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Antibody–bottlebrush prodrug conjugates for targeted cancer therapy

Nature Biotechnology (2025)Cite this article

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Abstract

Antibody–drug conjugates (ADCs) are effective targeted therapeutics but are limited in their ability to incorporate less-potent payloads, varied drug mechanisms of action, different drug release mechanisms and tunable drug-to-antibody ratios. Here we introduce a technology to overcome these limitations called ‘antibody–bottlebrush prodrug conjugates’ (ABCs). An ABC consists of an IgG1 monoclonal antibody covalently conjugated to the terminus of a compact bivalent bottlebrush prodrug that has payloads bound through cleavable linkers and polyethylene glycol branches. This design enables the synthesis of ABCs with tunable average drug-to-antibody ratios up to two orders of magnitude greater than those of traditional ADCs. We demonstrate the functional flexibility and manufacturing efficiency of this technology by synthesizing more than 10 different ABCs targeting either HER2 or MUC1 using drugs with potencies spanning several orders of magnitude as well as imaging agents for ABC visualization and photocatalysts for proximity-based labeling of the ABC interactome. ABCs display high target engagement, high cell uptake and improved efficacy in tumor models compared to conventional HER2-targeted ADCs, suggesting promise for clinical translation.

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Fig. 1: Construction and in vitro evaluation of ABCs.
Fig. 2: Pharmacokinetics and biodistribution of ABCs.
Fig. 3: Efficacy and safety of ABCs incorporating diverse payloads with different potencies in HER2+ BT-474 tumor-bearing mice.
Fig. 4: Efficacy of HER2-targeted ABCs with different payloads and commercial HER2-targeted ADCs T-DM1 and T-DXd in high and low HER2 tumors.
Fig. 5: Efficacy and safety of ABCs incorporating the PROTAC payload ARV771 in HER2+ BT-474 tumor-bearing mice.
Fig. 6: Biodistribution, efficacy and safety of MUC1-targeted ABCs in ovarian cancer (CAOV-3) tumor-bearing mice.

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Data availability

All data supporting the findings of this study are available within the article, in its supplementary information and on figshare (https://doi.org/10.6084/m9.figshare.29414048)90 and can be obtained from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the National Institutes of Health (2R01CA220468-06A1) and the Yosemite–American Cancer Society Award (YACS-24-1343208-01-YACS) for support. B.L. thanks the Ludwig Center of the Koch Institute for Integrative Cancer Research at MIT for a postdoctoral fellowship and the Koch Institute Frontier Research Program for support. P.M. would like to acknowledge support from the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP, grant no. 2141064). Z.H.B., P.A.R. and D.W.C.M. are thankful for the financial support provided by the National Institutes of Health, National Institute of General Medical Sciences (R35GM134897-04); the Princeton Catalysis Initiative; Princeton University; and kind gifts from Merck, Janssen, Bristol Myers Squibb, Genentech, Genmab and Pfizer. N.J. is supported by the US Department of Defense (DOD award W81XWH-22-1-1122). We are grateful for the use of open-access tools, including DIA-NN, Perseus and BioGRID.

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Bin Liu, Hung V.-T. Nguyen, Aiden X. Wang, Valerie Lensch, Yutong Dai, Piper L. MacNicol, Yuyan Wang, Wencong Wang, Sachin Bhagchandani, Peyton Shieh, Samantha L. Kristufek & Jeremiah A. Johnson

  2. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Bin Liu, Sachin Bhagchandani & Jeremiah A. Johnson

  3. Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA

    Bin Liu

  4. Window Therapeutics, Boston, MA, USA

    Hung V.-T. Nguyen, Yivan Jiang & Sanjana Hemdev

  5. Thayer School of Engineering, Dartmouth College, Hanover, NH, USA

    Hung V.-T. Nguyen

  6. Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, USA

    Zehao Sun

  7. Merck Center for Catalysis at Princeton University, Princeton, NJ, USA

    Zane H. Boyer, Philip A. Raftopoulos & David W. C. MacMillan

  8. Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA

    Nidhi Jyotsana, Michael Downes & Ronald M. Evans

  9. Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA, USA

    Magalie Boucher

  10. Broad Institute of MIT and Harvard, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jeremiah A. Johnson

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B.L. and J.A.J. conceived the project idea. B.L. designed and performed the polymerization and bioconjugation. B.L. and H.V.-T.N. synthesized and characterized the macromonomers. B.L. performed the in vitro experiments. B.L., H.V.-T.N. and Y.J. performed the in vivo experiments. A.X.W., V.L., Z.S., Y.D., P.L.M., Y.W., W.W., S.B., S.H., P.S., S.L.K. and M.B. assisted with the experiments. Z.H.B. and P.A.R. performed µMap optimization and proteomic analysis, under the guidance of D.W.C.M. N.J., M.D. and R.M.E. provided the site-specific thiol-mAb. J.A.J. supervised the research. B.L. and J.A.J. analyzed the data and wrote the manuscript, and all authors discussed and commented on it.

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Correspondence to Jeremiah A. Johnson.

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Competing interests

J.A.J., H.V.-T.N. and Y.J. are shareholders of Window Therapeutics. D.W.C.M. declares an ownership interest in Dexterity Pharma, which has commercialized materials used in this work. The remaining authors declare no competing interests.

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Nature Biotechnology thanks Charles Dumontet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 ABC synthesis using different stoichiometries of BP-Tz and Ab-TCO.

a. Non-reducing SDS-PAGE analysis of model IgG1-based ABCs as a function of synthesis stoichiometry. BAR = average brush–antibody ratio. b. Non-reducing SDS-PAGE analysis of Trastuzumab-based ABCs as a function of synthesis stoichiometry. c. Flow cytometry histograms showing Cy5.5-HER2 uptake into HER2 + BT-474 cells as a function of synthesis stoichiometry (that is, the constructs are present as a mixture of BAR values). The x-axis represents the Cy5.5 fluorescence intensity. Since the number of Cy5.5 dyes per ABC increases with BAR, histograms are normalized to the total Cy5.5 loading for each construct to enable comparison (normalized to 40 µg/mL Cy5.5-BP, 1 h). d. Flow cytometry histograms for the same constructs shown in panel c normalized by antibody dose (normalized to 10 µg/mL mAb, 1 h). The x-axis represents the Cy5.5 fluorescence intensity. e. and f. Flow cytometry histograms for BT-474 cell uptake of isolated Cy5.5-HER2 ABCs with different BAR values (25 µg/mL, 1 h) (that is, ABCs with each BAR were separated from the synthesis mixture). Results are presented as mean ± SEM (n = 3 biological replicates). Statistical analysis was done using a 2-tailed t-test. For these statistical tests, **** denotes P < 0.001.

Extended Data Fig. 2 HER2-targeted ABCs synthesized via site-specific cysteine conjugation.

a. Non-reducing SDS-PAGE analysis of site-specifically conjugated Cy5.5-HER ABCs. Two independent experiments were performed with similar results. b. Flow cytometry histograms showing enhanced BT474 cell uptake for site-specific Cy5.5-HER2 ABC compared to BPD alone. The x-axis represents the Cy5.5 fluorescence intensity. c. and d. Quantification of cell uptake based on mean fluorescence intensity from flow cytometry (n = 3 biological replicates). e. and f. Flow cytometry histograms (The x-axis represents the Cy5.5 fluorescence intensity) and quantification, respectively, for cell uptake studies comparing site-specific Cy5.5-HER2 to stochastically functionalized lysine-based Cy5.5-HER2 ABC with BAR of 1 (BT474 cells, 20 µg/mL, 60 min incubation). Both ABCs have the same 5% Cy5.5 concentration. ABCK is the lysine-conjugated Cy5.5-HER2 ABC prepared from commercial Trastuzumab. We note that this ABC was stored at 4 °C for ~1.5 years prior to this study, demonstrating excellent long-term storage stability. T-ABCK is a stochastic Lys conjugate prepared using the engineered Trastuzumab designed for cysteine conjugation; this construct is designed to rule out differences in cell uptake between Lys-conjugated commercial Trastuzumab and the engineered antibody. Finally, T-ABCC is the site-specific cysteine conjugate Cy5.5-HER2. Results are presented as mean ± SEM (n = 3 biological replicates). Statistical analysis was done using a 2-tailed t-test. For these statistical tests, NS denotes non-significant; **, P < 0.01.

Extended Data Fig. 3 HER2+BT474 cell uptake and toxicity experiments comparing C5.5-labeled constructs.

a. Cell uptake studies comparing Cy5.5-HER2 to non-targeted controls Cy5.5-IgG1 and Cy5.5-BP, each containing 1% Cy5.5 loading (40 µg/mL, 60 min incubation, BAR = 3 for ABCs). The x-axis represents the Cy5.5 fluorescence intensity. b. Cell uptake for similar constructs with 5% Cy5.5 loadings (40 µg/mL, 60 min incubation, BAR = 3 for ABCs). The x-axis represents the Cy5.5 fluorescence intensity. c. Cytotoxicity of PTX-HER2 ABC compared to non-targeted PTX-BP (BPD only), a mixture of PTX-BPD and Trastuzumab (aHER2), and Trastuzumab (aHER2) alone for 24 h incubation. Results are presented as mean ± SEM (n = 3 biological replicates). The ABC PTX-HER2 displays improved cytotoxicity compared to controls. d. Confocal fluorescence microscopy images showed greater cell engagement and uptake for Cy5.5-HER2 compared to non-targeted controls (50 μg/mL, 6 h incubation, 1% Cy5.5 labeled ABC). Two independent experiments were performed with similar results.

Extended Data Fig. 4 In vitro targeting abilities of ABCs across cell lines with different HER2 expression.

a) Flow cytometry histograms demonstrating that Cy5.5-HER2 ABC uptake is dependent on HER2 expression. The x-axes represents the Cy5.5 fluorescence intensity. Cell uptake was studied using cell lines with varied HER2 expression: MCF-10A (HER2–); SKOV-3 (HER2 medium); SKBR-3 (HER2 high). Cells were treated with Cy5.5-HER2 ABC or non-targeting Cy5.5-BP polymer (1% Cy5.5 labeling) under the conditions listed at the bottom of the figure (varied times and concentrations). (b-d) Cytotoxicity results for HER2-targeted ABCs comprising different payloads in cell lines with varied HER2 expression. Results are presented as mean ± SEM (n = 3 biological replicates). b. MTT assay results for PTX-HER2 compared to non-targeted PTX-BP in HER2 high, medium, and negative (from left to right), respectively, cell lines following 24 h and 72 h incubation. c. HER2 + SKBR3 cell viability results for ABCs with different payloads (from left to right: MMAE, SN-38, and DOX). d. HER2– MCF-10A cell viability results for ABCs with different payloads (from left to right: MMAE, SN-38, and DOX).

Extended Data Fig. 5 Imaging of cell uptake and payload release in BT-474 cells.

a. Confocal fluorescence images of BT-474 cells incubated with Cy5.5-HER2 (20 μg/mL) for different times. b. Confocal fluorescence images of BT-474 cells incubated with “theranostic” ABC SN38-Cy5.5-HER2 (50 μg/mL) for different times. White arrows point to cell nuclei where SN38 has localized following release from the ABC. In panels a and b, 4 h + 24 h and 4 h + 72 h mean that the cells were incubated with Cy5.5-HER2 or SN38-Cy5.5-HER2, respectively, for 4 h. Then, the cells were washed with PBS buffer three times and further incubated for an additional 24 h or 72 h before imaging. Two independent experiments were performed with similar results.

Extended Data Fig. 6 Proposed ABC cell uptake and drug release mechanism.

First, ABCs bind to the cell surface through antibody-antigen interactions (upper left). Then, bound ABCs enter the cells through receptor-mediated endocytosis. Inside the endosome or lysosome, covalently attached payloads are released and subsequently diffuse to regions of the cell (for example, the nucleus or cytosol) to perform their MoA. Graphic created with BioRender.com.

Extended Data Fig. 7 Time-dependent biodistribution (BD) studies.

a. Ex vivo images of organs from mice (n = 3) at different time points following administration of Cy5.5-HER2 and non-targeted controls Cy5.5-IgG1 and Cy5.5-BP. b. Quantification of time-dependent BD for targeted and non-targeted constructs as quantified by fluorescence imaging. The bottom bar graphs correspond to the tumor fluorescence signals for different treatment groups, showing substantially greater tumor accumulation for HER2-targeting Cy5.5-HER2. Results are presented as mean ± SEM (n = 3 biological replicates). Statistical analysis was done using a 2-tailed t-test. For these statistical tests, NS denotes non-significant; *, P < 0.05; **, P < 0.01.

Extended Data Fig. 8 In vitro and in vivo evaluation of the anticancer efficiency of ABCs with different payloads on BT-474 cells or xenograft tumor model.

a. Cell viability studies for ABCs with different payloads compared to their nontargeting BPs (top and middle row, from left to right: MMAE, SN-38, and DOX; top row: 2 days incubation; bottom row: 5 days incubation) and different ADCs (bottom row, from left to right: T-DM1 and T-DXd, 2 d or 5 d incubation). Each data point represents the mean of three independent replicates (n = 3 biological replicates). b. BT-474 tumor volumes at day 40 for mice given MMAE-based ABCs and non-targeted controls (n = 4 mice/group). c. Ex vivo images of the tumors at day 40 for mice given MMAE-based constructs. d. Enlarged Fig. 3d for tumor volume measurement with dose schedule illustrated via green arrows (n = 6 mice/group). e. SDS-PAGE analysis of ABCs with DOX as the payload. f. BT-474 tumor volumes at day 40 for mice given DOX-based ABCs and non-targeted controls (n = 6 mice/group). g. Ex vivo images of the tumors at day 40 for mice given DOX-based constructs. Results are presented as mean ± SEM. Statistical analysis was done using a 2-tailed t-test. For these statistical tests, NS denotes non-significant; *, P < 0.05; **, P < 0.01.

Extended Data Fig. 9 Images of mice organs and xenografts examined after ABC treatments in the BT-474 xenograft tumor model.

Tissue sections were formalin-fixed paraffin-embedded (FFPE), stained with hematoxylin and eosin (H&E), and evaluated by a board-certified veterinary pathologist. a. No sign of toxicity was observed in the heart, lung, liver, spleen, and kidney, supporting a good safety profile. b. Xenografts with different magnifications. Tumor cells were only visible in the PBS, MMAE-BP, and MMAE-IgG1 groups. Tumor-bearing mice were treated with MMAE-based constructs (5 mg/kg mAb; 1.7 mg/kg MMAE per dose). Mice were dosed once a week for a total of 4 doses as illustrated via green arrows in panel a of Fig. 3. Scale bar = 300 μm. For each representative histology image, tissue sections from 4 mice were analyzed independently, with 3 slices examined per mouse, yielding similar results across all samples.

Supplementary information

Supplementary Information

Supplementary Discussion and Supplementary Figs. 1–45; Synthesis and Bioconjugation; Spectral Data; Supplementary References

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Supplementary Data

Uncropped gels for Supplementary Figures

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Liu, B., Nguyen, H.VT., Jiang, Y. et al. Antibody–bottlebrush prodrug conjugates for targeted cancer therapy. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02772-z

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