 Receptor Alpha-2 Positive Gliomas

Kristen D. Pawlowski, Joseph T. Duffy, Arushi Tiwari, Markella Zannikou, Irina V. Balyasnikova*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Glioblastoma (GBM) is a lethal brain tumor with limited therapeutic options. Bi-specific killer cell engagers (BiKEs) are novel immunotherapies designed to engage natural killer (NK) cells against cancer. We designed a BiKE molecule consisting of a single-domain CD16 antibody, an interleukin-15 linker, and a single-chain variable antibody against the glioma-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2). Recombinant BiKE protein was expressed in HEK cells and purified. Flow cytometric analysis of co-cultures of peripheral blood-derived NK cells with GBM6 and GBM39 patient-derived xenograft lines revealed significantly increased activation of NK cells (CD25+CD69+) and increased glioma cell killing following BiKE treatment compared to controls (n = 4, p < 0.01). Glioma cell killing was also confirmed via immunofluorescence staining for cleaved caspase-3 (p < 0.05). In vivo, intracranial delivery of NK cells with BiKE extended median survival in mice bearing GBM6 (p < 0.01) and GBM12 (p < 0.01) tumors compared to controls. Finally, histological analysis of brain tissues revealed a higher frequency of peritumoral NK cells in mice treated with BiKE than with NK cells alone (p < 0.05). In conclusion, we demonstrate that a BiKE generated in a mammalian expression system is functional in augmenting NK cell targeting of IL13Rα2-positive gliomas.

Original languageEnglish (US)
Article number1716
Issue number13
StatePublished - Jul 2023


  • bi-specific antibody
  • glioblastoma
  • immunotherapy
  • natural killer cells

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)


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