Bivalent EGFR-Targeting DARPin-MMAE Conjugates

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).

[1]  U. Bornscheuer,et al.  Efficient Site‐Selective Immobilization of Aldehyde‐Tagged Peptides and Proteins by Knoevenagel Ligation , 2021, ChemCatChem.

[2]  T. Dierks,et al.  Site-Specific Conjugation Strategy for Dual Antibody-Drug Conjugates Using Aerobic Formylglycine-Generating Enzymes. , 2021, Bioconjugate chemistry.

[3]  N. Ueno,et al.  Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance , 2020, Nature Communications.

[4]  C. Dumontet,et al.  Antibody–Drug Conjugates: The Last Decade , 2020, Pharmaceuticals.

[5]  S. Kitamura,et al.  Vandetanib inhibits cell growth in EGFR-expressing cutaneous squamous cell carcinoma. , 2020, Biochemical and biophysical research communications.

[6]  T. Dierks,et al.  Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs , 2020, Chembiochem : a European journal of chemical biology.

[7]  A. Plückthun,et al.  Optimizing the anti-tumor efficacy of protein-drug conjugates by engineering the molecular size and half-life. , 2020, Journal of controlled release : official journal of the Controlled Release Society.

[8]  Zhaowu Ma,et al.  The Latest Battles Between EGFR Monoclonal Antibodies and Resistant Tumor Cells , 2020, Frontiers in Oncology.

[9]  W. Leenders,et al.  EpCAM-Binding DARPins for Targeted Photodynamic Therapy of Ovarian Cancer , 2020, Cancers.

[10]  H. Kolmar,et al.  Recent progress in transglutaminase-mediated assembly of antibody-drug conjugates. , 2020, Analytical biochemistry.

[11]  T. Dierks,et al.  Conversion of Serine‐Type Aldehyde Tags by the Radical SAM Protein AtsB from Methanosarcina mazei , 2019, Chembiochem : a European journal of chemical biology.

[12]  Aman P. Singh,et al.  A "Dual" Cell-Level Systems PK-PD Model to Characterize the Bystander Effect of ADC. , 2019, Journal of pharmaceutical sciences.

[13]  M. Weller,et al.  Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilization , 2019, EMBO molecular medicine.

[14]  K. Müller,et al.  EGF-mCherry Fusion Protein Expressed in E. coli Shows Product Heterogeneity but a High Biological Activity. , 2019, Biochemistry.

[15]  Xin Hu,et al.  An EGFR‐targeting antibody–drug conjugate LR004‐VC‐MMAE: potential in esophageal squamous cell carcinoma and other malignancies , 2018, Molecular oncology.

[16]  Judith A. Blake,et al.  Mouse Genome Database (MGD) 2019 , 2018, Nucleic Acids Res..

[17]  Ningyan Zhang,et al.  Glutamic acid–valine–citrulline linkers ensure stability and efficacy of antibody–drug conjugates in mice , 2018, Nature Communications.

[18]  T. Dierks,et al.  Two-fold Bioorthogonal Derivatization by Different Formylglycine-Generating Enzymes. , 2018, Angewandte Chemie.

[19]  Han-jie Zhang,et al.  Rapid, Stoichiometric, Site-Specific Modification of Aldehyde-Containing Proteins Using a Tandem Knoevenagel-Intra Michael Addition Reaction. , 2018, Bioconjugate chemistry.

[20]  A. Küchler,et al.  Dual, Site‐Specific Modification of Antibodies by Using Solid‐Phase Immobilized Microbial Transglutaminase , 2017, Chembiochem : a European journal of chemical biology.

[21]  T. Takayama,et al.  EGFR Downregulation after Anti-EGFR Therapy Predicts the Antitumor Effect in Colorectal Cancer , 2017, Molecular Cancer Research.

[22]  I. Maruyama,et al.  Activation of the EGF Receptor by Ligand Binding and Oncogenic Mutations: The “Rotation Model” , 2017, Cells.

[23]  Rebecca A. Ihrie,et al.  A Chimeric Egfr Protein Reporter Mouse Reveals Egfr Localization and Trafficking In Vivo. , 2017, Cell reports.

[24]  C. Dumontet,et al.  Strategies and challenges for the next generation of antibody–drug conjugates , 2017, Nature Reviews Drug Discovery.

[25]  Gunnar Jeschke,et al.  Copper is a Cofactor of the Formylglycine‐Generating Enzyme , 2016, Chembiochem : a European journal of chemical biology.

[26]  P. Burke,et al.  Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody–Drug Conjugates , 2016, Molecular Cancer Therapeutics.

[27]  P. Drake,et al.  Site-Specific Tandem Knoevenagel Condensation-Michael Addition To Generate Antibody-Drug Conjugates. , 2016, ACS medicinal chemistry letters.

[28]  K. Müller,et al.  Recent progress in protein-protein interaction study for EGFR-targeted therapeutics , 2016, Expert review of proteomics.

[29]  Jan Beck,et al.  Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site-Specific Conjugation. , 2015, Angewandte Chemie.

[30]  Yuyuan Tian,et al.  Quantification of epidermal growth factor receptor expression level and binding kinetics on cell surfaces by surface plasmon resonance imaging. , 2015, Analytical chemistry.

[31]  T. Dierks,et al.  Eukaryotic formylglycine‐generating enzyme catalyses a monooxygenase type of reaction , 2015, The FEBS journal.

[32]  R. Beerli,et al.  Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency , 2015, PloS one.

[33]  P. Burke,et al.  Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index , 2015, Nature Biotechnology.

[34]  P. Drake,et al.  Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion , 2015, The Journal of Biological Chemistry.

[35]  J. McFarland,et al.  Generating site-specifically modified proteins via a versatile and stable nucleophilic carbon ligation. , 2015, Chemistry & biology.

[36]  J. Bereiter-Hahn,et al.  Advanced 3D-Sonographic Imaging as a Precise Technique to Evaluate Tumor Volume , 2014, Translational oncology.

[37]  P. Chumakov,et al.  Apoptin enhances the oncolytic properties of vaccinia virus and modifies mechanisms of tumor regression , 2014, Oncotarget.

[38]  P. Drake,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 , 2014, Bioconjugate chemistry.

[39]  E. Fischer,et al.  Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. , 2014, Bioconjugate chemistry.

[40]  C. Futter,et al.  EGF receptor trafficking: consequences for signaling and cancer , 2014, Trends in cell biology.

[41]  A. Plückthun,et al.  Orthogonal assembly of a designed ankyrin repeat protein-cytotoxin conjugate with a clickable serum albumin module for half-life extension. , 2013, Bioconjugate chemistry.

[42]  A. Bader,et al.  Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif , 2013, Journal of Cell Science.

[43]  M. Distefano,et al.  Simultaneous dual protein labeling using a triorthogonal reagent. , 2013, Journal of the American Chemical Society.

[44]  P. Drake,et al.  Hydrazino-Pictet-Spengler ligation as a biocompatible method for the generation of stable protein conjugates. , 2013, Bioconjugate chemistry.

[45]  L. Gedda,et al.  Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach. , 2013, Molecular and clinical oncology.

[46]  B. Gorovits,et al.  Proposed mechanism of off-target toxicity for antibody–drug conjugates driven by mannose receptor uptake , 2013, Cancer Immunology, Immunotherapy.

[47]  A. Cardona,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[48]  H. Ploegh,et al.  Protein Ligation in Living Cells Using Sortase , 2012, Traffic.

[49]  A. Plückthun,et al.  Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting Epidermal Growth Factor Receptor Inhibit A431 Cell Proliferation and Receptor Recycling* , 2011, The Journal of Biological Chemistry.

[50]  A. Plückthun,et al.  A Novel Fusion Toxin Derived from an EpCAM-Specific Designed Ankyrin Repeat Protein Has Potent Antitumor Activity , 2010, Clinical Cancer Research.

[51]  Shigeyuki Yokoyama,et al.  Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor , 2010, Molecular and Cellular Biology.

[52]  P. Iversen,et al.  Interstitial fluid: the overlooked component of the tumor microenvironment? , 2010, Fibrogenesis & tissue repair.

[53]  Andreas Plückthun,et al.  Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size. , 2010, Cancer research.

[54]  A. Plückthun,et al.  Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display. , 2008, Journal of molecular biology.

[55]  Paul Polakis,et al.  Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index , 2008, Nature Biotechnology.

[56]  I. Chaiken,et al.  Enhanced EGFR inhibition and distinct epitope recognition by EGFR antagonistic MABS C225 and 425 , 2008, Cancer biology & therapy.

[57]  T. Knöchel,et al.  Matuzumab binding to EGFR prevents the conformational rearrangement required for dimerization. , 2008, Cancer cell.

[58]  A. Caflisch,et al.  Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments. , 2008, Journal of molecular biology.

[59]  Michael J Rust,et al.  Ligands for Clathrin-Mediated Endocytosis Are Differentially Sorted into Distinct Populations of Early Endosomes , 2006, Cell.

[60]  T. Dierks,et al.  The human SUMF1 gene, required for posttranslational sulfatase modification, defines a new gene family which is conserved from pro- to eukaryotes. , 2003, Gene.

[61]  T. Dierks,et al.  Multiple Sulfatase Deficiency Is Caused by Mutations in the Gene Encoding the Human Cα-Formylglycine Generating Enzyme , 2003, Cell.

[62]  Jae-Hoon Kim,et al.  Crystal Structure of the Complex of Human Epidermal Growth Factor and Receptor Extracellular Domains , 2002, Cell.

[63]  Donna Richardson,et al.  Tumour necrosis is an independent prognostic marker in non-small cell lung cancer: correlation with biological variables. , 2002, Lung cancer.

[64]  Chun Xing Li,et al.  Conjugation with (111)In-DTPA-poly(ethylene glycol) improves imaging of anti-EGF receptor antibody C225. , 2001, Journal of nuclear medicine : official publication, Society of Nuclear Medicine.

[65]  R. Jain,et al.  Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation. , 2000, Cancer research.

[66]  T. Dierks,et al.  Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases , 1999, The EMBO journal.

[67]  C. Davis,et al.  Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy. , 1999, Cancer research.

[68]  A. Plückthun,et al.  A dimeric bispecific miniantibody combines two specificities with avidity , 1998, FEBS letters.

[69]  T. Dierks,et al.  A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[70]  A. Plückthun,et al.  The first constant domain (CH1 and CL) of an antibody used as heterodimerization domain for bispecific miniantibodies , 1998, FEBS letters.

[71]  E. Padlan,et al.  Anatomy of the antibody molecule. , 1994, Molecular immunology.

[72]  E. Gelmann,et al.  Epidermal growth factor receptor gene expression in estrogen receptor-positive and negative human breast cancer cell lines. , 1987, Molecular endocrinology.

[73]  M. Herlyn,et al.  Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide. , 1987, Archives of biochemistry and biophysics.

[74]  S. Aaronson,et al.  In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. , 1973, Journal of the National Cancer Institute.

[75]  R. Roskoski.  The ErbB/HER family of protein-tyrosine kinases and cancer. , 2014, Pharmacological research.

[76]  J. Bereiter-Hahn,et al.  Ultrasonic Quantification of Tumor Interstitial Fluid Pressure Through Scanning Acoustic Microscopy , 2012 .