Antibody-drug conjugate (ADC) combines the tumor targeting specificity of monoclonal antibodies and the potent cell killing activity of cytotoxic drugs (warhead/payload), through a carefully designed chemical linker (linker). The two are coupled together to achieve precise targeting while exhibiting powerful anti-tumor activity to improve payload’s treatment window. Based on the clinical success of ADCs, researchers’ interest in designing new styles of ADCs has surged. In addition to ISAC, IM-ADC, PROTAC, LYTAC, etc., the construction of dual-loaded ADCs is also making continuous progress.
Dual-loaded ADCs can flexibly adjust their drug-antibody ratio (DAR) through different construction methods. The physical and chemical properties, efficacy and toxicity characteristics of ADCs can be fine-tuned according to the type of disease and treatment purpose, making full use of the advantages of dual-drug delivery of ADCs and improve ADC activity. At the same time, from the perspective of atomic economics, the molar dosage of the required reagents can be reduced to construct a highly homogeneous dual-loaded ADC drug, which can improve the efficacy while reducing production costs. Currently, the methods for constructing dual-loaded ADCs are mainly focused on the second-generation coupling technologies : engineered reactive cysteine residues, interchain disulfide bond remodeling technology, unnatural amino acid-mediated click chemistry, enzyme-assisted ligation, glycoform remodeling and glycoconjugation.
Engineered Reactive Cysteine Residue & Disulfide Re-Bridging
Due to the special thiol group (-SH) of the side chain group of cysteine (Cys), it can be coupled with payloads containing maleimide group (-Mal) to achieve site-selective modification of antibodies. The Michael addition reaction between the two is highly selective, fast, and the conditions are relatively mild. Currently, methods for constructing ADCs using Cys mainly include engineered reactive cysteine residue and antibody interchain disulfide bond re-bridging technology. The introduction of engineered reactive Cys has long been seen in Genentech’s ThioMab™ technology, which constructs ADC drugs with a DAR value of 2 through a three-step reduction-oxidation-coupling reaction (actual DAR value 1.7~1.9). Currently, many companies are optimizing this method based on it, such as Zymeworks, Byondis, etc. Disulfide bond reshaping technology mainly uses TCEP/DTT to reduce the interchain disulfide bonds of IgG, exposing Cys-SH that can be used for coupling reactions.
In order to make full use of the limited binding sites, Peter D. Senter’s research group at Seattle Genetics used a dual cysteine multiplexing carrier to introduce two protected cysteine groups [Cys (SiPr) + Cys (Acm)], combining different deprotection reactions to construct dual-loaded ADCs. At the same time, the vector also contains a PEG24 fragment, which can achieve high drug loading (DAR: 8+8) without hydrophobicity-induced ADC aggregation. cAC10-(2+3) constructed by this method showed good anti-tumor activity in a mouse xenograft model (DEL-BVR cell line) (Fig. 1).
Unnatural Amino Acids (UAA)
In addition to modifying Cys, site-specific conjugation of antibodies can also be achieved by introducing unnatural amino acids (UAA) through gene codon amplification technology. UAA is introduced using different orthogonal pairs [orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA], and then coupled with the target molecule to construct an ADC. This coupling method is controllable and quantitative, and can generate ADCs with uniform DAR values, high efficiency, stability and safety.
Abhishek Chatterjee’s research group at Boston College used the Escherichia coli tryptophan pair (EcTrp) as a TGA codon inhibitor, combined with other existing orthogonal pairs, to develop three new pathways for the introduction of double UAA. This method can be used to specifically introduce two different UAAs into antibodies with excellent efficiency and subsequently conjugate them with two different cytotoxic payloads. The EcTrp+EcLeu (TAG) system was used to introduce 5-HTP and LCA into the 121 and 198 positions of the Trastuzumab heavy chain, respectively, and then two different cytotoxic drugs (Diazo-MMAF+DBCO-PNU-159682) were coupled to the antibody by one-pot method (chemoselective rapid azo-coupling reaction and strain-promoted azide-alkyne cycloaddition). Preliminary cytotoxicity studies of the constructed ADC on NCI-N87 (high expression of HER2 receptor) and NCI-H520 (low expression of HER2 receptor) cell lines showed that the cytotoxicity of ADC to NCI-N87 was significantly higher. ADC labeled with PNU showed significantly higher toxicity compared with MMAF.
Glycan Remodeling and Glycoconjugation & Enzyme-assisted Ligation
Glycosylation is one of the most important post-translational modifications of antibodies, and site-selective modification using glycosylation sites is also a common method for constructing ADCs.
Professor Huang Wei’s research group at Shanghai Institute of Materia Medica developed two strategies to construct site-specific ADCs. One is a chemical enzymatic method to synthesize glycosyl-specific ADC through one-step glycoengineering catalyzed by endo-S2. The other one is to synthesize the K248 site-specific ADC in a traceless manner via ligand-directed chemistry of thioester-based acyl transfer reagents. Currently, the research group uses the above technical advantages to assemble homogeneous dual-loaded ADC at the glycosylation site (N297) and K248 site through three different routes (Fig. 3A) First introduce MMAE/MMAF at the glycosylation site through endo-S2 catalysis, and then couple another payload at K248 of the antibody in the presence of the FcBP linker-drug complex; B) Couple the payload first at K248 and then at the glycosylation site. However, the experimental results show that the payload at K248 affects the endo-S2-mediated transglycosylation activity to some extent and affects the coupling; C) Simultaneously assemble two different payloads at the glycosylation and K248 sites in a one-pot approach by simply adding all materials to the reaction mixture. Under this condition, the hydrolysis and transglycosylation activities of endo-S2 were completely inhibited, and the coupling efficiency of the payload at K248 was also affected. Finally, route A was chosen to prepare dual-loaded ADC, and the resulting ADC had excellent performance in terms of stability and anti-tumor activity.
In summary, the construction of dual-loaded ADCs and their analogues: one is to use two reaction sites to construct step by step; the other is to use a single reaction site to introduce branch linkers, and then assemble cytotoxic drugs through click chemistry. Although the dual-loaded ADC construction method is currently relatively mature, there is still a long way to go in terms of pharmacokinetics.
References
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