GENERATION OF SYNTHETIC ANTIBODIES AS POTENTIAL THERAPEUTICS TO BLOCK IL3–MEDIATED INNATE IMATINIB RESISTANCE IN CHRONIC MEYLOID LEUKEMIA
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This thesis was focused on generation of novel antibodies as therapeutics to block innate imatinib resistance in chronic myeloid leukemia (CML). Tyrosine kinase inhibitor (TKI), imatinib, is the standard therapy for CML. Although many patients respond to the conventional TKI chemotherapy, the majority relapse upon withdrawal of treatment (Ross et al., 2013) implicating the inability of TKIs in eradication of leukemic stem cells (LSCs). Accumulating evidence demonstrates that bone marrow stromal cell secreting factors, such as Interleukin 3 (IL3) hinder TKI activity and support CML–LSCs to survive the therapy with innate drug resistance. Therefore, the overall objective of this work was to target IL3 receptor as the biomarker of LSCs in CML. We generated anti–IL3Rα and anti–IL3Rβ antibodies against IL3R using solid phase phage display selection. The phage–displayed antibodies were affinity–matured human anti–mIL3Rα and –β antibodies with nanomolar dissociation for the target proteins that bound to IL3 cell surface receptor on multiple CML cell lines. Anti–IL3Rα and anti–IL3Rβ antibodies successfully impaired IL3 downstream signaling, inhibited IL3–mediated CML cell proliferation, and promoted cell apoptosis and targeted LSCs by disruption of colony formation in vitro. Engraftment of cells treated with anti–IL3Rα and anti–IL3Rβ antibodies showed a prolonged survival of C57BL/6 mouse models that received antibody–treated CML cells. Encouraged by anti–leukemic activity of anti–IL3Rα and anti–IL3Rβ antibodies on CML cells, we used our novel antibodies to engineer anti–IL3Rα–IL3Rα and anti–IL3Rβ–IL3Rβ bivalent and anti–IL3Rβ–IL3Rα bispecific antigen binding fragments (Fabs) to enhance targeting of CML cells. SpyCatcher-SpyTag system was used to join IL3Rα / IL3Rβ mono–Fabs by a covalent bond. The bivalent and bispecific Fab formats against IL3Rα, IL3Rβ, or IL3Rα and IL3Rβ elicited nanomolar dissociation for the targets, bound to CML cells, and impaired IL3 signaling in CML cells in vitro. Remarkably, bivalent Fabs and bispecific Fab induced direct cytotoxicity on CML cells in a dose–dependent manner in vitro. In testing the Synergy / additive effect of Fabs and TKIs, adding imatinib to anti–IL3Rβ–IL3Rβ bivalent or anti–IL3Rβ–IL3Rα bispecific (but not anti–IL3Rα mono–Fab or anti–IL3Rα–IL3Rα bivalent Fab) augmented their cytotoxic potential on CML cells in vitro, suggesting the potential key role of targeting IL3Rβ to deplete CML–LSC. This work provided novel anti–IL3Rα and anti–IL3Rβ antibodies, IL3Rα and IL3Rβ bivalent or bispecific Fabs to specifically target malignant CML blast cells and CML–LSCs. Future studies will advance preclinical strategies for targeted therapy and eradication of LSCs in CML patients.
DegreeDoctor of Philosophy (Ph.D.)
DepartmentPathology and Laboratory Medicine
CommitteeDeCoteau, John F; Vizeacoumar, Franco; Uppalapati, Maruti; Freywald, Andrew
Copyright DateOctober 2018
Synthetic antibodies, IL-3, CML, Chronic myeloid leukemia, Imatinib, Innate Drug Resistance, Therapeutics