Panel A shows a schematic of the workflow where IgG B cells from a birch allergic donor are transformed with EBV, screened by Bet v1 ELISA, and sequenced by RT-PCR and Sanger sequencing. Panel B shows a table listing four monoclonal antibodies mAb 5, 8, 10, and 11 with their HV, HJ, HD, LV, and LJ genes, CDRH3 and CDRL3 sequences, and ELISA EC50 values of 0.01137, 0.01033, 0.007264, and 0.004265 micrograms per milliliter respectively. Panel C shows two line graphs of absorbance at 450 nanometers up to 4 across mAb concentrations from 10 to the power of negative 5 to 10 to the power of 1 micrograms per milliliter, where mAb 5, 8, 10, and 11 show sigmoidal curves reaching approximately 3.5 to 4 between 10 to the power of negative 2 and 10 to the power of 0, while Regeneron antibodies 5713, 5714, and 5715 show right-shifted curves with 5713 reaching 4 at higher concentrations and 5714 and 5715 showing lower maximum absorbance. Panel D shows a heatmap of absorbance at 450 nanometers for seven antibodies against PR-10 and non PR-10 allergens, where Bet v1 shows the highest absorbance reaching 4 for all tested antibodies, Mal d1 shows high absorbance for mAb 10 and 11 and Cor a1 shows high absorbance for mAb 5 and 8, while non PR-10 allergens show minimal signal across all antibodies. Panel E shows biolayer interferometry binding curves for mAb 5, 8, 10, and 11 with KD values of 6.16 times 10 to the power of negative 10, 3.3 times 10 to the power of negative 10, 1.6 times 10 to the power of negative 9, and 3.61 times 10 to the power of negative 9 molars respectively, with interference shift reaching approximately 0.4 to 0.6 nanometers at maximum concentrations. Panel F shows line graphs of blocking efficiency percentage across log mAb concentrations, where the combination of 5713, 5714 and 5715 antibodies reaches approximately 70 to 80 percent, mAb 5, 8 show a blocking efficiency of approximately 50% and mAb 10, and 11 remain below 10 percent, confirmed by bar graphs showing maximum blocking efficiency with significant differences marked. Panel G shows a heatmap of competitive binding where antibodies 5 and 8 show the same competitive binding with themselves and 5713, antibody 10 shows competitive binding with 11 and 5715, and antibody 11 shows competitive binding with all antibodies except 5714. Antibodies 5713, 5714, and 5715 only show competitive binding with themselves except for 5713 that competes with antibodies 5 and 8. Panel H shows a molecular surface structure of Bet v1 with an electrostatic potential map scaled from 4.0 to 6.0. Panel I shows ribbon diagrams of Fab08 and Fab10 bound to Bet v1 from two orientations rotated 90 degrees, showing distinct binding positions on the antigen surface.
Discovery and characterization of four novel anti-Bet v1 human antibodies. (A) Experimental design of human anti-Bet v1 discovery. (B) Characteristics of four human anti-Bet v1 antibodies with variable domain genes (V), (D), and (J) for heavy (H) and light (L) chains, CDR3 amino acid sequences, and EC50 against Bet v1. (C) Anti-Bet v1 ELISA of the novel anti-Bet v1 antibodies (n = 3) and the three anti-Bet v1 Regeneron antibodies (REGN5713, REGN5714, and REGN5715) (n = 2). (D) Specificity of anti-Bet v1 antibodies to PR-10 and non-PR-10 allergens assessed by ELISA (samples with OD values exceeding the detection range of the plate reader were plotted at the maximal measurable absorbance [OD450 = 4.0]). (E) BLI kinetics of each novel antibody immobilized on a protein A–loaded biosensor and Bet v1 in solution. Red lines represent fittings to the sensogram traces. Bet v1 concentrations used for each antibody are reported in nM. (F) ELISA testing anti-Bet v1 antibody blocking Bet v1 binding to specific polyclonal IgE (n = 3 biologically independent samples). (G) Competitive binding study between antibodies binding Bet v1 protein–coupled beads. Competitors induced either strong blocking (red boxes), partial competition (gray boxes), or noncompetitive binding (white boxes) with corresponding mAb to Bet v1. (H) Cryo-EM reconstruction of the complex at 4.8 Å resolution. The map is colored according to local resolution. (I) Structural model derived from cryo-EM shown from side and top views. Bet v1 is in blue, Fab08 heavy chain in red, Fab08 light chain in orange, Fab10 heavy chain in green, and Fab10 light chain in cyan. Data in C and F are represented as the mean ± SEM. Exact P values were determined by one-way ANOVA with Tukey’s test in F. *P < 0.05; ****P < 0.0001. CDR3, third complementarity-determining region.