Antibody-drug conjugates (ADCs) stand out for their remarkable capacity to precisely ferry small molecule chemotherapeutic agents straight to malignant cells, evading any unintended repercussions in the systemic circulation. The US FDA has greenlit a total of 15 ADCs (refer to Table 1), with numerous others progressing through clinical trials. Despite the proven efficacy of ADC in tackling both solid and hematologic malignancies, the formidable adversaries of drug resistance and tumor heterogeneity loom large as primary culprits behind clinical setbacks. The intricate tapestry of tumor heterogeneity serves as the breeding ground for relapse, metastasis, and the insidious development of resistance against ADC and other therapeutic modalities. Diverse tumors, each harboring distinct sensitivities to drugs, fuel aggressive tumor proliferation, escalating recurrence rates, and diminishing survival prospects. To confront these formidable hurdles, most chemotherapy protocols embrace the synergy of multiple drugs. The simultaneous delivery of small molecules emerges as the linchpin in circumventing drug resistance, fostering either additive or synergistic effects, and elevating therapeutic outcomes. The advent of dual-toxin ADC heralds a promising amalgamation strategy.

Table 1. FDA approved antibody-drug conjugates (ADCs) by 2024.

How to Design Dual Payload ADC?

Install the Two Toxins on the Same Linker

In 2017, Levengood et al. disclosed the first dual-toxin ADC containing an auristatin derivative (MMAE), which is a short peptide linker containing orthogonally protected cysteine amino acids (Fig. 1). The interchain disulfides of native antibodies are first reduced with TCEP and then the dual Cys linker MMAF conjugates are coupled by adding free thiols to the linker via Michael. Next, the single-drug ADC was treated with aqueous mercury acetate to deprotect the acetamide methyl group on the second linker cysteine, and then purified with tetraaldehyde MP resin to remove any remaining mercury. Finally, the desired dual-drug ADC was obtained by reacting with the maleimide-MMAE drug structure, with a total drug-to-antibody ratio (DAR) of 16 and a drug ratio of 1:1.

Fig. 1. Two toxins attached to the same linker (Angew Chem Int Ed Engl. 2017, 56(3): 733-737).

Levengood et al. selected two tubulin-targeting auristatin derivatives, MMAE and MMAF, because their different physicochemical properties allow complementary activities in tumor cells. MMAE is an effective chemotherapy drug that has been validated in 5 approved ADCs. Of note, due to its cell permeability, resulting in bystander effects, neighboring cell types may not express high levels of the target antigen. However, it is a substrate for drug exporters and is often upregulated in drug-resistant tumor cells. In contrast, MMAF (used in current ADCs) has minimal cell permeability but is known to be active against multidrug-resistant cell lines. In conclusion, MMAE should be able to combat differential antigen expression in heterogeneous tumors, while MMAF remains effective in drug-resistant cells.

In 2018, Kumar et al. reported a second dual-toxin ADC. It contains MMAE and pyrrolebenzodiazepine (PBD) dimers, two small molecule drugs that target different cellular targets. They initially synthesized heterotrifunctional linkers, which allowed conjugation of two drugs to the same site on the antibody. Specifically, the linker contains a self-stabilizing N-arylmaleimide for site-specific binding to engineered thiols on the antibody.

Fig. 2. Heterotrifunctional linker for dual warheads ADC preparation (Bioorg Med Chem Lett. 2018, 28(23-24): 3617-3621).

For drug conjugation, the first drug (MMAE) was bound to the linker via an overnight reaction via oxime linkage and purified by CHT chromatography. Finally, PBD-SG3557 was installed via CuAAC reaction and purified by CHT chromatography. Overall, this strategy requires three synthesis steps for the antibody, each followed by purification. The MMAE/PBD dual-drug conjugate was evaluated in HER2-expressing MDA-MB-453 cells compared with single-drug controls. The dual-drug ADC and PBD single-drug ADC controls are equivalent, indicating that the higher potency of PBD drugs dominates the effect of ADC.

A third dual-drug ADC was constructed by Yamazazaki et al. in 2021 using an alternative heterotrifunctional linker. Specifically, a branched ADC linker constructed from a central lysine residue containing one or two azides was used to promote the azide dibenzocyclooctene (DBCO) cycloaddition, an orthogonal click methyltetrazine for trans-cyclooctene (TCO) cycloaddition.

In the Yamazazaki study, MMAE and MMAF were chosen to evaluate the impact of bystander effects and complementary mechanisms of drug resistance. In order to synthesize the dual-drug ADC, it is first installed on the antibody through MTGase-mediated conjugation to the glutamine side chain at the Q295 position of the N297A monoclonal antibody. Next, sequential reactions with TCO-MMAF and DBCO-MMAE produced the dual-drug ADC. Double-drug ADCs containing DAR MMAE/F 2+2, 4+2, and 2+4 and single-drug ADCs with DAR 2, 4, or 6 were synthesized.

Fig. 3. Molecular design and conjugation strategies for generating dual-drug ADCs (Nat Commun. 2021, 12: 3528).

The efficacy of the MMAE/F dual-agent ADC was evaluated in an in vitro established model of heterogeneous HER2 expression and drug resistance. As expected, DAR 2 MMAE was less potent than the DAR 2 MMAF single-agent conjugate in J1MT-1 cells, which are known to be resistant to hydrophobic drugs (half-inhibitory concentrations 1.023 and 0.213 nM, respectively). Notably, the DAR 6 dual-drug ADC (half inhibitory concentration 0.26 or 0.240 nM) had the highest efficacy in this cell line compared with the DAR 6 MMAE single-agent ADC (half inhibitory concentration 0.060 nM).

Different Antibody Sites Conjugate Different Toxins

In 2019, Nilchan et al. demonstrated this strategy through the sequential conjugation of MMAF and PNU-159682 on engineered selenocysteine and cysteine residues. PNU-159682 is a DNA damaging agent due to its high efficacy and activity against drug-resistant and non-dividing cancer cells. The selenocysteine residue-Kabat was first reduced under mild DTT conditions before reaction with PNU-159682 containing iodoacetamide. Next, the interchain and engineered cysteine residues on A114C were reduced with TCEP, followed by reoxidation of the interchain disulfide with DHAA. Finally, the free engineered thiol reacted with the MSODA functional group on MMAF to obtain a MMAF/PNU dual-drug ADC with a DARPNU of 1.9 and a dose of 1.5.

Fig. 4. Dual biotin/fluorescein conjugation (Antib Ther. 2019, 2(4): 71-78).

In order to further clarify the difference between dual-drug and single-drug ADC, the researchers analyzed the changes of cell cycle by flow cytometry to explore the mechanism of cell death. MMAF interferes with cell cycle progression, resulting in G2/M phase arrest. Analysis of MMAF monotherapy ADC showed an increase in the number of G2/M and G1 populations. In contrast, PNU single-agent ADC resulted in a sharp increase in S phase. The dual-drug ADC increased slightly in G2/M and G1 populations, but increased significantly in S phase. These data indicate that although PNU drives the toxicity of the dual-agent ADC, the mechanism effect of adding MMAF can be detected. These results further underscore the importance of matching drugs for complementary benefits to success.

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