A Versatile Chemo-Enzymatic Conjugation Approach Yields Homogeneous and Highly Potent Antibody-Drug Conjugates

Abstract

The therapeutic efficacy of antibodies can be successfully improved through targeted delivery of potent cytotoxic drugs in the form of antibody-drug conjugates. However, conventional conjugation strategies lead to heterogeneous conjugates with undefined stoichiometry and sites, even with considerable batch-to-batch variability. In this study, we have developed a chemo-enzymatic strategy by equipping the C-terminus of anti-CD20 ofatumumab with a click handle using Sortase A, followed by ligation of the payload based on a strain-promoted azide-alkyne cycloaddition to produce homogeneous conjugates. The resulting antibody-drug conjugates fully retained their antigen binding capability and proved to be internalized and trafficked to the lysosome, which released the payload with a favorable efficacy in vitro and in vivo. Thus, this reported method is a versatile tool with maximum flexibility for development of antibody-drug conjugates and protein modification.

TCR-mimic antibody-drug conjugates targeting intracellular tumor-specific mutant antigen KRAS G12V mutation

“…the IC 50 of 2A5-MMAE (112.8 nM) was nearly half of the 2E8-MMAE (214.6 nM), suggesting that antitumor activity of TCRm-ADCs can be enhanced through increasing their payload. Although TCRm-ADCs were still much weaker than other TAA-associated ADCs, that their IC 50 was 700-fold higher compared under the same DAR [20] , it can be well tolerated by negative cells (K562) even at a very high concentration of 400 nM ( Fig. 6 ).…”

“…The random conjugation via lysine amines would lead to the aggregates or binding affinity loss [18] . Site-specific conjugation methods such as sortase A-mediated conjugation, cysteine-maleimide conjugation, chemo-enzymatic-mediated conjugation have been tried in antibody conjugations [19] , [20] . Therefore, we chose the cysteine-maleimide conjugation to generate homogeneous TCRm-ADCs.…”

The Antitumor Activity of TCR-Mimic Antibody-Drug Conjugates (TCRm-ADCs) Targeting the Intracellular Wilms Tumor 1 (WT1) Oncoprotein

“…The mean tumor volume of the mice reached 700 mm 3 (3.5–7 fold of tumor volume compared to the regular group which reached 100–200 mm 3 ) [11,26] when we initiated administration. Anti-CD20 ADC (ofatumumab (OFA)-MMAE) was used as a negative control and was prepared at a DAR of 3.3 via a previous method [27]. As shown in Figure 4B, the mean tumor volume of PBS group and OFA-MMAE group (15 mg/kg) reached 3000 mm 3 rapidly, and there was no significant difference of mean tumor volume between two groups ( p = 0.6745).…”

Antibody-drug conjugates for the treatment of lymphoma: clinical advances and latest progress

“…Several site-specific conjugation approaches that utilize integrated non-natural amino acids, engineered cysteine residues [ 37 ] or enzymatic modifications, including transglutaminases [ 38 ], sortase A [ 39 ], glycosyltransferase [ 40 ] and formylglycine-generating enzyme [ 41 ], have been reported. The chemoenzymatic approach can harvest a high yield of ADCs with suitable DARs and attractive conjugation efficiency by achieving site-specific conjugation and generating precisely controlled modification sites [ 42 ].…”