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

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Abstract

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 (51Cr) 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.

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Acknowledgments

We acknowledge the excellent technical assistance of Elizabeth Taras, Valerie McCuller, Bin Zhang, Deborah Todhunter, Andy Sicheneder, and Seunguk Oh.

Compliance with Ethical Standards

This study was funded in part by the US Public Health Service Grant R01-CA36725 awarded by the NCI and the NIAID, DHHS, the Mayo Partnership Award, the HERA Women’s Cancer Foundation, Minnesota Ovarian Cancer Alliance, the Lion Fund, William Lawrence and Blanche Hughes Fund the Randy Shaver Foundation, the Atwater Cancer Drug Development Award, the Deutsche Krebshilfe (JU.S. 111548), and a CETI translational award from the University of Minnesota Masonic Cancer Center.

Conflict of Interest

Schmohl JU, Dougherty PR, Miller JS, Vallera DA declare, that there are no financial conflicts in regards to this work. Gleason MK is an employee of Sandoz Inc.

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Authors and Affiliations

  1. University of Minnesota Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN, USA

    J. U. Schmohl, P. R. Dougherty & D. A. Vallera

  2. Department for Hematology and Oncology, Medicine Department 2, University Hospital of Tuebingen, Tuebingen, 72076, Germany

    J. U. Schmohl

  3. University of Minnesota Masonic Cancer Center, Adult Division of Hematology, Oncology and Transplantation Oncology, University of Minnesota, Minneapolis, MN, USA

    M. K. Gleason & J. S. Miller

  4. Medical Science Liaison, Immuno-Oncology, Novartis Oncology/Sandoz, Minneapolis, MN, USA

    M. K. Gleason

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  1. J. U. Schmohl
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  5. D. A. Vallera
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Correspondence to D. A. Vallera.

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Schmohl, J.U., Gleason, M.K., Dougherty, P.R. et al. Heterodimeric Bispecific Single Chain Variable Fragments (scFv) Killer Engagers (BiKEs) Enhance NK-cell Activity Against CD133+ Colorectal Cancer Cells. Targ Oncol 11, 353–361 (2016). https://doi.org/10.1007/s11523-015-0391-8

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