title: 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.
authors.affiliation: National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Shima
authors.initials: S
authors.lastname: Moradi-Kalbolandi
authors.affiliation: Sunnybrook Research Institute, Department of Immunology, University of Toronto, Toronto, ON, M5S 1A8, Canada.
authors.forename: Dariush
authors.initials: D
authors.lastname: Davani
authors.affiliation: Parasitology Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Majid
authors.initials: M
authors.lastname: Golkar
authors.affiliation: National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran. habibi_m@pasteur.ac.ir.
authors.forename: Mahdi
authors.initials: M
authors.lastname: Habibi-Anbouhi
authors.affiliation: Immunology Department, Hybridoma Laboratory, Pasteur Institute of Iran, Tehran, Iran. mabolhassani@yahoo.com.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Abolhassani
authors.affiliation: National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran. mashokrgozar@pasteur.ac.ir.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Shokrgozar
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000627:therapeutic use; D000918:Antibody Specificity; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D007167:Immunotherapy; D053650:Interleukin-3 Receptor alpha Subunit / Q000276:immunology* / Q000627:therapeutic use; D015470:Leukemia, Myeloid, Acute / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D051379:Mice; D058990:Molecular Targeted Therapy; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000627:therapeutic use
chemical_list: D000911:Antibodies, Monoclonal; C115022:IL3RA protein, human; D053650:Interleukin-3 Receptor alpha Subunit; D019151:Peptide Library; D057127:Single-Chain Antibodies
keywords: AML; CD123; Leukemic cancer stem cell; Phage display; scFv