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Homogeneous multi-payload antibody–drug conjugates

Nature Chemistry (2024)Cite this article

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Abstract

Many systemic cancer chemotherapies comprise a combination of drugs, yet all clinically used antibody–drug conjugates (ADCs) contain a single-drug payload. These combination regimens improve treatment outcomes by producing synergistic anticancer effects and slowing the development of drug-resistant cell populations. In an attempt to replicate these regimens and improve the efficacy of targeted therapy, the field of ADCs has moved towards developing techniques that allow for multiple unique payloads to be attached to a single antibody molecule with high homogeneity. However, the methods for generating such constructs—homogeneous multi-payload ADCs—are both numerous and complex owing to the plethora of reactive functional groups that make up the surface of an antibody. Here, by summarizing and comparing the methods of both single- and multi-payload ADC generation and their key preclinical and clinical results, we provide a timely overview of this relatively new area of research. The methods discussed range from branched linker installation to the incorporation of unnatural amino acids, with a generalized comparison tool of the most promising modification strategies also provided. Finally, the successes and challenges of this rapidly growing field are critically evaluated, and from this, future areas of research and development are proposed.

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Fig. 1: Schematic summarizing the therapeutic positioning and concept of multi-payload ADCs.
Fig. 2: Overview of bioconjugation methods used to generate homogeneous single-payload ADCs.
Fig. 3: Overview of the methods used to generate homogeneous multi-payload ADCs.
Fig. 4: Overview of the methods used to generate homogeneous multi-site multi-payload ADCs.

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References

  1. Colombo, R. & Rich, J. R. The therapeutic window of antibody drug conjugates: a dogma in need of revision. Cancer Cell 40, 1255–1263 (2022).

    Article  CAS  PubMed  Google Scholar 

  2. Strebhardt, K. & Ullrich, A. Paul Ehrlich’s magic bullet concept: 100 years of progress. Nat. Rev. Cancer 8, 473–480 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Bross, P. F. et al. Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin. Cancer Res. 7, 1490–1496 (2001).

    CAS  PubMed  Google Scholar 

  4. Rowe, J. M. & Lowenberg, B. Gemtuzumab ozogamicin in acute myeloid leukemia: a remarkable saga about an active drug. Blood 121, 4838–4841 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. ClinicalTrials.gov. Search terms ‘cancer’, ‘antibody drug conjugate’ with phase 1–3 trials of ‘not yet recruiting’, ‘recruiting’, ‘enrolling by invitation’ and ‘active, not recruiting’ status (US National Library of Medicine, accessed 13 March 2022).

  6. Levengood, M. R. et al. Orthogonal cysteine protection enables homogeneous multi-drug antibody-drug conjugates. Angew. Chem. Int. Ed. 56, 733–737 (2017).

    Article  CAS  Google Scholar 

  7. Tijink, B. M. et al. A phase I dose escalation study with anti-CD44v6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck or esophagus. Clin. Cancer Res. 12, 6064–6072 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Desnoyers, L. R. et al. Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index. Sci. Transl. Med. 5, 207ra144 (2013).

    Article  PubMed  Google Scholar 

  9. Razzaghdoust, A. et al. Data-driven discovery of molecular targets for antibody-drug conjugates in cancer treatment. Biomed. Res. Int 2021, 2670573 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Su, D. & Zhang, D. Linker design impacts antibody-drug conjugate pharmacokinetics and efficacy via modulating the stability and payload release efficiency. Front. Pharmacol. 12, 687926 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Collins, D. M., Bossenmaier, B., Kollmorgen, G. & Niederfellner, G. Acquired resistance to antibody-drug conjugates. Cancers 11, 394 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Abelman, R. O., Wu, B., Spring, L. M., Ellisen, L. W. & Bardia, A. Mechanisms of resistance to antibody-drug conjugates. Cancers 15, 1278 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Marei, H. E., Cenciarelli, C. & Hasan, A. Potential of antibody-drug conjugates (ADCs) for cancer therapy. Cancer Cell Int. 22, 255 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Dagogo-Jack, I. & Shaw, A. T. Tumour heterogeneity and resistance to cancer therapies. Nat. Rev. Clin. Oncol. 15, 81–94 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Mokhtari, R. B. et al. Combination therapy in combating cancer. Oncotarget 8, 38022 (2017).

    Article  PubMed Central  Google Scholar 

  16. Barok, M., Joensuu, H. & Isola, J. Trastuzumab emtansine: mechanisms of action and drug resistance. Breast Cancer Res. 16, 209 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Fuentes-Antrás, J., Genta, S., Vijenthira, A. & Siu, L. L. Antibody-drug conjugates: in search of partners of choice. Trends Cancer 9, 339–354 (2023).

    Article  PubMed  Google Scholar 

  18. Nicol’o, E. et al. Combining antibody-drug conjugates with immunotherapy in solid tumors: current landscape and future perspectives. Cancer Treat. Rev. 106, 102395 (2022).

    Article  Google Scholar 

  19. Wang, A.-J., Gao, Y., Shi, Y.-Y., Dai, M.-Y. & Cai, H.-B. A review of recent advances on single use of antibody-drug conjugates or combination with tumor immunology therapy for gynecologic cancer. Front. Pharmacol. 13, 1093666 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Martin, M. et al. Trastuzumab emtansine (T-DM1) plus docetaxel with or without pertuzumab in patients with HER2-positive locally advanced or metastatic breast cancer: results from a phase Ib/IIa study. Ann. Oncol. 27, 1249–1256 (2016).

    Article  CAS  PubMed  Google Scholar 

  21. Krop, I. E. et al. Phase 1b/2a study of trastuzumab emtansine (T-DM1), paclitaxel, and pertuzumab in HER2-positive metastatic breast cancer. Breast Cancer Res. 18, 34 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Nilchan, N. et al. Dual-mechanistic antibody-drug conjugate via site-specific selenocysteine/cysteine conjugation. Antib. Ther. 2, 71–78 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Tang, F. et al. One-pot N-glycosylation remodeling of IgG with non-natural sialylglycopeptides enables glycosite-specific and dual-payload antibody-drug conjugates. Org. Biomol. Chem. 14, 9501–9518 (2016).

    Article  CAS  PubMed  Google Scholar 

  24. Tang, Y. et al. Real-time analysis on drug-antibody ratio of antibody-drug conjugates for synthesis, process optimization and quality control. Sci. Rep. 7, 7763 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Le, L. N. et al. Profiling antibody drug conjugate positional isomers: a system-of-equations approach. Anal. Chem. 84, 7479–7486 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Matsuda, Y. et al. Chemical site-specific conjugation platform to improve the pharmacokinetics and therapeutic index of antibody-drug conjugates. Mol. Pharm. 18, 4058–4066 (2021).

    Article  CAS  PubMed  Google Scholar 

  27. Herrera, A. F. et al. Anti-CD79B antibody-drug conjugate DCDS0780A in patients with B-cell non-Hodgkin lymphoma: phase 1 dose-escalation study. Clin. Cancer Res. 28, 1294–1301 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Seki, T. et al. Biological evaluation of maytansinoid-based site-specific antibody-drug conjugate produced by fully chemical conjugation approach: Ajicap®. Front. Biosci. Landmark 27, 234 (2022).

    Article  CAS  Google Scholar 

  29. Junutula, J. R. et al. Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res. 16, 4769–4778 (2010).

    Article  CAS  PubMed  Google Scholar 

  30. Behrens, C. R. et al. Antibody-drug conjugates (ADCs) derived from interchain cysteine cross-linking demonstrate improved homogeneity and other pharmacological properties over conventional heterogeneous ADCs. Mol. Pharmaceutics 12, 3986–3998 (2015).

    Article  CAS  Google Scholar 

  31. Bryant, P. et al. In vitro and in vivo evaluation of cysteine rebridged trastuzumab-MMAE antibody drug conjugates with defined drug-to-antibody ratios. Mol. Pharmaceutics 12, 1872–1879 (2015).

    Article  CAS  Google Scholar 

  32. Yamazaki, C. M. et al. Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance. Nat. Commun. 12, 3528 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hoyt, E. A., Cal, P. M., Oliveira, B. L. & Bernardes, G. J. Contemporary approaches to site-selective protein modification. Nat. Rev. Chem. 3, 147–171 (2019).

    Article  CAS  Google Scholar 

  34. Boutureira, O. & Bernardes, G. J. Advances in chemical protein modification. Chem. Rev. 115, 2174–2195 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. Rawale, D. G., Thakur, K., Adusumalli, S. R. & Rai, V. Chemical methods for selective labeling of proteins. Eur. J. Org. Chem. 2019, 6749–6763 (2019).

    Article  CAS  Google Scholar 

  36. Agarwal, P. & Bertozzi, C. R. Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering and drug development. Bioconjug. Chem. 26, 176–192 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tumey, L. N. et al. Site selection: a case study in the identification of optimal cysteine engineered antibody drug conjugates. AAPS J. 19, 1123–1135 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. Shen, B.-Q. et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 30, 184–189 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Junutula, J. R. et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26, 925–932 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Sussman, D. et al. Engineered cysteine antibodies: an improved antibody-drug conjugate platform with a novel mechanism of drug-linker stability. Protein Eng. Des. Sel. 31, 47–54 (2018).

    Article  CAS  PubMed  Google Scholar 

  42. Kovtun, Y. et al. A CD123-targeting antibody-drug conjugate, IMGN632, designed to eradicate AML while sparing normal bone marrow cells. Blood Adv. 2, 848–858 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Pemmaraju, N. et al. Clinical profile of IMGN632, a novel CD123-targeting antibody-drug conjugate (ADC), in patients with relapsed/refractory (R/R) blastic plasmacytoid dendritic cell neoplasm (BPDCN). Blood 136, 11–13 (2020).

    Article  Google Scholar 

  44. Daver, N. et al. Safety and efficacy from a phase 1b/2 study of IMGN632 in combination with azacitidine and venetoclax for patients with CD123-positive acute myeloid leukemia. Blood 138, 372 (2021).

    Article  Google Scholar 

  45. Kaplon, H., Crescioli, S., Chenoweth, A., Visweswaraiah, J. & Reichert, J. M. Antibodies to watch in 2023. MAbs 15, 2153410 (2023).

    Article  PubMed  Google Scholar 

  46. Lyon, R. P. et al. Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. Nat. Biotechnol. 32, 1059–1062 (2014).

    Article  CAS  PubMed  Google Scholar 

  47. Young, T. S. & Schultz, P. G. Beyond the canonical 20 amino acids: expanding the genetic lexicon. J. Biol. Chem. 285, 11039–11044 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C. & Schultz, P. G. A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188 (1989).

    Article  CAS  PubMed  Google Scholar 

  49. Ambrx Biopharma Inc. news page. https://ir.ambrx.com/news/news-details/2022/ambrxbiopharma-inc.-announces-strategic-reprioritization-and-provides-corporateupdate/default.aspx (accessed 13 March 2023).

  50. Snyder, J. T. et al. Metabolism of an oxime-linked antibody drug conjugate, AGS62P1, and characterization of its identified metabolite. Mol. Pharm. 15, 2384–2390 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Skidmore, L. et al. ARX788, a site-specific anti-HER2 antibody-drug conjugate, demonstrates potent and selective activity in HER2-low and T-DM1-resistant breast and gastric cancers. Mol. Cancer Ther. 19, 1833–1843 (2020).

    Article  CAS  PubMed  Google Scholar 

  52. Sutro Biopharma: company overview. https://www.sutrobio.com/wp-content/uploads/2022/09/sutro-corporate-presentation-september-2022-final.pdf (accessed 13 March 2023).

  53. Hallam, T. J., Wold, E., Wahl, A. & Smider, V. V. Antibody conjugates with unnatural amino acids. Mol. Pharm. 12, 1848–1862 (2015).

    Article  CAS  PubMed  Google Scholar 

  54. Drake, P. M. et al. Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes. Bioconjug. Chem. 25, 1331–1341 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Dennler, P. et al. Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconjug. Chem. 25, 569–578 (2014).

    Article  CAS  PubMed  Google Scholar 

  56. Zheng, K., Bantog, C. & Bayer, R. The impact of glycosylation on monoclonal antibody conformation and stability. MAbs 3, 568–576 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Dickgiesser, S. et al. Site-specific conjugation of native antibodies using engineered microbial transglutaminases. Bioconjug. Chem. 31, 1070–1076 (2020).

    Article  CAS  PubMed  Google Scholar 

  58. Strop, P. et al. Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem. Biol. 20, 161–167 (2013).

    Article  CAS  PubMed  Google Scholar 

  59. Farias, S. E. et al. Mass spectrometric characterization of transglutaminase based site-specific antibody-drug conjugates. Bioconjug. Chem. 25, 240–250 (2014).

    Article  CAS  PubMed  Google Scholar 

  60. Dorywalska, M. et al. Effect of attachment site on stability of cleavable antibody drug conjugates. Bioconjug. Chem. 26, 650–659 (2015).

    Article  CAS  PubMed  Google Scholar 

  61. King, G. T. et al. A phase 1, dose-escalation study of PF-06664178, an anti-Trop-2/Aur0101 antibody-drug conjugate in patients with advanced or metastatic solid tumors. Invest. New Drugs 36, 836–847 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Strop, P. et al. RN927C, a site-specific Trop-2 antibody-drug conjugate (ADC) with enhanced stability, is highly efficacious in preclinical solid tumor models. Mol. Cancer Ther. 15, 2698–2708 (2016).

    Article  CAS  PubMed  Google Scholar 

  63. Beerli, R. R., Hell, T., Merkel, A. S. & Grawunder, U. Sortase enzyme-mediated generation of site-specifically conjugated antibody drug conjugates with high in vitro and in vivo potency. PLoS ONE 10, e0131177 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Tolcher, A. W. et al. NBE-002: a novel anthracycline-based antibody-drug conjugate (ADC) targeting ROR1 for the treatment of advanced solid tumors-a phase 1/2 clinical trial. J. Clin. Oncol. 39, TPS1108 (2021).

    Article  Google Scholar 

  65. Zhang, Y. et al. Simultaneous site-specific dual protein labeling using protein prenyltransferases. Bioconjug. Chem. 26, 2542–2553 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Lee, B. I. et al. Quantification of an antibody-conjugated drug in fat plasma by an affinity capture LC-MS/MS method for a novel prenyl transferase-mediated site-specific antibody-drug conjugate. Molecules 25, 1515 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Lee, J.-j et al. Enzymatic prenylation and oxime ligation for the synthesis of stable and homogeneous protein-drug conjugates for targeted therapy. Angew. Chem. 127, 12188–12192 (2015).

    Article  Google Scholar 

  68. Kim, Y. et al. Antibody-active agent conjugates and methods of use. US patent US20150105541A1 (2017).

  69. LegoChem Biosciences news report. http://www.businesskorea.co.kr/news/articleview.html?idxno=100127 (accessed 27 April 2023).

  70. von Witting, E., Hober, S. & Kanje, S. Affinity-based methods for site-specific conjugation of antibodies. Bioconjug. Chem. 32, 1515–1524 (2021).

    Article  Google Scholar 

  71. Matsuda, Y., Leung, M., Okuzumi, T. & Mendelsohn, B. A purification strategy utilizing hydrophobic interaction chromatography to obtain homogeneous species from a site-specific antibody drug conjugate produced by AJICAP™ first generation. Antibodies 9, 16 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Yamada, K. et al. AJICAP: affinity peptide mediated regiodivergent functionalization of native antibodies. Angew. Chem. 131, 5648–5653 (2019).

    Article  Google Scholar 

  73. Matsuda, Y., Yamada, K., Okuzumi, T. & Mendelsohn, B. A. Gram-scale antibody-drug conjugate synthesis by site-specific chemical conjugation: AJICAP first generation. Org. Process Res. Dev. 23, 2647–2654 (2019).

    Article  CAS  Google Scholar 

  74. Fujii, T. et al. AJICAP second generation: improved chemical site-specific conjugation technology for antibody-drug conjugate production. Bioconjug. Chem. 34, 728–738 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Van Geel, R. et al. Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates. Bioconjug. Chem. 26, 2233–2242 (2015).

    Article  PubMed  Google Scholar 

  76. Verkade, J. M. et al. A polar sulfamide spacer significantly enhances the manufacturability, stability and therapeutic index of antibody-drug conjugates. Antibodies 7, 12 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  77. Fessler, S. et al. XMT-1592, a site-specific Dolasynthen-based NaPi2b-targeted antibody-drug conjugate for the treatment of ovarian cancer and lung adenocarcinoma. Cancer Res. 80, 2894 (2020).

    Article  Google Scholar 

  78. Wijdeven, M. A. et al. Enzymatic glycan remodeling-metal free click (GlycoConnectT) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering. mAbs 14, 2078456 (2022).

    Article  Google Scholar 

  79. Dannheim, F. M. et al. All-in-one disulfide bridging enables the generation of antibody conjugates with modular cargo loading. Chem. Sci. 13, 8781–8790 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Badescu, G. et al. Bridging disulfides for stable and defined antibody drug conjugates. Bioconjug. Chem. 25, 1124–1136 (2014).

    Article  CAS  PubMed  Google Scholar 

  81. Schumacher, F. F. et al. Next generation maleimides enable the controlled assembly of antibody-drug conjugates via native disulfide bond bridging. Org. Biomol. Chem. 12, 7261–7269 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Robinson, E. et al. Pyridazinediones deliver potent, stable, targeted and efficacious antibody-drug conjugates (ADCs) with a controlled loading of 4 drugs per antibody. RSC Adv. 7, 9073–9077 (2017).

    Article  CAS  Google Scholar 

  83. Yang, M.-C. et al. Preclinical studies of OBI-999: a novel globo H-targeting antibody-drug conjugate. Mol. Cancer Ther. 20, 1121–1132 (2021).

    Article  CAS  PubMed  Google Scholar 

  84. Chakraborty, R., Yan, Y. & Royal, M. A phase 1, open-label, dose-escalation study of the safety and efficacy of anti-CD38 antibody drug conjugate (STI-6129) in patients with relapsed or refractory multiple myeloma. Blood 138, 4763 (2021).

    Article  Google Scholar 

  85. Li, L. et al. Preclinical development and characterization of STI-6129, an anti-CD38 antibody drug conjugate, as a new therapeutic agent for multiple myeloma. Cancer Res. 80, LB-227 (2020).

  86. Kumar, A. et al. Synthesis of a heterotrifunctional linker for the site-specific preparation of antibody-drug conjugates with two distinct warheads. Bioorg. Med. Chem. Lett. 28, 3617–3621 (2018).

    Article  CAS  PubMed  Google Scholar 

  87. Walker, J. A. et al. Substrate design enables heterobifunctional, dual ‘click’ antibody modification via microbial transglutaminase. Bioconjug. Chem. 30, 2452–2457 (2019).

    Article  CAS  PubMed  Google Scholar 

  88. Maruani, A. et al. A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy. Nat. Commun. 6, 6645 (2015).

    Article  CAS  PubMed  Google Scholar 

  89. Hanby, A. R. et al. Antibody dual-functionalisation enabled through a modular divinylpyrimidine disulfide rebridging strategy. Chem. Commun. 58, 9401–9404 (2022).

    Article  CAS  Google Scholar 

  90. King, H. D. et al. Monoclonal antibody conjugates of doxorubicin prepared with branched peptide linkers: inhibition of aggregation by methoxytriethyleneglycol chains. J. Med. Chem. 45, 4336–4343 (2002).

    Article  CAS  PubMed  Google Scholar 

  91. Hamblett, K. J. et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 10, 7063–7070 (2004).

    Article  CAS  PubMed  Google Scholar 

  92. Sun, X. et al. Effects of drug-antibody ratio on pharmacokinetics, biodistribution, efficacy, and tolerability of antibody-maytansinoid conjugates. Bioconjug. Chem. 28, 1371–1381 (2017).

    Article  CAS  PubMed  Google Scholar 

  93. Ausserwöger, H. et al. Non-specificity as the sticky problem in therapeutic antibody development. Nat. Rev. Chem. 6, 844–861 (2022).

    Article  PubMed  Google Scholar 

  94. Burke, P. J. et al. Optimization of a pegylated glucuronide-monomethylauristatin E linker for antibody-drug conjugates optimized glucuronide-monomethylauristatin E linker for ADCs. Mol. Cancer Ther. 16, 116–123 (2017).

    Article  CAS  PubMed  Google Scholar 

  95. Lyon, R. P. et al. Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index. Nat. Biotechnol. 33, 733–735 (2015).

    Article  CAS  PubMed  Google Scholar 

  96. Tedeschini, T. et al. Polyethylene glycol-based linkers as hydrophilicity reservoir for antibody-drug conjugates. J. Controlled Release 337, 431–447 (2021).

    Article  CAS  Google Scholar 

  97. Viricel, W. et al. Monodisperse polysarcosine-based highly-loaded antibody-drug conjugates. Chem. Sci. 10, 4048–4053 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Chen, B. et al. Design, synthesis and in vitro evaluation of multivalent drug linkers for high-drug-load antibody-drug conjugates. ChemMedChem 13, 790–794 (2018).

    Article  CAS  PubMed  Google Scholar 

  99. Yang, Q. & Lai, S. K. Anti-PEG immunity: emergence, characteristics and unaddressed questions. Wiley Interdiscip. Rev. Nanomed. 7, 655–677 (2015).

    Article  Google Scholar 

  100. Garay, R. P., El-Gewely, R., Armstrong, J. K., Garratty, G. & Richette, P. Antibodies against polyethylene glycol in healthy subjects and in patients treated with PEG-conjugated agents. Expert Opin. Drug Deliv. 9, 1319–1323 (2012).

    Article  CAS  PubMed  Google Scholar 

  101. Pabst, M. et al. Modulation of drug-linker design to enhance in vivo potency of homogeneous antibody-drug conjugates. J. Control. Release 253, 160–164 (2017).

    Article  CAS  PubMed  Google Scholar 

  102. Wang, X., Ishida, T. & Kiwada, H. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J. Control. Release 119, 236–244 (2007).

    Article  CAS  PubMed  Google Scholar 

  103. Dams, E. T. et al. Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes. J. Pharmacol. Exp. Ther. 292, 1071–1079 (2000).

    CAS  PubMed  Google Scholar 

  104. Judge, A., McClintock, K., Phelps, J. R. & MacLachlan, I. Hypersensitivity and loss of disease site targeting caused by antibody responses to PEGylated liposomes. Mol. Ther. 13, 328–337 (2006).

    Article  CAS  PubMed  Google Scholar 

  105. Sroda, K. et al. Repeated injections of PEG-PE liposomes generate anti-PEG antibodies. Cell. Mol. Biol. Lett. 10, 37–47 (2005).

    CAS  PubMed  Google Scholar 

  106. Lin, W. et al. Biocompatible long-circulating star carboxybetaine polymers. J. Mater. Chem. B 3, 440–448 (2015).

    Article  CAS  PubMed  Google Scholar 

  107. Xiao, H. et al. Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew. Chem. 125, 14330–14333 (2013).

    Article  Google Scholar 

  108. Osgood, A. O. et al. An efficient opal-suppressor tryptophanyl pair creates new routes for simultaneously incorporating up to three distinct noncanonical amino acids into proteins in mammalian cells. Angew. Chem. Int. Ed. 62, e202219269 (2023).

    Article  CAS  Google Scholar 

  109. Zhang, L. et al. A simple and efficient method to generate dual site-specific conjugation ADCs with cysteine residue and an unnatural amino acid. Bioconjug. Chem. 32, 1094–1104 (2021).

    Article  CAS  PubMed  Google Scholar 

  110. Byun, B. J. & Kang, Y. K. Conformational preferences and pKa value of selenocysteine residue. Biopolymers 95, 345–353 (2011).

    Article  CAS  PubMed  Google Scholar 

  111. Li, X. et al. Site-specific dual antibody conjugation via engineered cysteine and selenocysteine residues. Bioconjug. Chem. 26, 2243–2248 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Harmand, T. J. et al. One-pot dual labeling of IgG 1 and preparation of C-to-C fusion proteins through a combination of sortase A and butelase 1. Bioconjug. Chem. 29, 3245–3249 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Thornlow, D. N. et al. Dual site-specific antibody conjugates for sequential and orthogonal cargo release. Bioconjug. Chem. 30, 1702–1710 (2019).

    Article  CAS  PubMed  Google Scholar 

  114. Le Gall, C. M. et al. Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering. Bioconjug. Chem. 32, 301–310 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  115. Pfizer Inc. Antibodies and antibody fragments for site-specific conjugation. PCT patent US 2020/06764_A1 (2020).

  116. Spycher, P. R. et al. Dual, site-specific modification of antibodies by using solid-phase immobilized microbial transglutaminase. ChemBioChem 18, 1923–1927 (2017).

    Article  CAS  PubMed  Google Scholar 

  117. Tang, C. et al. One-pot assembly of dual-site-specific antibody-drug conjugates via glycan remodeling and affinity-directed traceless conjugation. Bioconjug. Chem. 34, 748–755 (2023).

    CAS  Google Scholar 

  118. Zhang, C. et al. π-clamp-mediated cysteine conjugation. Nat. Chem. 8, 120–128 (2016).

    Article  PubMed  Google Scholar 

  119. Zhang, C., Dai, P., Vinogradov, A. A., Gates, Z. P. & Pentelute, B. L. Site-selective cysteine-cyclooctyne conjugation. Angew. Chem. 130, 6569–6573 (2018).

    Article  Google Scholar 

  120. Murray, T. V. et al. An efficient system for bioconjugation based on a widely applicable engineered O-glycosylation tag. MAbs 13, 1992068 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  121. Palmer, A. C. & Sorger, P. K. Combination cancer therapy can confer benefit via patient-to patient variability without drug additivity or synergy. Cell 171, 1678–1691 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Xiong, M. et al. An innovative site-specific dual-payload antibody drug conjugate (dpADC) combining a novel Topo1 inhibitor and an immune agonist delivers a strong immunogenic cell death (ICD) and antitumor response in vitro and in vivo. Cancer Res. 83, 2640 (2023).

    Article  Google Scholar 

  123. Batist, G. et al. Safety, pharmacokinetics and efficacy of CPX-1 liposome injection in patients with advanced solid tumors. Clin. Cancer Res. 15, 692–700 (2009).

    Article  CAS  PubMed  Google Scholar 

  124. Wei, X. et al. Codelivery of a π-π stacked dual anticancer drug combination with nanocarriers for overcoming multidrug resistance and tumor metastasis. Adv. Funct. Mater. 26, 8266–8280 (2016).

    Article  CAS  Google Scholar 

  125. Wang, H. et al. Enhanced anti-tumor efficacy by co-delivery of doxorubicin and paclitaxel with amphiphilic methoxy PEG-PLGA copolymer nanoparticles. Biomater. 32, 8281–8290 (2011).

    Article  CAS  Google Scholar 

  126. Lancet, J. E. et al. Phase 2 trial of CPX-351, a fixed 5:1 molar ratio of cytarabine/daunorubicin, vs cytarabine/daunorubicin in older adults with untreated AML. Am. J. Hematol. 123, 3239–3246 (2014).

    CAS  Google Scholar 

  127. Matsuda, Y. & Mendelsohn, B. A. An overview of process development for antibody-drug conjugates produced by chemical conjugation technology. Expert Opin. Biol. Ther. 21, 963–975 (2021).

    Article  CAS  PubMed  Google Scholar 

  128. Matsuda, Y. Current approaches for the purification of antibody-drug conjugates. J. Sep. Sci. 45, 27–37 (2022).

    Article  CAS  PubMed  Google Scholar 

  129. Zhou, Q. et al. Site-specific antibody conjugation to engineered double cysteine residues. Pharmaceuticals 14, 672 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Botzanowski, T. et al. Insights from native mass spectrometry approaches for top- and middle-level characterization of site-specific antibody-drug conjugates. MAbs 9, 801–811 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Strop, P. et al. Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading. Nat. Biotechnol. 33, 694–696 (2015).

    Article  CAS  PubMed  Google Scholar 

  132. Stefan, N. et al. Highly potent, anthracycline-based antibody-drug conjugates generated by enzymatic, site-specific conjugation. Mol. Cancer Ther. 16, 879–892 (2017).

    Article  CAS  PubMed  Google Scholar 

  133. Pretto, F. & FitzGerald, R. E. In vivo safety testing of antibody drug conjugates. Regul. Toxicol. Pharmacol. 122, 104890 (2021).

    Article  CAS  PubMed  Google Scholar 

  134. Dingman, R. & Balu-Iyer, S. V. Immunogenicity of protein pharmaceuticals. J. Pharm. Sci. 108, 1637–1654 (2019).

    Article  CAS  PubMed  Google Scholar 

  135. Grünewald, J. et al. Immunochemical termination of self-tolerance. Proc. Natl Acad. Sci. USA 105, 11276–11280 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  136. Grünewald, J. et al. Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids. Proc. Natl Acad. Sci. USA 106, 4337–4342 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  137. Benkirane, N. et al. Antigenicity and immunogenicity of modified synthetic peptides containing D-amino acid residues. antibodies to a D-enantiomer do recognize the parent l-hexapeptide and reciprocally. J. Biol. Chem. 268, 26279–26285 (1993).

    Article  CAS  PubMed  Google Scholar 

  138. Buse, J. B. et al. Liraglutide treatment is associated with a low frequency and magnitude of antibody formation with no apparent impact on glycemic response or increased frequency of adverse events: results from the liraglutide effect and action in diabetes (lead) trials. J. Clin. Endocrinol. Metab. 96, 1695–1702 (2011).

    Article  CAS  PubMed  Google Scholar 

  139. Azam, A. et al. Introduction of non-natural amino acids into T-cell epitopes to mitigate peptide-specific T-cell responses. Front. Immunol. 12, 637963 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  140. Subedi, G. P. & Barb, A. W. The structural role of antibody N-glycosylation in receptor interactions. Structure 23, 1573–1583 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Ackerman, S. E. et al. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nat. Cancer 2, 18–33 (2021).

    Article  CAS  PubMed  Google Scholar 

  142. Uppal, H. et al. Potential mechanisms for thrombocytopenia development with trastuzumab emtansine (T-DM1). Clin. Cancer Res. 21, 123–133 (2015).

    Article  CAS  PubMed  Google Scholar 

  143. Mimura, Y. et al. The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: properties of a series of truncated glycoforms. Mol. Immunol. 37, 697–706 (2000).

    Article  CAS  PubMed  Google Scholar 

  144. Mimura, Y. et al. Role of oligosaccharide residues of IgG1-Fc in FcγRIIb binding. J. Biol. Chem. 276, 45539–45547 (2001).

    Article  CAS  PubMed  Google Scholar 

  145. Irvine, E. B. & Alter, G. Understanding the role of antibody glycosylation through the lens of severe viral and bacterial diseases. Glycobiology 30, 241–253 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  146. Bahou, C. et al. Disulfide modified IgG1: an investigation of biophysical profile and clinically relevant Fc interactions. Bioconjug. Chem. 30, 1048–1054 (2019).

    Article  CAS  PubMed  Google Scholar 

  147. Duvall, J. R. et al. An antibody-drug conjugate carrying a microtubule inhibitor and a DNA alkylator exerts both mechanisms of action on tumor cells. Cancer Res. 79, 232 (2019).

    Article  Google Scholar 

  148. Morais, M. et al. Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis. Org. Biomol. Chem. 15, 2947–2952 (2017).

    Article  CAS  PubMed  Google Scholar 

  149. Shi, B. et al. Antitumor activity of a 5T4 targeting antibody drug conjugate with a novel payload derived from MMAF via C-lock linker. Cancer Med. 8, 1793–1805 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  150. Yin, G. et al. RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates. Sci. Rep. 7, 3026 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  151. Sadiki, A. et al. Site-specific bioconjugation and convergent click chemistry enhances antibody-chromophore conjugate binding efficiency. Photochem. Photobiol. 96, 596–603 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Harris, L. et al. Serimabs: N-terminal serine modification enables modular, site-specific payload incorporation into antibody-drug conjugates (ADCs). Cancer Res. 75, 647 (2015).

    Article  Google Scholar 

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Acknowledgements

We thank AstraZeneca (grant 10045723, to T.J.) for funding.

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Authors and Affiliations

  1. Department of Chemistry, University of Cambridge, Cambridge, UK

    Toby Journeaux & Gonçalo J. L. Bernardes

  2. Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal

    Gonçalo J. L. Bernardes

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T.J. wrote the original manuscript. G.J.L.B. contributed to manuscript revisions and editing.

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Journeaux, T., Bernardes, G.J.L. Homogeneous multi-payload antibody–drug conjugates. Nat. Chem. (2024). https://doi.org/10.1038/s41557-024-01507-y

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