Investigators Report Series of Substates that Reprogram IgG to 1-Step Synthesis of gsADCs

In an article published in Acta Pharmaceutica Sinica B, the authors, discussed a series of novel disaccharide-based substrates, which reprogram the IgG glycoengineering to 1-step synthesis of gsADCs, catalyzed by an endo-N-acetylglucosaminidase (ENGase) of Endo-S2.

“These data demonstrated that the modifications on 6-position of Gal is excellently tolerated by Endo-S2 catalysis. After the optimization of reaction conditions, encouragingly, LacNAc-ox 2c could be quantitatively transferred onto the N-glycosylation site of both the deglycosylated and native trastuzumab in 1 step,” investigators said in the study.²

Investigators reported a series of novel disaccharide-based substrates, which reprogram the IgG glycoengineering to 1-step synthesis of gsADCsm catalyzed by an endo-N-acetylglucosaminidase (ENGase) of Endo-S2.¹

IgG glycoengineering via ENGases usually has 2 steps, which include deglycosylation by wild-type WT ENGases and transglycosylation by mutated ENGases.¹

In the current method, the investigators found that disaccharide LacNAc oxazoline can be efficiently assembled onto IgG by WT Endo-S2 without hydrolysis of the product, which enables the 1-step glycoengineering directly from native antibodies.¹

“Efficient 1-step IgG glycoengineering and glycosite-specific ADCs synthesis were achieved. The LacNAc-based substrates and WT Endo-S2 demonstrated a reprogrammed procedure for IgG glycoengineering, which avoided the multi-enzymatic treatment and payload conjugation in previous approaches,” investigators wrote in the study.²

They added that gsADCs do not rely on the bioorthogonal reactions for payload assembly, which could reduce the risk of triazole-induced immunogenicity.²

They concluded that this method is comparable to other therapeutic antibodies, which include biospecific antibodies, Fc-fused nanobodies, and rituximab.²

Further studies on substrate specificity showed that the approach has excellent tolerance on various modification of 6-Gal motif of LacNAc, investigators said.

Within 1 hour, 1-step synthesis of gsADC was achieved using the LacNAc-toxin substrates including structures free of bioorthogonal groups.¹

gsADCs demonstrated good homogeneity, buffer stability, and both in vitro and in vivo anti-tumor activity, the investigators said.¹

There are a total of 12 ADCs that have been approved by the FDA and are synthesized majorly by random conjugation of the payloads, according to the study.²

However, in the past decade, site-specific conjugation has been a feasible strategy to improve ADCs, such as through the THIOMAB technology and unnatural-amino-acids incorporation, including chemoenzymatic litigations with sortase A or transglutaminase.²

“Multiple steps, multiple enzymes, functionalized sugars, modified drugs, and biorthogonal reactions are usually indispensable in IgG glycoremodeling and payload conjugation, which limited the application of gsADCs. A more robust synthetic process for gsADCs bearing diversified linkages is required for gain of functions,” investigators said in the study.²

Reference

1. One-step synthesis of site-specific antibody–drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates. EurekAlert. News release. June 2, 2022. Accessed June 3, 2022. https://www.eurekalert.org/news-releases/954630

2. Shi W, Li W, Zhang J, Li T, et al. One-step synthesis of site-specific antibody–drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates. Acta Pharmaceutica Sinica B. 2022;2417-2428. doi: 10.1016/j.apsb.2021.12.013