(M1430-01-01) Engineering Novel Antibody-Drug Conjugates with Dual Payloads to Improve the Incidence of Drug Resistance Associated with the Use of Antibody-Drug Conjugates in Oncology

Purpose: Antibody-drug conjugates (ADCs) are a promising group of therapeutics that allow delivery of drug payloads to specific cells expressing a target antigen, while sparing healthy cells. ADCs are also beneficial for delivering small molecule drugs that cannot be administered systemically due to toxic side effects or a narrow therapeutic window. Despite an increase in the number of ADCs being developed, these therapeutics still require improvement, as the success of clinically approved versions continues to suffer from the development of resistance and off-target side effects caused by nonspecific release of the payload. Most cytotoxic payloads used in clinically approved ADCs for oncology are substrates for drug-efflux pumps, and as a result, many patients develop drug resistance and experience disease relapse. To address this issue, we have developed ADCs bearing two different payloads conjugated to separate sites on the protein. Dual-drug ADCs permit delivery of payloads with different cellular targets, payloads with varying degrees of tumor bystander effect, and combinations of payloads where one is a substrate for drug efflux pumps and the other is not. This will be especially useful in tumors with heterogeneous expression of transport proteins. Ultimately, development of ADCs bearing novel combinations of two payloads with complementary mechanisms of action is necessary to overcome the issues of resistance observed with single-drug ADCs.

Methods: Selected cytotoxic drug payloads were conjugated to peptide linkers bearing a maleimide functional group for attachment to native cysteine residues or cyclopentadiene containing non-canonical amino acids in the antibody. For these studies, the non-canonical amino acid spiro[2.4]hepta-4,6-diene (SCpHK) was selected and supplemented into cell culture media during antibody expression. Expression of an antibody with SCpHK also required transfecting human embryonic kidney cells with the plasmid vector encoding the heavy and light chain sequences of the antibody, as well as plasmids for expression of an amber suppressor tRNA and associated tRNA synthetase for incorporation of the non-canonical amino acid at desired conjugation sites. Antibody was purified from cell media using fast protein liquid chromatography (FPLC) and mass spectrometry was used to confirm correct molecular weight, glycosylation pattern, and disulfide bond formation. Synthesis of dual-drug ADCs occurred in a two-step process. The first payload was attached to the antibody via a Diels-Alder reaction between the maleimide present in the linker and the SCpHK amino acid (Figure 2). Following conjugation, the single drug ADC was purified by desalting column to remove unreacted drug-linker complex, followed by dialysis. The purified single drug ADC was then reduced using tris(2-carboxyethyl)phosphine (TCEP) to give free cysteine thiols for conjugation. The second payload was then conjugated to the antibody through a thiol-maleimide reaction following the scheme in figure 2. This complex was again purified using a desalting column and dialysis. The final drug-to-antibody ratio of all dual-drug ADCs was determined using mass spectrometry.

Results: The data presented is representative of mass spectra and cell viability assays of dual-drug ADCs. ADC27 (Figure 3) is a dual-drug ADC built on a trastuzumab antibody (Herceptin®) scaffold with payloads vinblastine (VBL) and geldanamycin (DMAG). Characterization of this dual-drug ADC by mass spectrometry revealed complete conversion of the Diels-Alder reaction to give a drug-to-antibody ratio (DAR) of 2 for the vinblastine (Figure 3A-C). Conjugation to native cysteine residues gave a DAR of 1.5 for DMAG, which was determined as the weighted average of the peak heights from the mass spectra from the heavy or light chain. Given that trastuzumab targets HER-2 overexpression, cell viability assays were performed in a cell line, MDA-MB-453, expressing lower HER-2 levels, to determine whether the dual-drug combination could also show improvement in HER-2 low cancers. When compared to the single-drug ADCs of vinblastine and geldanamycin, the dual-drug ADC had significantly greater potency when compared to either single-drug ADC alone (Figure 3C).

Conclusion: Development of drug resistance remains a significant obstacle preventing the long-term clinical success of antibody-drug conjugates (ADCs) for oncology. To circumvent this, dual-drug ADCs bearing complimentary mechanisms of action were designed. The two-step synthesis approach presented here provides a strategy for conjugating diverse cytotoxic payloads onto antibodies using well-established Diels-Alder and thiol-maleimide reaction chemistries. This approach enables the targeted delivery of a large variety of drug combinations to address several issues observed in current single-drug ADCs. Representative results illustrate that when compared to single-drug ADCs, dual-drug ADCs have a greater potency, thus suggesting a potential synergistic effect of dual-drug ADCs.

Acknowledgements: The work was partially completed by funding from the National Institutes of Health R21CA256460.


Figure 1. Graphical Abstract: Dual-drug antibody-drug conjugates for improving incidence of drug resistance and treatment efficacy in oncology.


Figure 2. Reaction scheme for producing dual-drug ADCs beginning with a Diels-Alder conjugation to non-canonical amino acid SCpHK, followed by reduction of disulfide bonds in the antibody to permit conjugation of the second drug through a thiol-maleimide reaction.


Figure 3. Characterization and cell viability evaluation of a dual-drug ADC built on a trastuzumab antibody scaffold bearing payloads vinblastine and geldanamycin. Cell studies were conducted in MDA-MB-453 cells, which were classified as a HER-2-mid expressing cell line.