Format chain exchange (FORCE) for high-throughput generation of bispecific antibodies in combinatorial binder-format matrices

Generation of bispecific antibodies (bsAbs) requires a combination of compatible binders in formats that support desired functionalities. Here, we report that bsAb-matrices can be generated by Format Chain Exchange (FORCE), enabling screening of combinatorial binder/format spaces. Input molecules for generation of bi/multi-valent bsAbs are monospecific entities similar to knob-into-hole half-antibodies, yet with complementary CH3-interface-modulated and affinity-tagged dummy-chains. These contain mutations that lead to limited interface repulsions without compromising expression or biophysical properties of educts. Mild reduction of combinations of educts triggers spontaneous chain-exchange reactions driven by partially flawed CH3-educt interfaces resolving to perfect complementarity. This generates large bsAb matrices harboring different binders in multiple formats. Benign biophysical properties and good expression yields of educts, combined with simplicity of purification enables process automation. Examples that demonstrate the relevance of screening binder/format combinations are provided as a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5 without encountering binder or format-inflicted interferences.

[1]  Jinke Cheng,et al.  Structural basis of a novel heterodimeric Fc for bispecific antibody production , 2017, Oncotarget.

[2]  Camellia W. Adams,et al.  An efficient route to human bispecific IgG , 1998, Nature Biotechnology.

[3]  Randy J. Read,et al.  Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix , 2019, Acta crystallographica. Section D, Structural biology.

[4]  Giulia Bianchi,et al.  Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced, inflammatory, or early HER2-positive breast cancer (NeoSphere): a randomised multicentre, open-label, phase 2 trial. , 2012, The Lancet. Oncology.

[5]  T. Singer,et al.  Brain Shuttle Antibody for Alzheimer's Disease with Attenuated Peripheral Effector Function due to an Inverted Binding Mode. , 2018, Cell reports.

[6]  Charles Eigenbrot,et al.  Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. , 2014, Journal of molecular biology.

[7]  S. Zolla-Pazner,et al.  Statistical Approaches to Analyzing HIV-1 Neutralizing Antibody Assay Data , 2012, Statistics in biopharmaceutical research.

[8]  A. M. Stanley,et al.  Structure of the extracellular region of HER 2 alone and in complex with the Herceptin Fab , 2022 .

[9]  Diego Ellerman,et al.  Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies , 2013, Nature Biotechnology.

[10]  I. Hiles,et al.  ‘In-Format’ screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules , 2017, mAbs.

[11]  I. Kasman,et al.  MetMAb, the one-armed 5D5 anti-c-Met antibody, inhibits orthotopic pancreatic tumor growth and improves survival. , 2007, Cancer research.

[12]  J. Wells,et al.  Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. , 1997, Journal of molecular biology.

[13]  J. Posey,et al.  Phase I trial of weekly tigatuzumab, an agonistic humanized monoclonal antibody targeting death receptor 5 (DR5). , 2010, Cancer biotherapy & radiopharmaceuticals.

[14]  E. Van Cutsem,et al.  Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. , 2009, The New England journal of medicine.

[15]  P. Parren,et al.  Controlled Fab-arm exchange for the generation of stable bispecific IgG1 , 2014, Nature Protocols.

[16]  J. Elstrott,et al.  Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants , 2014, The Journal of experimental medicine.

[17]  Anirvan Ghosh,et al.  Increased Brain Penetration and Potency of a Therapeutic Antibody Using a Monovalent Molecular Shuttle , 2014, Neuron.

[18]  U. Brinkmann,et al.  TriFabs—Trivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery , 2015, International journal of molecular sciences.

[19]  Yongping Song,et al.  Recent advances on blinatumomab for acute lymphoblastic leukemia , 2019, Experimental Hematology & Oncology.

[20]  Haiyan Wu,et al.  A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line. , 2017, The Journal of biological chemistry.

[21]  G. V. Vande Woude,et al.  Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent , 2013, Proceedings of the National Academy of Sciences.

[22]  Herren Wu,et al.  Fab-Arm Exchange Combined with Selective Protein A Purification Results in a Platform for Rapid Preparation of Monovalent Bispecific Antibodies Directly from Culture Media , 2019, Pharmaceutics.

[23]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[24]  Y. Yonezawa,et al.  TRAIL-R2 Superoligomerization Induced by Human Monoclonal Agonistic Antibody KMTR2 , 2015, Scientific Reports.

[25]  R. Kontermann,et al.  Diabody-Ig: a novel platform for the generation of multivalent and multispecific antibody molecules , 2019, mAbs.

[26]  W. Sandoval,et al.  Reorienting the Fab Domains of Trastuzumab Results in Potent HER2 Activators , 2012, PloS one.

[27]  C. Klein,et al.  DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity , 2019, mAbs.

[28]  Ulrich Brinkmann,et al.  The making of bispecific antibodies , 2017, mAbs.

[29]  C. Klein,et al.  Engineering therapeutic bispecific antibodies using CrossMab technology. , 2019, Methods.

[30]  Michael J. Gramer,et al.  Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange , 2013, Proceedings of the National Academy of Sciences.

[31]  Tsukasa Suzuki,et al.  A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model , 2012, Nature Medicine.

[32]  Steffen Dickopf,et al.  Format and geometries matter: Structure-based design defines the functionality of bispecific antibodies , 2020, Computational and structural biotechnology journal.

[33]  L. Presta,et al.  'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. , 1996, Protein engineering.

[34]  Chunning Yang,et al.  Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence , 2017, Scientific Reports.

[35]  R. Herbst,et al.  A First-in-Human Study of Conatumumab in Adult Patients with Advanced Solid Tumors , 2010, Clinical Cancer Research.

[36]  C. Klein,et al.  A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors , 2016, Clinical Cancer Research.

[37]  Shi-Yong Sun,et al.  Drozitumab, a Human Antibody to Death Receptor 5, Has Potent Antitumor Activity against Rhabdomyosarcoma with the Expression of Caspase-8 Predictive of Response , 2011, Clinical Cancer Research.

[38]  U. Brinkmann,et al.  Engineered hapten‐binding antibody derivatives for modulation of pharmacokinetic properties of small molecules and targeted payload delivery , 2016, Immunological reviews.

[39]  G. A. Lazar,et al.  Next generation antibody drugs: pursuit of the 'high-hanging fruit' , 2017, Nature Reviews Drug Discovery.

[40]  T. Igawa,et al.  Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity , 2013, PloS one.

[41]  U. Brinkmann,et al.  Corrigendum to "Bispecific antibodies" [Drug Discov. Today 20 (July (7)) (2015) 838-847]. , 2018, Drug discovery today.

[42]  Jihong Wang,et al.  Improving target cell specificity using a novel monovalent bispecific IgG design , 2015, mAbs.

[43]  U. Brinkmann,et al.  Highly flexible, IgG-shaped, trivalent antibodies effectively target tumor cells and induce T cell-mediated killing , 2019, Biological chemistry.

[44]  Meric A. Ovacik,et al.  Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells , 2016, mAbs.

[45]  P. Zwart,et al.  Towards automated crystallographic structure refinement with phenix.refine , 2012, Acta crystallographica. Section D, Biological crystallography.

[46]  Sebastian Raschka,et al.  Protein–ligand interfaces are polarized: discovery of a strong trend for intermolecular hydrogen bonds to favor donors on the protein side with implications for predicting and designing ligand complexes , 2018, Journal of Computer-Aided Molecular Design.

[47]  C. Klein,et al.  Structural differences between glycosylated, disulfide-linked heterodimeric Knob-into-Hole Fc fragment and its homodimeric Knob–Knob and Hole–Hole side products , 2017, Protein engineering, design & selection : PEDS.

[48]  C. Ries,et al.  GA201 (RG7160): A Novel, Humanized, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior In Vivo Efficacy Compared with Cetuximab , 2012, Clinical Cancer Research.

[49]  C. Klein,et al.  RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis , 2016, Molecular Cancer Therapeutics.

[50]  U. Brinkmann,et al.  The Contorsbody, an antibody format for agonism: Design, structure, and function , 2020, Computational and structural biotechnology journal.

[51]  P. Parren,et al.  Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo , 2009, Nature Biotechnology.

[52]  M. Wittekind,et al.  Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects , 2010, The Journal of Biological Chemistry.

[53]  U. Brinkmann,et al.  Transcytosis of payloads that are non-covalently complexed to bispecific antibodies across the hCMEC/D3 blood-brain barrier model , 2018, Biological chemistry.