Abstract The genetic and phenotypic heterogeneity of human cancers is a primary driver of drug resistance, posing a major challenge to achieving durable therapeutic responses. To overcome this, Sutro has developed a dual-payload antibody-drug conjugate (dpADC) platform that enables precise co-delivery of two cytotoxic payloads via a single, homogeneous molecule. Sutro’s HER2-targeting dpADC demonstrates that simultaneous delivery of two cytotoxic payloads provides greater anti-tumor activity than single-payload ADCs and overcomes preclinical, in-vivo-derived treatment resistance. Combining Sutro Biopharma’s XpressCF+® cell-free expression system with site-specific conjugation technology, we engineered a HER2-targeting dpADC combining exatecan (topoisomerase I inhibitor; TOPO1i) and monomethyl auristatin E (MMAE) (microtubule inhibitor; MTi) payloads at an 8:4 ratio. The resulting dpADC exhibited favorable pharmacokinetics in vivo and minimal linker-payload loss over a 21-day study. In in vitro cell killing assays, the dpADC performed better than Enhertu and DAR8 exatecan ADC across multiple tumor cell lines. In in vivo efficacy studies, the dpADC exhibited greater anti-tumor activity than both single-payload ADC comparators across multiple xenograft models. To further evaluate the dpADC concept’s potential, we examined its utility in addressing resistance induced by ADC treatment. To mimic resistances to single payload ADCs observed in the clinic, we continuously dosed xenograft tumors with Enhertu on a weekly dosing schedule until the tumors progressed. These Enhertu-resistant tumors were then continuously dosed with a MTi ADC until de novo resistance developed. Dual-resistant tumors were then treated with a dpADC resulting in deep and durable anti-tumor responses. Even in tumors that were rendered resistant to both payloads, the dpADC treatment was able to achieve substantial tumor regression, demonstrating the potential for benefit in mono payload ADC refractory settings. Overall, these results indicate that Sutro’s HER2 dpADC can enhance anti-tumor activity beyond that of single-payload ADCs and can overcome prior treatment-induced resistance in a preclinical setting. Citation Format: Angela Matcham, Robert Yuan, Brian Vuillemenot, Rhoneil Pena, Young Park, Abigail Yu, Jeffrey Hanson, Cuong Tran, Xiaofan Li, Miao Wen, Daniel Calarese, Werner Rubas, Krishna Bajjuri, Guifen Xu, Alice Yam, Hanspeter Gerber, . The HER2-targeting dual-payload antibody-drug conjugate combining a topoisomerase I inhibitor and a microtubule inhibitor demonstrates superior efficacy and overcomes resistance to single-payload ADCs in xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1685.

Supplementary Fig. S1 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

PF-0259 did not induce platelet sequestration in spleen. Monkeys were dosed intravenously with vehicle or PF-0259 at 6 mg/m2/dose once every 3 weeks and were necropsied on Day 3 (at the time of platelet nadirs) or on Day 63 (at the end of the 3rd cycle). CD41 IHC for platelets was performed on spleen samples from vehicle control (A, C) and PF-0259-dosed (B, D) monkeys on Day 3 and Day 63. There was no evidence of increased CD41 immunostaining in PF-0259-dosed monkeys on Day 3 (B) or Day 63 (D) as compared with vehicle control monkeys (A and C, respectively), indicating lack of splenic sequestration of platelets at both time points. Noteworthy was a reduced CD41 immunostaining in PF-0259-dosed monkeys as compared with the control monkey on Day 3, indicating release of platelets from the spleen storage pool secondary to PF-0259-related acute thrombocytopenia. Scale bar = 60 µm.

In vitro cytotoxicity of PF-06804103 across a panel of cancer cell lines.

ADC retention times by hydrophobic interaction chromatography (HIC).

Supplementary Table S4 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

The commentary by Colombo and Rich recently published in Cancer Cell provides a timely and comprehensive review of the clinical maximum tolerated doses (MTDs) of antibody-drug conjugates (ADCs) and their corresponding small molecules/chemotherapies. The authors identified similarities between their MTDs and therefore question the historic assumptions made for ADCs, namely, that they increase the MTDs of their corresponding cytotoxic molecules. However, the authors did not address the superior anti-tumor responses of ADCs compared to their corresponding chemotherapies, as reported in clinical trials. In this point of view, we propose a revised model wherein the anti-tumor activities of ADCs and consequently their therapeutic indexes (TIs) are not solely associated with changes not only in their MTDs but also in their minimal effective doses (MEDs). In addition, when using an exposure-based TI calculation method, the superior anti-tumor activities of ADCs relative to their corresponding chemotherapy can readily be explained. We discussed the clinical and preclinical data in support of lower MEDs of ADCs and generated a revised graph illustrating the TI improvements of ADCs vs chemotherapy more accurately. We believe that our revised model can provide a blueprint for future improvements in protein engineering and chemical engineering of toxins to further advance ADC research and development.

In vivo efficacy of site-specific anti-HER2 vc0101 4 DAR ADCs in N87 xenograft tumor model.

Supplementary Fig. S2 from Anti-CD30 diabody-drug conjugates with potent antitumor activity

Immunohistochemical evaluation of HER2 expression in vivo cell line and patient-derived xenograft models.

Supplementary Table S3 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Supplementary Figure S1: N87 and N87-TM cells are similarly sensitive to unconjugated DM1-SMe; Supplementary Figure S2: Trastuzumab binding to N87 and N87-TM cells; Supplementary Figure S3: Generation and characterization of T-DM1-resistant HCC1954-TM and BT474-TM cells; Supplementary Figure S4: Relative resistance profiles of N87-TM to N87 cells to T-ADCs, unconjugated payloads and standard-of-care chemotherapeutics; Supplementary Figure S5: Distribution of T-ADCs in N87-TM tumors; Supplementary Figure S6: N87-TM cells are cross-resistant to a T-ADC conjugated to a cleavable linker with a cysteine-capped auristatin analog; Supplementary Figure S7: N87-TM cells internalize T-ADC into non-low pH intracellular compartments; Supplementary Figure S8: CAV1-GFP expression causes intracellular caveolae formation capable of internalizing T-ADCs in N87 cells; Supplementary Figure S9: CAV1 knockdown is not sufficient to re-sensitize N87-TM cells to T-DM1; Supplementary Figure S10: Non-cleavable and cleavable T-ADCs co-localize with CAV1 similarly in N87 and N87-TM cells; Supplementary Table S1. Cytotoxicity values of Trastuzumab and isotype-control ADCs in HT29 cells, a HER2-negative cell line

Supplementary Methods, Table S1 S2, and Figure Legends. Supplementary Table S1: Properties of cell lines made resistant to TM-conjugate. Supplementary Table S2: Relative resistance of selected free payloads and Cys-capped-released species in H69 (no ABCC1) and H69AR (high ABCC1 expressing) cell lines.

Supplementary Figures 1-4. Supplementary Figure 1. Analysis of EFNA4 copy number in breast and hepatocellular carcinoma. Supplementary Figure 2. Characterization of PF-06647263 conjugates. Supplementary Figure 3. Biomarkers of PF-06647263 activity in breast PDX tumors. Supplementary Figure 4. Characterization of EFNA4 affinity for EphA and EphB receptors.

PF-0259 did not induce platelet sequestration in kidney or lung. Monkeys were necropsied 48 hours after a single intravenous administration of vehicle or PF-0259 at 6 mg/m2. CD41 IHC for platelets was performed on kidney and lung samples from vehicle control (A and C, respectively) and PF-0259-dosed (B and D, respectively) monkeys. Contrary to what was observed in the liver, there was no increase in CD41 immunostaining and therefore no evidence of platelet sequestration in the kidney and lung vasculature following PF-0259 administration. Scale bar = 60 µm.

Legends of supplementary figures 1, 2 and 3

Supplementary Data, Materials and Methods

Conjugation of vc0101 does not alter anti-HER2 mAb binding.

Effects of a Conventional and a Site-Specific Conjugate Targeting HER2 on Indicators of Bone Marrow Toxicity and Serum ADC/Payload Exposures in Rats

PDF file-458KB, Supplementary Figure S1. Representation of A1mcMMAF and its proposed processing., Supplementary Figure S2. Chromatographic characterization of A1mcMMAF., Supplementary Figure S3. LC-MS analysis of A1mcMMAF ADC., Supplementary Figure S4. Anti-5T4 A1 antibody binding to 5T4 orthologs., Supplementary Figure S5. Anti-5T4 A1 antibody binding to cancer cell lines., Supplementary Figure S6. Cytotoxic activity of A1mcMMAF on TIC-enriched TUM622 spheroids., Supplementary Figure S7. In vivo pharmacology in MDAMB435/5T4., Supplementary Figure S8. No activity of A1mcMMAF in a 5T4-negative tumor. Supplementary Table S1. Humanized A1 BIAcore Affinity Constants for Human and Cynomolgus Monkey 5T4 at pH 7.4 Supplementary Table S2. Representative internalization in MDAMB435/5T4. Supplementary Table S3. Comparison of in vitro cytotoxicities of ADC vs. free payload (IC50 values expressed in nM auristatin).