Fig. 4: Antigenicity barrier of highly stabilized trimers affords highly mutated binders.

a DANA 6–9: Overview of recombinant HIV-1 Env trimer used for panning (I) and type and number of ribosome display selection rounds (II). Modifications to ribosome display are indicated. Quat-pur: panning primer purified with PGT145 to ascertain conformation; SS: super-stabilized SOSIP trimer; PP: pre-panning of DARPin library with V3 peptide to remove DARPins highly reactive with V3 before DANA. b–e DARPin clones with valid ORF derived from randomly picked 190 clones of DANA 6–9 were analyzed for binding, neutralizing and sequence properties (n = 108–134). Data are derived from single experiments. b Binding properties of DARPins derived in DANA 1–5 based on ELISA using Env trimer probes and the V3-crown mimetic peptide V3-IF (BG505) as specified in Supplementary Table 3. Trimer binding is categorized as binding to at least one of several trimers but not the V3-mimetic. V3 specificity includes solely V3-reactive and V3- and trimer dual-reactive clones. Low-level binders categorize DARPins with no trimer- and V3-reactivity >3-fold over background. c Neutralization score against a multi-clade 5-pseudovirus panel reflecting breadth and potency. The max. score is 75, a score 5–14 indicates low neutralizing activity, a score of >15 indicates high neutralizing activity. d Frequency of DARPins without mutations (i.e., typical DARPins), with insertions or deletions and with >5% mutations in the framework. e Distribution of DARPin types.