Annabel Kitowski

Despite recent advances in targeted drug delivery, approved Antibody-Drug-Conjugates (ADCs) are still limited by the delivery of a restricted set of payloads with limited modes of action (MOA). Versatile linkers, applicable to functional groups prevalent across diverse pharmacophores are needed to expand this space. We present phosphoramidate-based self-immolative linker-units that facilitate stable attachment in serum and traceless drug release in the target cell of aliphatic and aromatic alcohols. Studies with camptothecins show that stability and release are tunable and that various intracellular trigger events can be exploited to ensure traceless drug delivery. Superior stability, in vivo efficacy, and pharmacokinetics (PK) compared to approved camptothecin ADCs are demonstrated. Moreover, we report targeted delivery of 10 different hydroxy-containing cytotoxins with different intracellular MOAs. In vivo studies with gemcitabine show excellent PK and efficacy, unlocking gemcitabine’s full potential and illustrating the ability of the phosphoramidate-based linker system to expand the payload space for ADCs.

Supplementary Figure 5 shows cross-reactivity of TUB-040 to cynomolgus and rat NaPi2b

TUB-040 is a highly homogeneous and hydrophilic antibody–drug conjugate (ADC) targeting sodium-dependent phosphate transport protein 2b (NaPi2b), a surface receptor overexpressed in ovarian cancer and non–small cell lung adenocarcinoma. Previous NaPi2b-directed therapies have shown target-mediated and expression-dependent clinical activity. However, none of the previous tubulin inhibitor-based ADCs have been able to leverage the full therapeutic potential of the target. TUB-040 was constructed with a drug-to-antibody ratio of 8 using the Tubutecan linker-payload technology based on ethynylphosphonamidates (P5 conjugation chemistry), a protease cleavage site, and exatecan, a potent topoisomerase 1 inhibitor. TUB-040 induces potent antigen-specific cytotoxicity against NaPi2b-expressing cancer cells and demonstrates strong bystander activity. It displays favorable pharmacokinetic behavior, showing dose proportionality and superimposable total antibody and intact ADC curves, as well as low free payload levels, reflecting the high stability of TUB-040 enabled by the P5 conjugation platform. This specific feature also ensures sustained delivery of exatecan to tumor sites, which translates into excellent in vivo efficacy and tolerability. In cell line– and patient-derived xenograft models, including those with low target expression, single-dose TUB-040 administration leads to prolonged tumor growth inhibition and significant rates of complete remission, with a minimally effective dose level of 1 mg/kg in the OVCAR-3 model. Repeated-dose toxicologic assessment in rats indicates that TUB-040 is well tolerated, with no evidence of lung toxicity or thrombocytopenia. Taken together, TUB-040 is designed to enable long-lasting, durable tumor responses and to optimize both efficacy and tolerability, supporting the advancement of TUB-040 into clinical trials.

Supplementary Methods 1 includes descriptions about chemicals, LC/MS methods, sample preparation for MS, DARav determination, TUB-040 conjugation and free exatecan quantification

Supplementary Figure 1 shows healthy tissue expression of NaPi2b

Supplementary Figure 3 shows high-affinity binding of NaPi2b mAb and TUB-040 ADC to HCC-78 cells

Supplementary Table 2 shows toxicokinetic parameters of TUB-040 in rats

Supplementary Figure 8 shows mode of action of TUB-040

Supplementary Figure 9 shows binding of TUB-040 to FcgRs

Supplementary Figure 4 shows no binding of NaPi2b mAb and TUB-040 ADC to NaPi2a and NaPi2c