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Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab

Nature Structural & Molecular Biology volume 22, pages 953–958 (2015)Cite this article

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Abstract

Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility.

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Figure 1: Overall pembrolizumab structure.
Figure 2: Pembrolizumab linker region.
Figure 3: Pembrolizumab Fc conformation.
Figure 4: Model of binding of the FcγRIII and of the FcRn receptors to pembrolizumab.

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Acknowledgements

The authors would like to thank J. Chapman and R. Ruzanski for performing receptor binding and capillary electrophoresis assays and M. Hohn for help in generation of the N15 labeled antibody. Research described in this paper was performed at the Canadian Light Source, which is supported by the Canadian Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada and the Canadian Institutes of Health Research. The X-ray diffraction data were collected by Shamrock, and we thank G. Ranieri, J. Carter and R. Walter for collecting the data.

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Authors and Affiliations

  1. Structural Chemistry, Merck & Co., Inc., Kenilworth, New Jersey, USA

    Giovanna Scapin, Winifred W Prosise, Mark McCoy, Paul Reichert, Jennifer M Johnston & Corey Strickland

  2. Biologics and Vaccines Formulation, Analytical Method Development, Merck & Co, Inc., Kenilworth, New Jersey, USA

    Xiaoyu Yang

  3. Bioprocess Technology and Expression, Merck & Co., Inc., Kenilworth, New Jersey, USA

    Ramesh S Kashi

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  1. Giovanna Scapin
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Contributions

G.S. carried out crystallographic experiments and structure determination. X.Y. prepared pembrolizumab and Fab fragments for crystallization and NMR experiments, designed and carried out the deglycosylation experiments, designed the receptor binding experiments and analyzed the resulting data. W.W.P. and P.R. established the crystallization procedures. M.McC. carried out NMR experiments and analyzed the resulting data. J.M.J. performed molecular dynamics simulations and analyzed the resulting data. R.S.K. supervised antibody sourcing and formulation for crystallization studies. G.S. and C.S. wrote the paper, and all authors contributed to editing of the manuscript.

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Correspondence to Giovanna Scapin.

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Competing interests

All authors are Merck & Co. Inc. employees, and pembrolizumab is a Merck marketed product called KEYTRUDA®.

Integrated supplementary information

Supplementary Figure 1 Surface representation of the upper linker region in pembrolizumab.

The surface view of the upper linker region in pembrolizumab, including the hinge peptide (residues Val218–Pro225) and the nearby peptides Pro130–Thr138 and Leu196–Gly197 clearly shows the interdigitation occurring in this area. In the IgG1 structures 1HZH (Saphire, E.O., et al., Science. 293, 1155-1159, 2001) and 1IGY (Harris, L.J., et al., J. Mol. Biol. 275, 861-872, 1998) there are no contacts between the two upper linker regions. The compact hinge seen in the crystal structure differs from the extended hinge conformation proposed for the solution structure of human IgG4, both wild type and S228P mutant (Rayner, L.E., et al., J Biol Chem. 289, 20740–20756, 2014). The models generated in the paper to fit the BioSAXs data were calculated from the IgG1 structure, constraining the full hinge region (Val218–Pro238) to be of a minimum length of between 50 and 73.5 Å (or between 22 to 31.5 Å for the Val218–Cys226 peptide), values derived from the 1HZH structure. From the pembrolizumab structure the length (Ca to CA) of the very well ordered Val218–Cys226 peptide is about 13 Å, while the distance between Val218 and Pro238 is 24 Å in one chain and 34 Å in the other, much shorter than the values used for the modeling of the bioSAXs data.

Supplementary Figure 2 Distribution of phi and psi angles for the wild-type and S228P pembrolizumab linker peptides Val218–Gly236.

Top panel: Phi/Psi populations (frequency versus angle) plotted for residues P227 and P(S)228 of the linker region. Phi/Psi populations derived from last 36 nanoseconds of simulations of linker peptide dimers (20 residues, Val218–Gly236), with either a Pro or a Ser at position 228. The missing portions of the linker (230PAP232 in chain B and 230PAPEFL235 in chain G) were built and minimized in the context of the entire protein using the ab initio loop builder functionality of MOE 2013.08. The linker region was extracted and prepared using the prep-wizard in Maestro (Schrodinger) and to generate the WT linker, P228 was mutated back to S. Both WT and S228P pembrolizumab linker dimers were simulated for 48 ns each, in cubic boxes of ~8000 water molecules using the Schrodinger implementation of Desmond molecular dynamics. After the standard restrained equilibration procedure (default settings in Schrodinger 2014-4), the first 12 nanoseconds were also discarded as ‘equilibration’ from both simulations. Bottom panel: Distribution of the S-S distances between residues C226 and C229 during the two simulations for the S228P (left) and S228 (right) peptide. Both S228P and WT (S228) linkers were simulated as single chains, in exactly the same way as described above, but with the Cysteine S atoms protonated. The dual distribution in the S228 peptide suggests that there is a tendency for Cys226 and Cys229 to move closer to each other thus facilitating the formation of an intra-chain disulfide bond.

Supplementary Figure 3 Structural conservation between the Fc domains in pembrolizumab.

A) Overlay of the two CH2 domains observed in pembrolizumab: residues 240–245 and 257–340 of the conventional CH2 (Chain A, yellow trace) and the flipped CH2 (chain B, cyan trace) align with an RMSD on CA of 0.76 Å (1.24 Å if calculated for all atoms). The major difference is for the loop spanning residues 246–256 which in the flipped CH2 assumes a different conformation because of crystal contacts. The sugar residues also align well: the RMSD for the core residues is 0.1 Å. B) Overlay of the glycans in chain A (black carbon), PDB entry 4C54 (yellow carbons, RMSD with chain A is 0.2 Ang) and 1HZH (magenta carbon, rmsd with Chain A is 0.4 Ang).

Supplementary Figure 4 Representative sensorgrams from the single-cycle kinetic SPR measurements.

Left Panel: representative sensorgrams for the single cycle kinetic (SCK) SPR method used to measure the affinity (KD) of pembrolizumab (A) and IgG1 (B) to FcγRI; Right panel: Representative sensorgrams for the multi-cycle steady state affinity method used to measure the affinity of pembrolizumab (C) and IgG1 (D) to FcγRIIAH131

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Scapin, G., Yang, X., Prosise, W. et al. Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab. Nat Struct Mol Biol 22, 953–958 (2015). https://doi.org/10.1038/nsmb.3129

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