Heterodimeric Bispecific Single Chain Variable Fragments (scFv) Killer Engagers (BiKEs) Enhance NK-cell Activity Against CD133+ Colorectal Cancer Cells

Target Oncol. 2016 Jun;11(3):353-61. doi: 10.1007/s11523-015-0391-8.


Background: Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse, and express the well-known surface marker CD133.

Methods: DNA fragments from two fully humanized single chain fragment variable (scFv) antibodies recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigens to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated a unique antibody that recognized the molecular framework and was species cross-reactive.

Results: In vitro chromium-51 ((51)Cr) release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5 % CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug.

Conclusion: These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133 and that this anti-CD133 scFv may be useful in this bispecific platform or perhaps in the design of more complex trispecific molecules for carcinoma therapy.

Publication types

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

MeSH terms

  • AC133 Antigen / immunology*
  • Animals
  • Antigens, Neoplasm / immunology*
  • Colorectal Neoplasms / immunology*
  • Humans
  • Killer Cells, Natural / immunology*
  • Mice
  • Single-Chain Antibodies / immunology*


  • AC133 Antigen
  • Antigens, Neoplasm
  • Prom1 protein, mouse
  • Single-Chain Antibodies