Detailed Protocols for the Selection of Antiviral Human Antibodies from Combinatorial Immune Phage Display Libraries

Diebolder, Philipp; Krawczyk, Adalbert

doi:10.5772/intechopen.70139

Cited by 1 publication

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“…The yield of the scFv-CD44 after purification was 3.3 mg per liter of culture bacteria with high purity (>98%) (Supplemental Fig. 2) (15). A truncated version of human CD44(Q21-P220) was expressed and purified to demonstrate binding of different recombinant formats and to demonstrate no cross-reactivity toward murine CD44, despite sharing 76% sequence homology on the amino acid level, or other proteins (Supplemental Figures 3 and 4).…”

Section: Antibody Generationmentioning

confidence: 99%

Preclinical Evaluation of an Engineered Single-Chain Fragment Variable-Fragment Crystallizable Targeting Human CD44

et al. 2020

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Glycoprotein CD44 and alternative splice variants are overexpressed in many cancers and cancer stem cells. Binding of hyaluronic acid (HA) to CD44 activates cell signaling pathways, inducing cell proliferation, cell survival, and invasion. As such, CD44 is regarded an excellent target for cancer therapy when this interaction can be blocked. In this study, we developed a CD44-specific antibody fragment and evaluated it for imaging CD44-positive cancers utilizing positron emission tomography (PET).Methods: A human single-chain antibody fragment (scFv) was generated by phage display, employing the extracellular domain (ECD) of recombinant human CD44. Specificity and affinity of the scFv-CD44 were evaluated using recombinant and tumor cell-expressed CD44. Epitope mapping of the putative CD44 binding site was performed via overlapping peptide microarray. The scFv-CD44 was reformatted into a bivalent scFv-Fc-CD44, based on human IgG 1 -Fc (fragment crystallizable). The scFv-Fc-CD44 was radiolabeled with copper-64 ( 64 Cu) and zirconium-89 ( 89 Zr). The purified reagents were injected into athymic nude mice bearing CD44positive human tumors (MDA-MB-231, breast cancer, triple negative). Biodistribution studies were performed at different time points after injection of [ 64 Cu]Cu-NOTA-scFv-Fc-CD44 or [ 89 Zr]Zr-DFO-scFv-Fc-CD44. PET/computerized tomography (CT) imaging was conducted with [ 89 Zr]Zr-DFO-scFv-Fc-CD44 on days one and seven post-injection and compared to a scFv-Fc control antibody construct targeting glycophorin A (GPA). Results: Epitope mapping of the scFv binding site revealed a linear epitope within the ECD of human CD44, capable of blocking binding to native HA. Switching from a monovalent scFv to a bivalent scFv-Fc format improved its binding affinity toward native CD44 on human breast cancer cells by nearly 200-fold. In vivo biodistribution data showed the highest tumor uptake and tumorto-blood (TTB) ratios of [ 89 Zr]Zr-DFO-scFv-Fc-CD44 between days five and seven. PET imaging 3 confirmed excellent tumor specificity of [ 89 Zr]Zr-DFO-scFv-Fc-CD44 when compared to the control scFv-Fc. Conclusion: We developed a CD44-specific scFv-Fc construct that binds with nanomolar affinity to human CD44. When radiolabeled with 64 Cu or 89 Zr, it demonstrated specific uptake in CD44 expressing MDA-MB-231 tumors. The high tumor uptake (~56%ID/g) warrants clinical investigation of [ 89 Zr]Zr-DFO-scFv-Fc-CD44 as a versatile PET imaging agent for patients with CD44-positive tumors.

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Section: Antibody Generationmentioning

confidence: 99%