Soluble Expression and Characterization of a New scFv Directed to Human CD123

Abstract

Leukemic cancer stem cells (LSCs), as a unique cell population in acute myeloid leukemia (AML) marked by CD123 overexpression, are thought to play a key role in relapsed AML after chemotherapy. Thus, CD123 is considered as a particularly important target candidate for antibody-derived diagnosis and therapy. In the present work, we constructed an immunized murine antibody phage display library and isolated the functional anti-CD123 Single-chain fragment variable (scFv) clones. We also introduced fusing variable light (VL) and heavy (VH) chains with a new 18-amino acid residue linker as an alternative to conventional linkers. CD123-specific phage clones were progressively enriched through 4 rounds of biopanning, validated by phage ELISA, and anti-CD123 scFv clones with highest affinity were produced in Escherichia coli. The expression and purification of soluble scFv were verified by Western blot, and the results were indicative of the functionality of our proposed linker. The purified scFv specifically recognized CD123 by ELISA and flow cytometry, without any cross-reactivity with other related cell markers. Affinity of anti-CD123 scFv was measured to be 6.9 × 10(-7) M, using the competitive ELISA. Our work, therefore, provides a framework for future studies involving biological functions and applications of our anti-CD123 scFv. It also reveals the feasibility of high throughput methods to isolate biomarker-specific scFvs.

Development of a new anti‐CD123 monoclonal antibody to target the human CD123 antigen as an acute myeloid leukemia cancer stem cell biomarker

“…Consistent with our study, Qiyu Sun et al showed that 7G3, raised against CD123, specifically binds to the IL-3R a-chain; more importantly, the mAb 7G3 was demonstrated to inhibit IL-3-mediated proliferation of leukemic cell lines [3]. Consistent with our findings, Moradi-Kalbolandi et al showed that purified scFv could specifically recognize CD123 by ELISA and flow cytometry, with no cross-reactivity with related cell markers [1]. The absence of cross-reactivity and high binding capacity of our mAb make it a potential tool for detection or even clinical applications.…”

“…Acute myeloid leukemia (AML), a cancer of the myeloid line of blood cells, is an aggressive malignancy of the bone marrow . AML is characterized by a cell maturation arrest, uncontrolled proliferation, and subsequent accumulation of undifferentiated myeloid blasts in bone marrow, blood, and other tissues, interfering with the production of normal blood cells . Although most patients with AML can be effectively treated with a standard chemotherapy regimen, relapsed AML remains a major problem .…”

“…This unique marker is composed of three extracellular domains, a transmembrane domain, and a short intracellular domain. The N‐terminal domain of this molecule plays a key role in binding to IL‐3, making it an ideal candidate for targeted therapy .…”

“…This unique marker is composed of three extracellular domains, a transmembrane domain, and a short intracellular domain. The N-terminal domain of this molecule plays a key role in binding to IL-3, making it an ideal candidate for targeted therapy [1]. Applied Biochemistry Importantly, high-level CD123 expression on AML cells correlates with a more primitive cell-surface phenotype, higher blast counts at diagnosis, resistance to apoptosis, and a lower complete remission rate, which lead to reduced survival [5].…”

Adenovirus-mediated anti-AEG-1 ScFv expression driven by stathmin promoter inhibits tumor growth in cervical cancer

“…In our study, we creatively developed an adenovirusmediated anti-AEG-1 ScFv expression system driven by stathmin promoter. Actually, due to its antigen-targeting and high-affinity, ScFv is supposed to be widely deployed involving various aspects, especially tumor targeting therapy [13,14,18]. A recent study has displayed a good result of ScFv in AML that ScFv against EphA2 have specific cytotoxicity effect on AML with EphA2 overexpression [13].…”