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Binding and functional profiling of antibody mutants guides selection of optimal candidates as antibody drug conjugates


Autoři: John C. Zwaagstra aff001;  Traian Sulea aff001;  Jason Baardsnes aff001;  Stevo Radinovic aff001;  Yuneivy Cepero-Donates aff001;  Alma Robert aff001;  Maureen D. O’Connor-McCourt aff002;  Ilia A. Tikhomirov aff002;  Maria Luz. Jaramillo aff001
Působiště autorů: Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, Canada aff001;  Forbius, Montreal, Quebec, Canada aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0226593

Souhrn

An increasingly appreciated conundrum in the discovery of antibody drug conjugates (ADCs) is that an antibody that was selected primarily for strong binding to its cancer target may not serve as an optimal ADC. In this study, we performed mechanistic cell-based experiments to determine the correlation between antibody affinity, avidity, internalization and ADC efficacy. We used structure-guided design to assemble a panel of antibody mutants with predicted Her2 affinities ranging from higher to lower relative to the parent antibody, Herceptin. These antibodies were ranked for binding via SPR and via flow-cytometry on high-Her2 SKOV3 cells and low-Her2 MCF7 cells, the latter acting as a surrogate for low-Her2 normal cells. A subpanel of variants, representative of different Her2-binding affinities (2 strong, 2 moderate and 3 weak), were further screened via high-content imaging for internalization efficacies in high versus low-Her2 cells. Finally, these antibodies were evaluated in ADC cytotoxicity screening assays (using DM1 and MMAE secondary antibodies) and as antibody-drug conjugates (DM1 and PNU159682). Our results identified specific but weak Her2-binding variants as optimal candidates for developing DM1 and PNU ADCs since they exhibited high potencies (low to sub-nM) in high-Her2 SKOV3 cells and low toxicities in low-Her2 cells. The 2 strong-affinity variants were highly potent in SKOV3 cells but also showed significant toxicities in low-Her2 cells and therefore are predicted to be toxic in normal tissues. Our findings show that pharmacological profiling of an antibody library in multiple binding and functional assays allows for selection of optimal ADCs.

Klíčová slova:

Adenocarcinomas – Cell binding – Cell binding assay – Cytotoxicity – Flow cytometry – Fluorescence competition – Fluorescence imaging – Toxicity


Zdroje

1. Beck A, Goetsch L, Dumontet C, Corvaïa N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov. 2017; 16(5):315–337. doi: 10.1038/nrd.2016.268 28303026

2. Chalouni C, Doll S. Fate of Antibody-Drug Conjugates in Cancer Cells. J Exp Clin Cancer Res. 2018; 37(1):20. doi: 10.1186/s13046-017-0667-1 29409507

3. Litvak-Greenfeld D, Benhar I. Risks and untoward toxicities of antibody-based immunoconjugates. Adv Drug Deliv Rev. 2012; 64(15):1782–1799. doi: 10.1016/j.addr.2012.05.013 22659123

4. Stagg NJ, Shen BQ, Brunstein F, Li C, Kamath AV, Zhong F, et al. Peripheral neuropathy with microtubule inhibitor containing antibody drug conjugates: Challenges and perspectives in translatability from nonclinical toxicology studies to the clinic. Regul Toxicol Pharmacol. 2016; 82:1–13. doi: 10.1016/j.yrtph.2016.10.012 27773754

5. Tolcher AW. Antibody drug conjugates: lessons from 20 years of clinical experience. Ann Oncol. 2016; 27(12):2168–2172. doi: 10.1093/annonc/mdw424 27733376

6. Donaghy H. Effects of antibody, drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates. MAbs. 2016; 8(4):659–671. doi: 10.1080/19420862.2016.1156829 27045800

7. Sievers EL, Appelbaum FR, Spielberger RT, Forman SJ, Flowers D, Smith FO, et al. Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-CD33 calicheamicin immunoconjugate. Blood. 1999; 93(11):3678–3684. 10339474

8. Tijink BM, Buter J, de Bree R, Giaccone G, Lang MS, Staab A, 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. 2006; 12(20 Pt 1):6064–6072. doi: 10.1158/1078-0432.CCR-06-0910 17062682

9. Annunziata CM, Kohn EC, LoRusso P, Houston ND, Coleman RL, Buzoianu M, et al. Phase I, open-label study of MEDI-547 in patients with relapsed or refractory solid tumors. Invest New Drugs. 2013; 31(1):77–84. doi: 10.1007/s10637-012-9801-2 22370972

10. Stefan N, Gébleux R, Waldmeier L, Hell T, Escher M, Wolter FI, et al. Highly Potent, Anthracycline-based Antibody-Drug Conjugates Generated by Enzymatic, Site-specific Conjugation. Mol Cancer Ther. 2017; 16(5):879–892. doi: 10.1158/1535-7163.MCT-16-0688 28258164

11. Harms BD, Kearns JD, Su SV, Kohli N, Nielsen UB, Schoeberl B. Optimizing properties of antireceptor antibodies using kinetic computational models and experiments. Methods Enzymol. 2012; 502:67–87. doi: 10.1016/B978-0-12-416039-2.00004-5 22208982

12. Zhou Y, Goenaga AL, Harms BD, Zou H, Lou J, Conrad F, et al. Impact of intrinsic affinity on functional binding and biological activity of EGFR antibodies. Mol Cancer Ther. 2012; 11(7):1467–1476. doi: 10.1158/1535-7163.MCT-11-1038 22564724

13. Rudnick SI, Lou J, Shaller CC, Tang Y, Klein-Szanto AJ, Weiner LM, et al. Influence of affinity and antigen internalization on the uptake and penetration of Anti-HER2 antibodies in solid tumors. Cancer Res. 2011; 71(6):2250–2259. doi: 10.1158/0008-5472.CAN-10-2277 21406401

14. Slaga D, Ellerman D, Lombana TN, Vij R, Li J, Hristopoulos M, et al. Avidity-based binding to HER2 results in selective killing of HER2-overexpressing cells by anti-HER2/CD3. Sci Transl Med. 2018; 10 (463). pii: eaat5775.

15. Bostrom J, Haber L, Koenig P, Kelley RF, Fuh G. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity. PLoS One. 2011; 6(4):e17887. doi: 10.1371/journal.pone.0017887 21526167

16. Vivcharuk V, Baardsnes J, Deprez C, Sulea T, Jaramillo M, Corbeil CR, et al. Assisted Design of Antibody and Protein Therapeutics (ADAPT). PLoS One. 2017; 12(7):e0181490. doi: 10.1371/journal.pone.0181490 28750054

17. Drake AW, Myszka DG, Klakamp SL. Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods. Anal Biochem. 2004; 328(1):35–43. doi: 10.1016/j.ab.2003.12.025 15081905

18. de Goeij BE, Satijn D, Freitag CM, Wubbolts R, Bleeker WK, Khasanov A, et al. High turnover of tissue factor enables efficient intracellular delivery of antibody-drug conjugates. Mol Cancer Ther. 2015; 14(5):1130–1140. doi: 10.1158/1535-7163.MCT-14-0798 25724665

19. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, et al. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res. 2008; 68(22):9280–9290. doi: 10.1158/0008-5472.CAN-08-1776 19010901

20. Juweid M, Neumann R, Paik C, Perez-Bacete MJ, Sato J, van Osdol W, et al. Micropharmacology of monoclonal antibodies in solid tumors: direct experimental evidence for a binding site barrier. Cancer Res. 1992; 52(19):5144–5153. 1327501

21. Rudnick SI, Adams GP. Affinity and avidity in antibody-based tumor targeting. Cancer Biother Radiopharm. 2009; 24(2):155–161. doi: 10.1089/cbr.2009.0627 19409036

22. Charlton SJ. Agonist efficacy and receptor desensitization: from partial truths to a fuller picture. Br J Pharmacol. 2009; 158(1):165–168. doi: 10.1111/j.1476-5381.2009.00352.x 19719779

23. Yan H, Endo Y, Shen Y, Rotstein D, Dokmanovic M, Mohan N, et al. Ado-Trastuzumab Emtansine Targets Hepatocytes Via Human Epidermal Growth Factor Receptor 2 to Induce Hepatotoxicity. Mol Cancer Ther. 2016; 15(3):480–490. doi: 10.1158/1535-7163.MCT-15-0580 26712117

24. Peddi PF, Hurvitz SA. Ado-trastuzumab emtansine (T-DM1) in human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer: latest evidence and clinical potential. Ther Adv Med Oncol. 2014; 6(5):202–209. doi: 10.1177/1758834014539183 25342987

25. Yan H, Yu K, Zhang K, Liu L, Li Y. Efficacy and safety of trastuzumab emtansine (T-DM1) in the treatment of HER2-positive metastatic breast cancer (MBC): a meta-analysis of randomized controlled trial. Oncotarget. 2017; 8(60):102458–102467. doi: 10.18632/oncotarget.22270 29254261

26. Wolska-Washer A, Robak T. Safety and Tolerability of Antibody-Drug Conjugates in Cancer. Drug Saf. 2019; Jan 16.

27. Endo Y, Takeda K, Mohan N, Shen Y, Jiang J, Rotstein D, et al. Payload of T-DM1 binds to cell surface cytoskeleton-associated protein 5 to mediate cytotoxicity of hepatocytes. Oncotarget. 2018; 9(98):37200–37215. doi: 10.18632/oncotarget.26461 30647854

28. Zhao H, Gulesserian S, Ganesan SK, Ou J, Morrison K, Zeng Z, et al. Inhibition of Megakaryocyte Differentiation by Antibody-Drug Conjugates (ADCs) is Mediated by Macropinocytosis: Implications for ADC-induced Thrombocytopenia. Mol Cancer Ther. 2017; 16(9):1877–1886. doi: 10.1158/1535-7163.MCT-16-0710 28655784

29. Tsumura R, Manabe S, Takashima H, Koga Y, Yasunaga M, Matsumura Y. Influence of the dissociation rate constant on the intra-tumor distribution of antibody-drug conjugate against tissue factor. J Control Release. 2018; 284:49–56. doi: 10.1016/j.jconrel.2018.06.016 29906553

30. Cho HS, Mason K, Ramyar KX, Stanley AM, Gabelli SB, Denney DW Jr, et al. Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature. 2003; 421(6924):756–760. doi: 10.1038/nature01392 12610629

31. Bostrom J, Yu SF, Kan D, Appleton BA, Lee CV, Billeci K, et al. Variants of the antibody herceptin that interact with HER2 and VEGF at the antigen binding site. Science. 2009; 323(5921):1610–1614. doi: 10.1126/science.1165480 19299620

32. Krivov GG, Shapovalov MV, Dunbrack RL Jr. Improved prediction of protein side-chain conformations with SCWRL4. Proteins. 2009; 77(4):778–795. doi: 10.1002/prot.22488 19603484

33. Naïm M, Bhat S, Rankin KN, Dennis S, Chowdhury SF, Siddiqi I, et al. Solvated interaction energy (SIE) for scoring protein-ligand binding affinities. 1. Exploring the parameter space. J Chem Inf Model. 2007; 47(1):122–133. doi: 10.1021/ci600406v 17238257

34. Sulea T, Purisima EO. The solvated interaction energy method for scoring binding affinities. Methods Mol Biol. 2012; 819:295–303. doi: 10.1007/978-1-61779-465-0_19 22183544

35. Guerois R, Nielsen JE, Serrano L. Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J Mol Biol. 2002; 320(2):369–387. doi: 10.1016/S0022-2836(02)00442-4 12079393

36. Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. The FoldX web server: an online force field. Nucleic Acids Res. 2005; 33(Web Server issue):W382–388. doi: 10.1093/nar/gki387 15980494

37. Ó Conchúir S, Barlow KA, Pache RA, Ollikainen N, Kundert K, O’Meara MJ, et al. Web Resource for Standardized Benchmark Datasets, Metrics, and Rosetta Protocols for Macromolecular Modeling and Design. PLoS One. 2015; 10(9):e0130433. doi: 10.1371/journal.pone.0130433 26335248

38. Rohl CA, Strauss CE, Misura KM, Baker D. Protein structure prediction using Rosetta. Methods Enzymol. 2004; 383:66–93. doi: 10.1016/S0076-6879(04)83004-0 15063647

39. Sulea T, Vivcharuk V, Corbeil CR, Deprez C, Purisima EO. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities. J Chem Inf Model. 2016; 56(7):1292–1303. doi: 10.1021/acs.jcim.6b00043 27367467


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