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

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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.

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Abbreviations

AML:

acute myeloid leukemia

CD123:

cluster of differentiation 123

LSCs:

leukemic cancer stem cells

scFv:

single-chain fragment variable

VH:

variable heavy chains of an antibody

VL:

variable light chains of an antibody

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Acknowledgments

This work was supported by Postgraduate Office, Pasteur Institute of Iran, Tehran, Iran.

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Authors and Affiliations

  1. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

    Shima Moradi-Kalbolandi, Mahdi Habibi-Anbouhi & Mohammad Ali Shokrgozar

  2. Sunnybrook Research Institute, Department of Immunology, University of Toronto, Toronto, ON, M5S 1A8, Canada

    Dariush Davani

  3. Parasitology Department, Pasteur Institute of Iran, Tehran, Iran

    Majid Golkar

  4. Immunology Department, Hybridoma Laboratory, Pasteur Institute of Iran, Tehran, Iran

    Mohsen Abolhassani

Authors
  1. Shima Moradi-Kalbolandi
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  2. Dariush Davani
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  3. Majid Golkar
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  4. Mahdi Habibi-Anbouhi
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  5. Mohsen Abolhassani
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  6. Mohammad Ali Shokrgozar
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Correspondence to Mahdi Habibi-Anbouhi, Mohsen Abolhassani or Mohammad Ali Shokrgozar.

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Moradi-Kalbolandi, S., Davani, D., Golkar, M. et al. Soluble Expression and Characterization of a New scFv Directed to Human CD123. Appl Biochem Biotechnol 178, 1390–1406 (2016). https://doi.org/10.1007/s12010-015-1954-y

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  • DOI: https://doi.org/10.1007/s12010-015-1954-y

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