J U Schmohl1,2, M K Gleason3,4, P R Dougherty1, J S Miller3, D A Vallera5. 1. University of Minnesota Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN, USA. 2. Department for Hematology and Oncology, Medicine Department 2, University Hospital of Tuebingen, Tuebingen, 72076, Germany. 3. University of Minnesota Masonic Cancer Center, Adult Division of Hematology, Oncology and Transplantation Oncology, University of Minnesota, Minneapolis, MN, USA. 4. Medical Science Liaison, Immuno-Oncology, Novartis Oncology/Sandoz, Minneapolis, MN, USA. 5. University of Minnesota Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN, USA. valle001@umn.edu.
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 ((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.
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 humanCaco-2colorectal 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 humanBurkitt's lymphomaDaudi 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-CD133scFv may be useful in this bispecific platform or perhaps in the design of more complex trispecific molecules for carcinoma therapy.
Authors: Konstantinos A Papadakis; John L Prehn; Carol Landers; Qiwei Han; Xia Luo; Stephanie C Cha; Ping Wei; Stephan R Targan Journal: J Immunol Date: 2004-06-01 Impact factor: 5.422
Authors: Jianhua Huang; Chonghui Li; Yao Wang; Haiyan Lv; Yelei Guo; Hanren Dai; Max S Wicha; Alfred E Chang; Qiao Li Journal: Clin Immunol Date: 2013-08-07 Impact factor: 3.969
Authors: Michael F Clarke; John E Dick; Peter B Dirks; Connie J Eaves; Catriona H M Jamieson; D Leanne Jones; Jane Visvader; Irving L Weissman; Geoffrey M Wahl Journal: Cancer Res Date: 2006-09-21 Impact factor: 12.701
Authors: M Peichev; A J Naiyer; D Pereira; Z Zhu; W J Lane; M Williams; M C Oz; D J Hicklin; L Witte; M A Moore; S Rafii Journal: Blood Date: 2000-02-01 Impact factor: 22.113
Authors: Michelle K Gleason; Michael R Verneris; Deborah A Todhunter; Bin Zhang; Valarie McCullar; Sophia X Zhou; Angela Panoskaltsis-Mortari; Louis M Weiner; Daniel A Vallera; Jeffrey S Miller Journal: Mol Cancer Ther Date: 2012-10-17 Impact factor: 6.261
Authors: Amy P N Skubitz; Elizabeth P Taras; Kristin L M Boylan; Nate N Waldron; Seunguk Oh; Angela Panoskaltsis-Mortari; Daniel A Vallera Journal: Gynecol Oncol Date: 2013-05-27 Impact factor: 5.482
Authors: Nate N Waldron; Dan S Kaufman; Seunguk Oh; Zintis Inde; Melinda K Hexum; John R Ohlfest; Daniel A Vallera Journal: Mol Cancer Ther Date: 2011-08-23 Impact factor: 6.009
Authors: Christoph Stein; Christian Kellner; Markus Kügler; Nina Reiff; Kristin Mentz; Michael Schwenkert; Bernhard Stockmeyer; Andreas Mackensen; Georg H Fey Journal: Br J Haematol Date: 2010-01-08 Impact factor: 8.615