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. 2024 Aug 30;15:7553. doi: 10.1038/s41467-024-51781-0

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 1 — a Overview of the phage display process, illustrating key molecules involved at each step (1-panning, 2-counter panning, 3-phage selection, 4-polyclonal ELISA, 5-clone sequencing and monoclonal ELISA, 6-cloning, 7-recombinant production of positive candidates, 8-resulting antibody fragments) of the panning strategies performed on naive LiAb-SFMAXTM (scFv) and synthetic HuCAL (Fab) (gray boxes) or ALTHEA Gold semi-synthetic (scFv) library (purple boxes). b Sensograms depicting the interaction of resulting recombinant antibody fragments (F3 scFv², F4 scFv², F8 scFv², F11 scFv², C9 scFv², B1 Fab², and B3 scFv²) to ofCS, purified from human placenta and immobilized on a quartz crystal microbalance biosensor chip. Red curves correspond to experimental points and rate constants (association rate k_on, dissociation rate k_off, binding constant K_D) were determined using a 1:2 fitting model with fitted curves in black. c Flow cytometry binding of V5-tagged-scFv² (F4, F11, B3, C9, F8, and F3) and FLAG-tagged-Fab² (B1) to cancer cell lines (hematological leukemic NALM-6 (top), epithelial colorectal Colo205 (middle) and mesenchymal glioblastoma U2OS (bottom)). The binding was measured as relative GeoMFI probed with FITC-labeled secondary antibodies without (colored) or with (gray) chABC treatment, with number of data point (n) indicated for each group. d C9 scFv² binding to a larger panel of human cancer cell lines (hematological (red), epithelial (blue), and mesenchymal (green)) and to healthy purified white blood cells (purple), at 150 nM concentration, compared to the secondary antibody control (gray). e Immunofluorescence staining of A549 lung cancer cells spiked into healthy donor blood with PE-labeled scFv² (F4, F8) or Fab² (B1) (yellow), anti-CD45-Alexa647 (pink) and DAPI (blue). Top panel is a merged image of the three individuals shown below. These scans are representative of an image dimension of 2286 tiles and 41.06 mm × 14.03 mm per image (B1/F4) or 130 tiles and 5.12 mm × 2.91 mm (F8). Source data are provided as a Source data file.