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

Glycosite-specific antibody‒drug conjugates (gsADCs), harnessing Asn297 N-glycan of IgG Fc as the conjugation site for drug payloads, usually require multi-step glycoengineering with 2 or more enzymes, which limits the substrate diversification and complicates the preparation process.

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