Abstract 1222A: Identification and targeting of CD34+CD176+IL1RAP+ chronic myeloid leukemia stem cells with bi-specific antibodies

Abstract

Abstract Leukemia Stem Cells (LSCs) quiescence in Chronic Myeloid Leukemia (CML) plays a major role in therapeutic resistance and disease progression calling for the need for identifying and targeting such cells (1). LSCs belong to the primitive population; CD34+CD38-Lin-, which can’t distinguish normal hematopoietic stem cells (HSCs) from CML LSCs. IL1RAP was successfully identified as a marker for BCR-ABL+ CD34+ LSCs, but was not specific as it was also expressed peripherally on platelets and monocytes (2). Another marker, Thomsen-Friedenreich antigen (TF, or CD176), was targeted by anti-CD176 mAb in CD176+ leukemic cell lines and induced Fas-mediated apoptosis not only in LSCs but also in all CD176+ cells (3). Because CD34 molecule is a major carrier of CD176 antigen (4) and IL1RAP is tightly correlated to BCR-ABL expression (5), we evaluated the co-expression of IL1RAP and CD176 on hematopoietic progenitor CD34+ CML stem cells. Peripheral blood mononuclear cells (PBMCs) from patients with CML (n = 4) or healthy volunteers (n = 1) were analyzed for BCR-ABL expression and stained with monoclonal anti-human CD176 and anti-human IL1RAP antibodies for analysis by flow cytometry analysis. CD34+HSCs displayed highly significant co-expression of these markers (P &It; 0.01). Additionally, flow-sorted CD34+CD176+IL1RAP+ cells displayed colony forming potential compared to CD34+CD176+IL1RAP- cells (CML-2, P &It; 0.01). Therefore, we generated a bi-specifıc antibody (bis-Ab); TF/RAP, that binds both antigens simultaneously. One Fab contained the VH and VL specific for CD176 and the other Fab was specific for IL1RAP. Site-directed mutagenesis was used to induce knob-in-hole mutations. Either a duel-positive cell line or CML samples were treated with bis-Ab for one hour, and increasing binding was observed (p &It; 0.001). Linear regression analysis has shown cooperative binding of the bis-Ab as compared to monoclonal antibodies. Complement-dependent cytotoxicity assay (CDC) was used to demonstrate killing of CML stem cells or duel+ cell lines. Our results have shown that our TF/RAP selectively targeted IL1RAP+ and CD176+ cell population among CML PBMCs, but not corresponding normal cells; providing a novel therapeutic strategy for the depletion of CML stem cells from the bulk population in clinical HSC transplantation. 1. Zhou H & Xu R (2015). Leukemia stem cells: the root of CML. Protein & cell, 6(6), 403-12. 2. Järås M et al. (2010). Isolation and killing of candidate chronic myeloid leukemia stem cells.... PNAS, 107(37), 16280-5. 3. Yi B et al. (2011). Mechanisms of the apoptosis induced by CD176 antibody in human leukemic cells. Int. J of Onc, 38, 1565-1573. 4. Karsten U & Goletz S(2013). What makes cancer stem cell markers different? Springer Plus, 2(1), 301. 5. Zhao K et al. (2014). IL1RAP as a surface marker for leukemia stem cells ... Int. J .of clinical and experimental medicine, 7(12), 4787-98. Citation Format: Raghda Elsawi, ChengXiang Wu, Soha Younes, Eman Abdel-Momen Mohammed, James E. Robinson, Fouad Mohammed Badr, Stephen E. Braun. Identification and targeting of CD34+CD176+IL1RAP+ chronic myeloid leukemia stem cells with bi-specific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1222A.