Fig. 2: Agonistic activity of novel clones driven by the interplay between epitope, affinity, and IgG subclass. | Nature Communications

Fig. 2: Agonistic activity of novel clones driven by the interplay between epitope, affinity, and IgG subclass.

From: A bispecific CD40 agonistic antibody allowing for antibody-peptide conjugate formation to enable cancer-specific peptide delivery, resulting in improved T Cell proliferation and anti-tumor immunity in mice

Fig. 2

A The agonistic activity of the anti-CD40 mAbs clones incubated for 48 h with immature moDCs evaluated for upregulation of markers MHC-II, CD86, and CD83 clustered in heatmap together with IL-12 secretion. B Epitope mapping of the top agonistic clone A9 and non-agonistic clone B1 determined by HDX-MS illustrated in a CD40L-CD40 crystal structure (PDB 3QD6). C Agonistic activity of the mAbs was performed by incubation with immature moDCs for 48 h and evaluated by flow cytometry of CD86 MFI levels (illustrated as background-subtracted log-transformed values) n = 3 (independent donors) and IL-12p40 levels n = 5 (independent donors pooled from two individual experiments) in the supernatant were quantified by ELISA. P-values are shown in the graph (ns = non-significant) and calculated with Kruskal-Wallis with Dunn´s multiple (CD86) and one-way ANOVA with Dunnett´s multiple comparisons test (hIL12p40). D Agonistic activity of mAbs evaluated by upregulation of CD86 on isolated CD19+ B cells via flow cytometry 24 h post-stimulation. n = 2 (independent donors, illustrated as the mean of two technical replicates). E Immature htgCD40 BMDCs were stimulated for 48 h with the mAbs thereafter, IL-12p40 was quantified by ELISA in the supernatant. n = 1 (mean value from two technical replicates). Data is shown as mean ± SEM.

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