Cryo-electron microscopy reveals a single domain antibody with a unique binding epitope on fibroblast activation protein alpha

Abstract

Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues but is overexpressed in a variety of pathologies, including cancer. Despite this unique expression profile, designing functional diagnostic and therapeutic agents that effectively target this biomarker remains elusive. Here we report the structural characterization of the interaction between a novel single domain antibody (sdAb), I3, and FAP using cryo-electron microscopy. The reconstructions were determined to a resolution of 2.7 Å and contained two distinct populations; one I3 bound and two I3 molecules bound to the FAP dimer. In both cases, the sdAb bound a unique epitope that was distinct from the active site of the enzyme. Furthermore, this report describes the rational mutation of specific residues within the complementarity determining region 3 (CDR3) loop to enhance affinity and selectivity of the I3 molecule for FAP. This report represents the first sdAb-FAP structure to be described in the literature.

Fig. 1. Mass photometry analysis of FAP with SUMO-I3 complexes. (A) Mass distribution of 15 nM FAP alone. The molecular weight (MW) observed by MP for FAP is 198 ± 8.3 kDa, which agrees well with the predicted MW of the dimeric FAP (170 kDa) with glycosylation. (B) Sensorgram from biolayer interferometry data showing binding of FAP with increasing concentrations of MBP-I3. Data are representative of triplicate measurements. (C) Mass distribution of 15 nM BS3 cross-linked FAP with SUMO-I3 in 1 : 5 molar ratio. The MWs observed are 57 ± 7.8 kDa, 200 ± 10.2 kDa, 227 ± 9.5 kDa, and 253 ± 8.8 kDa, which correspond to the expected MWs of two SUMO-I3 (54 kDa), glycosylated FAP alone (198 kDa, panel 1A), and FAP with one SUMO-I3 (225 kDa) or 2 SUMO-I3 (252 kDa) molecules bound, respectively.

Fig. 2. Cryo-EM structures of FAP-I3 complexes. (A) FAP-I3 reconstruction with a local resolution map. (B) FAP-I3 model. (C) FSC curve for the 3D reconstruction of the cryo-EM map of FAP-I3. The average resolution is estimated to be 2.7 Å based on the FSC value of 0.143. (D) FAP-(I3)₂ reconstruction with local resolution map. (E) FAP-(I3)₂ model. (F) FSC curve for the 3D reconstruction of the cryo-EM map of FAP-(I3)₂. The average resolution is estimated to be 2.7 Å based on the FSC value of 0.143. Cartoon models in (B) and (E) show the FAP dimer in two shades of blue and I3 in green. Yellow sticks represent sites of glycosylation on FAP.

Fig. 3. Interactions of FAP with I3. (A) Overview of I3 (green) bound to FAP (blue) showing common VHH regions involved in paratope formation. Blue and red spheres correspond to the first and last residues modeled for I3. Only half of the dimer is shown for clarity. (B) Residues involved in specific interactions at the FAP-I3 interface. Residues were manually selected to highlight interactions or proximity to the interface. (C) An electrostatic surface map of I3 and residues from FAP involved in epitope formation in blue sticks. FAP Y274 sticks into a hydrophobic pocket on I3 as a critical residue in the epitope. (D) A surface representation of the FAP monomer highlighting the I3 epitope region and its location relative to the active site.

Fig. 4. In silico affinity maturation of I3. (A) An electrostatic surface map of FAP and selected residues of I3 near the surface in green sticks. I3 residues selected for mutations are in cyan sticks. (B) Change in affinity and stability results for V107 variants compared with the original I3 sequence. (C) Change in affinity and stability results for S109 variants compared with the original I3 sequence. (D) Data for I3, V107R, and S109R showing the number of hydrogen bonds (HB), salt bridges (SB), and pi stacking interactions present at the interface of FAP and I3.

Fig. 5. FAP epitope comparison with DPP4. (A) Overall structure-based alignment of FAP with DPP4. (B) Highlight of FAP epitope region comparison shows conformational differences in DPP4. (C) Structure based sequence alignment of the FAP epitope region with DPP4. Two key residue differences are indicated by red asterisks. (D) BLI binding data showing MBP-I3 (10 mM) specifically interacts with FAP and not DPP4.