Fig 1.
SEC profiles showing the formation of HER2-trastuzumab-pertuzumab ternary complex.
A. HER2 is first complexed with trastuzumab Fab and the binary complex elutes earlier than HER2 alone. The purified complex is further complexed with pertuzumab Fab and the ternary complex elutes earlier than the binary complex. The UV absorption is normalized. B. HER2 is first complexed with pertuzumab Fab and the binary complex elutes earlier than HER2 alone. The purified complex is further complexed with trastuzumab Fab and the ternary complex elutes earlier than the dimer. The UV absorption is normalized.
Fig 2.
Brief summary of cryo-EM data processing.
1,032,611 particles were picked automatically and extracted in cisTEM software [28]. Three rounds of 2D classification were carried out and classes with clearer structure features were selected. Representative classes are shown here. A total of 398,409 particles were used in 3D Auto Refine and the final reconstruction was generated. The resolution was determined with FSC cutoff at 0.143.
Fig 3.
Cryo-EM map of HER2-trastuzumab-pertuzumab.
A. Cryo-EM map showing the three components of the ternary complex: HER2 (sky blue), trastuzumab Fab (pink), and pertuzumab Fab (orange). B. Local resolution analysis of the final construction calculated using ResMap. C. Final construction (grey surface) with HER2 (sky blue), trastuzumab Fab (pink), and pertuzumab Fab (orange) fitted in the map. Glycans on HER2 are shown as sticks.
Fig 4.
Overall structure of HER2-trastuzumab-pertuzumab and comparison with HER2-pertuzumab (PDB: 1S78) and HER2-trastuzumab (PDB: 1N8Z) structures.
A. Overall structure of HER2-trastuzumab-pertuzumab. Domain I through domain IV of HER2 are shown in green, teal, cyan, and blue respectively. Trastuzumab Fab and pertuzumab Fab are shown in pink and yellow respectively. The previously proposed pertuzumab-induced new trastuzumab epitope is highlighted in magenta. B. Structure comparison of HER2-trastuzumab-pertuzumab and HER2-pertuzumab. In HER2-trastuzumab-pertuzumab, HER2, pertuzumab and trastuzumab are shown in cyan, yellow and pink respectively. In HER2-pertuzumab, HER2 and pertuzumab are shown in blue and orange respectively. C. Structure comparison of HER2-trastuzumab-pertuzumab and HER2-trastuzumab. In HER2-trastuzumab-pertuzumab, HER2, trastuzumab and pertuzumab are shown in cyan, pink and yellow respectively. In HER2- trastuzumab, HER2 and trastuzumab are shown in blue and magenta respectively. D. Distances between the C-terminus of trastuzumab VL and N-terminus of pertuzumab VL (68.6 Å), between C-terminus of trastuzumab VH and N-terminus of pertuzumab VH (56.2 Å), and between the C-termini of trastuzumab CH1 and pertuzumab CH1 (99.7 Å) were measured. The residues used for measurement are highlighted in spheres. Light chain and heavy chain of trastuzumab Fab are shown in pink and magenta. Light chain and heavy chain of pertuzumab Fab are shown in yellow and orange. The sequences used as nine-residue linkers in TPL bispecific antibody are highlighted in teal (light chain) and blue (heavy chain).
Fig 5.
Schematic representation of bispecific molecules targeting HER2.
A. Bispecific antibody with knob-into-hole Fc. B. Bispecific molecules with engineered hinge region of IgG3. C. Bispecific molecules connected by a dimeric coiled coil.