Raghda E Eldesouki,1,2 Chengxiang Wu,2 Fayez M Saleh,2,3 Eman Abdel-Moemen Mohammed,1 Soha Younes,4 Naglaa Elsayed Hassan,5 Theresa C Brown,6 Eckhard U Alt,7 James E Robinson,8 Fouad Mohamed Badr,1 Stephen E Braun2,9

1Genetics Unit, Department of Histology and Cell Biology, School of Medicine, Suez Canal University, Ismailia, Egypt; 2Division of Immunology, Tulane National Primate Research Center, Covington, LA, USA; 3Department of Medical Microbiology, Faculty of Medicine, University of Tabuk, Tabuk, Kingdom of Saudi Arabia; 4Department of Clinical pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt; 5Womens Hospital, Hamad Medical Corporation, Doha, Qatar; 6Hayward Genetics Center, Tulane University School of Medicine, New Orleans, LA, USA; 7Applied Stem Cell Laboratory, Departments of Medicine, Tulane University School of Medicine, New Orleans, LA, USA; 8Sections of Infectious Disease, Departments of Pediatrics and Internal Medicine, Tulane University School of Medicine, New Orleans, LA, USA; 9Departments of Pharmacology, Tulane University School of Medicine, New Orleans, LA, USA

Correspondence: Raghda E Eldesouki Email rag.eldesouki@gmail.com

Introduction: Quiescent leukemia stem cells (LSCs) play a major role in therapeutic resistance and disease progression of chronic myeloid leukemia (CML). LSCs belong to the primitive population; CD34+CD38-Lin-, which does not distinguish normal hematopoietic stem cells (HSC) from CML LSCs. Because ThomsenFriedenreich/CD176 antigen is expressed on CD34+ HSC and IL1RAP is tightly correlated to BCR-ABL expression, we sought to increase the specificity towards LSC by using additional biomarkers. Methods: We evaluated the co-expression of both antigens on CD34+ peripheral blood mononuclear cells (PBMCs) from both healthy volunteers and CML patients, using flow cytometry. Then, we used site-directed mutagenesis to induce knob-in-hole mutations in the human IgG heavy chain and the human lambda light chain to generate the bi-specific antibody (Bis-Ab) TF/RAP that binds both antigens simultaneously. We measured complement-directed cytotoxicity (CDC) in CML samples with the Bis-Ab by flow cytometry. Results: In contrast to healthy volunteers, CML samples displayed a highly significant co-expression of CD176 and IL1RAP. When either a double-positive cell line or CML samples were treated with increasing doses of Bis-Ab, increased binding and CDC was observed indicating co-operative binding of the Bis-Ab as compared to monoclonal antibodies. Discussion: These results show that the bi-specific antibody is capable of targeting IL1RAP+ and CD176+ cell population among CML PBMCs, but not corresponding normal cells in CDC assay. We hereby offer a novel strategy for the depletion of CML stem cells from the bulk population in clinical hematopoietic stem cell transplantation.

Keywords: TF antigen, ThomsenFriedenreich/CD176 antigen, IL1RAP, chronic myeloid leukemia, bi-specific antibodies, complement-dependent cell cytotoxicity, CDC

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