Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates

Abstract

Glycoengineered therapeutic antibodies and glycosite-specific antibody-drug conjugates (gsADCs) have generated great interest among researchers because of their therapeutic potential. Endoglycosidase-catalyzed in vitro glycoengineering technology is a powerful tool for IgG Fc (fragment cystallizable) N-glycosylation remodeling. In this protocol, native heterogeneously glycosylated IgG N-glycans are first deglycosylated with a wild-type endoglycosidase. Next, a homogeneous N-glycan substrate, presynthesized as described here, is attached to the remaining N-acetylglucosamine (GlcNAc) of IgG, using a mutant endoglycosidase (also called endoglycosynthase) that lacks hydrolytic activity but possesses transglycosylation activity for glycoengineering. Compared with in vivo glycoengineering technologies and the glycosyltransferase-enabled in vitro engineering method, the current approach is robust and features quantitative yield, homogeneous glycoforms of produced antibodies and ADCs, compatibility with diverse natural and non-natural glycan structures, convenient exploitation of native IgG as the starting material, and a well-defined conjugation site for antibody modifications. Potential applications of this method cover a broad scope of antibody-related research, including the development of novel glycoengineered therapeutic antibodies with enhanced efficacy, site-specific antibody-drug conjugation, and site-specific modification of antibodies for fluorescent labeling, PEGylation, protein cross-linking, immunoliposome formation, and so on, without loss of antigen-binding affinity. It takes 5-8 d to prepare the natural or modified N-glycan substrates, 3-4 d to engineer the IgG N-glycosylation, and 2-5 d to synthesize the small-molecule toxins and prepare the gsADCs.

Figure 1

General scheme of chemoenzymatic synthesis of glycoengineered IgGs and gsADCs. EndoS, endo-β-N-acetylglucosaminidase from Streptococcus pyogenes; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; KVANKT, Lys-Val-Ala-Asn-Lys-Thr; Man, mannose; NeuAc, N-acetylneuraminic acid.

Figure 2

Flowchart of the preparation procedure for glycoengineered IgG and gsADCs.

Figure 3

Synthesis of DBCO-tagged small molecules (Box 3).

Figure 4

Semisynthesis of N-glycan oxazolines (Steps 1–44). (i) 15 mM NaIO₄, PB, pH 7.0; (ii) N₃(CH₂)₂ONH₃⁺Cl⁻ (21a), PB, pH 7.0; (iii) N₃(CH₂)₃NH₂, NaCNBH₃, PB pH 6.0, MeOH; (iv) Endo-M, PB, pH 6.2–6.5; (v) DMC, Et₃N; (vi) neuraminidase, PB, pH 5.0; (vii) β-1,4-galactosidase, PB, pH 6.5; (viii) β-N-acetylglucosaminidase, PB, pH 6.5.

Figure 5

One-pot chemoenzymatic synthesis of glycoengineered Herceptin 5a–e with in situ oxazolines derived from SGP analogs 4a–e (Steps 74–85).

Figure 6

Synthesis of dual-payload ADC (28) (Steps 96–108). (i) DM1-SMCC 10, PB, pH 7.5; (ii) 6c, 50 mM PB, pH 7.5.

Figure 7

LC–MS profiles (deconvolution data) from IgG deglycosylation monitoring. (a) Native rituximab. (b) Rituximab after treatment with WT for 5 min. (c) Results after defucosylation of Fucα1,6GlcNAc-rituximab with AlfC for 4 h. the MS peak at 144,894.20 was designated as 2F (two fucoses); the MS peak at 144,749.13 was designated as 1F (one fucose); the MS peak at 144,602.67 was designated as 0F (no fucose). (d) Results after defucosylation of Fucα1,6GlcNAc-rituximab with AlfC for 16 h.

Figure 8

SDS-PAGE and LC–MS characterization of glycoengineered Herceptin (5a–e) bearing non-natural N-glycans26. (a) SDS-PAGE analysis of 5a–e. Lane 0: marker, lane 1: commercial Herceptin, lane 2: Herceptin-Fucα1,6GlcNAc (2a), lanes 3–7, 5a–e; (b) LC–MS profiles of 5a. The multiple charged m/z data are labeled with charge numbers. The deconvolution mass spectrum is shown in the embedded box. (c) SDS-PAGE analysis of PNGase-F digestion of 5a. Lane 8: 5a, Lane 9: 5a after PNGase-F digestion; (d) MS profile of the released non-natural glycan (shown in glycan symbol) from 5a by PNGase-F digestion. The ion flow chromatography of the PNGase-F digested sample is shown in the embedded box, and the peak for the glycan is marked with an arrow.

Figure 9

SDS-PAGE and LC–MS characterization of gsADC (7) (ref. 26). (a) SDS-PAGE analysis of 7b and 7c. Lane 0: marker, lane 1: commercial Herceptin, lane 2: Herceptin-Fucα1,6GlcNAc (2a), lane 3: glycoengineered Herceptin (5a), lanes 4 and 5: gsADC (7b and 7c). (b) LC–MS profiles for 7b. The multiple charged m/z data are labeled with charge numbers. The deconvolution mass spectrum is shown in the embedded box. (c) SDS-PAGE analysis of PNGase-F digestion of 7c. Lane 0: marker, lane 1: commercial Herceptin, lane 2: 7c, lane 3: 7c after PNGase-F digestion. (d) MS profile of the glycan–drug complex (shown with glycan symbol along with MMAE structure) released from 7c by PNGase-F digestion.