Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123

Abstract

Current therapies for acute myeloid leukemia (AML), are associated with high relapse rates. Hence, development of new therapeutic strategies is crucial to circumvent this problem. Bivalent antibody technology has been used to engineer novel antibody fragments with increased avidity, by assembling two scFv in a single molecule. Here, we present accompanying data from construction and characterization experiments of a biscFv antibody targeting CD123, the most important biomarker of leukemic cancer stem cells which play a key role in relapsed AML after chemotherapy. Data in this article are related to the research paper "Development of a novel engineered antibody targeting human CD123" Moradi-Kalbolandi S. et al. (2016) [1].

Fig. 1

Schematic generation of biscFv. A) dsDNA coding sequence for (Gly₄Ser)₃ linker results from annealing of synthesized overlapping oligonucleotides L-1 and L-2, after heating for 10 min at 94 °C and cooling slowly to room temperature. Overhangs of BamHI and EcoRI were formed after annealing for cloning purpose without digestion requirement. B) scFv-1, by PCR using primer scFv1-F, scFv2-R with NcoI, BamHI restriction sites applied. C) scFv-2, by PCR using primer scFv2-F, scFv2-R with EcoRI, NotI restriction sites applied. D) BiscFv cassette, results from cloning of scFv-1, linker, scFv-2 in to pET22-b vector respectively. E) Protein form of biscFv.

Fig. 2

Construction of biscFv, colony PCR following insertion of each steps. A) Colony PCR following insertion of linker within the plasmid. Lane M: GenRuler DNA ladder (Thermo fisher scientific, USA), Lane V, the empty plasmid, Lane V+L ~450 bp-band, confirmed the insertion of 50 bp linker (L) in to vector (V). B) Colony PCR after second cloning procedure. The ~1250 bp-band indicates the insertion of scFv1 in to the vector contains the linker (V+L+ scFv-1). C) Colony PCR of last step cloning. The ~2000 bp-band indicates of biscFv (scFv1+L+scFv2).

Fig. 3

Expression and purification of biscFv. A) SDS-PAGE of expression, Coomassie brilliant blue staining. Lane M1 and M2: PageRuler prestained protein ladder (Thermo fisher scientific, USA), Lane1: periplasmic extract of E.coli BL21 without pET22-biscFv plasmids (negative control), Lane2: periplasmic extract before passing into the HisTrap column and lane3: purified biscFv. B) Western blot of purified biscFv using anti-His monoclonal antibody and HRP conjugated goat-anti mouse. Lane M1 and M2: PageRuler prestained protein ladder, lane 1: periplasmic extract of E.coli BL21 without pET22-biscFv plasmid as negative control, lane 2: periplasmic extract before passing into the HisTrap column, and lane3: purified biscFv.

Fig. 4

Competitive binding assays by ELISA and flow cytometry. A. Apparent (Relative) affinity determination of dimeric/monomeric scFvs formats with the competitive-ELISA method. Antibody affinity was obtained through measuring the IC₅₀. These results demonstrate that the concentration required for 50% inhibition of the binding was similar for both scFv and biscFv (red and green dashes, respectively), indicating the same intrinsic affinity.But more biscFv remained bound to immobilized CD123 obtained by ELISA, indicating increase in avidity of biscFv. B) Competitive binding assay of purified biscFv and commercial anti-CD123 mAb by flow cytometry. As shown, no obvious shift in fluorescence value were observed with different concentration of biscFv (added to the cells prior to incubating commercial anti-CD123 mAb compared to background staining in the absence of biscFv.

Fig. 5

Proliferation -Apoptosis assays by flow cytometry. TF-1 cells were cultured in RPMI with 10% FBS and treated as describes bellow prior to addition of IL-3 and examined for Annexin V-FITC binding and 7-AAD uptake with flow cytometry after 24 h treatment. The percentage of cells in the lower right (LR, Annexin V⁺/7-AAD⁻) and upper right (UR, Annexin V⁺/7-ADD⁺) regions, indicate early apoptotic cells and late apoptotic cells respectively. A) Treatment with commercial anti-CD123 mAb. B) Treatment with anti-CD123 biscFv. C) Treatment with anti-CD123 scFv. D) Negative control, Treatment with PBS. E) Positive control, cells cultured in the absence of IL-3.