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

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.

Room temperature structure of human IgG4-Fc from crystals analysed in situ

“…However, in these chains, both loop conformations are involved in crystal packing interactions (with an average area of ∼147 Å 2 ). More recently, a crystal structure of intact IgG4, solved under cryogenic conditions, revealed an IgG1-like Cγ2 FG loop conformation in one chain, in the absence of crystal packing interactions (Scapin et al, 2015). …”

“…The recent crystal structure of intact IgG4 (Scapin et al, 2015) revealed an unusual position for one of the Cγ2 domains, which was rotated by ∼120°, exposing the carbohydrate moiety covalently attached to Asn297. In the FG loop from this domain, torsion angles for Lys326 (ψ) (103°), Gly327 (φ) (132°) and Ser331 (φ) (−133°) are comparable to the range of torsion angle values for the unique IgG4-like conformation in the cryogenic and RT IgG4-Fc structures [Lys326 (ψ), 102 to 125°; Gly327 (φ) 77 to 128°; Ser331(φ), −143 to −160°], which differ from the range of typical torsion angle values found in high resolution IgG1-Fc structures [Lys326 (ψ), −3 to −41°; Ala327 (φ), −66 to −103°; Pro331 (φ), −41 to −71°].…”

“…While the conformation of the Cγ2 domain FG loop is conserved in IgG1, high resolution cryogenic crystal structures of IgG4-Fc revealed a different, unique conformation for the Cγ2 FG loop, which would disrupt the interaction with Fcγ receptors (Davies et al, 2014b). Subsequent cryogenic crystal structures of IgG4-Fc (Davies et al, 2014a) and intact IgG4 (Scapin et al, 2015) revealed that the IgG4 Cγ2 FG loop could also adopt the conserved IgG1-like conformation. However, the role of the unique loop conformation in modulating the biological properties of IgG4, and whether one, or both, conformations could be adopted at physiological temperature, and in solution, remains unclear.…”

Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671

“…6 plots the 498 human Fab and four mammalian mAb crystal structures in the PDB database by resolution [9], release date from the PDB, and technical application. The symbol, “Y”, marks the publication of four intact mammalian IgG structures (1HzH, 1IGT, 1IGY, and 5DK3) [32–35]. Solid squares represent the 164 apo-Fabs (Fabs with no bound antigen).…”

Pembrolizumab for the treatment of advanced melanoma

“…19 Unlike other immunoglobulin G subclasses (IgG1, IgG2 and IgG3), the IgG4 isotype has a low affinity for C1q and Fc receptors which allows pembrolizumab to bind to PD-1 on T cells without activating the complement system. 20 Thus, pembrolizumab is capable of blocking the PD-1/PD-L1 interaction while simultaneously preserving the host T-cell antitumor functions.…”