Journal: Cancer Research
Article Title: A Tetravalent Bispecific Antibody Selectively Inhibits Diverse FGFR3 Oncogenic Variants
doi: 10.1158/0008-5472.can-23-3195
Figure Lengend Snippet: Figure 2. The bispecific arms of R3-Altibody form cis complexes with FGFR3-ECD. A, HDX epitope mapping shows that Ab1 (Fab component of R3-Altibody) interacts with Ig-like domain 3, whereas Ab3 (N-term scFv component of R3-Altibody) interacts with Ig-like domain 1. Magenta, peptides that showed differences in deuterium uptake ΔD% ≥ 25%; blue, 25% >ΔD ≥ 20%. Structure of FGFR3 D2–D3 was adopted from PDB 1RY7, model of FGFR3 D1 was adopted from AlphaFold structure prediction (AF-P22607-F1- model_v4). B, R3-Altibody predominantly forms a 1:2 complex (calculated molecular weight 302 kDa) with monomeric FGFR3b-ECD when mixed at 1:2 µmol/L ratio (left). The molar masses of free FGFR3-ECD, R3-Altibody, and R3-Altibody in complex with FGFR3b-ECD were determined by A4F-MALS. The experimentally determined molar masses are indicated by horizon lines. Left, two models of the 1:2 complex are depicted as cis vs. trans binding. Right, the molar masses of the free FGFR3-ECD, scFv-Fab arm from R3-Altibody, and the scFv-Fab arm in complex with FGFR3b-ECD were determined by SEC-MALS. The chromatograms show traces representing scFv-Fab:hFGFR3b- ECD mixed at ratios of 1:3 (green), 1:1 (red), and 3:1 (blue). Molecular mass and pictogram of free scFv-Fab, FGFR3b-ECD, and the complex of scFv-Fab bound to FGFR3-ECD are shown above the corresponding peaks. The minor species of ∼247 kDa is consistent with a 1:2 complex (in cis mode) between residual unreduced 2 + 2 (scFv-Fabʹ)2 in our preparation and FGFR3b-ECD. (A, Created with PyMOL.)
Article Snippet: All samples were incubated at ambient temperature for 2170 Cancer Res; 84(13) July 1, 2024 CANCER RESEARCH ow nloaded from http://aacrjournals.org/cancerres/article-pdf/84/13/2169/3469387/can-23-3195.pdf by guest on 13 Septem ber 2024 2 hours and injected into an Eclipse DualTec A4F separation system coupled to an Agilent 1200 HPLC with DAWN laser light scattering instrument and an Optilab T-rEX differential refractometer detector (Wyatt Technology).
Techniques: Molecular Weight, Binding Assay
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