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1.
J Immunol ; 211(11): 1725-1735, 2023 12 01.
Article in English | MEDLINE | ID: mdl-37843500

ABSTRACT

Complement activation via the classical pathway is initiated when oligomeric Igs on target surfaces are recognized by C1 of the complement cascade. The strength of this interaction and activation of the complement system are influenced by structural variation of the Ab, including Ab isotype, subclass, and glycosylation profile. Polymorphic variants of IgG have also been described to influence Fc-dependent effector functions. Therefore, we assessed complement binding, deposition, and complement-dependent cytotoxicity (CDC) of 27 known IgG allotypes with anti-trinitrophenyl specificity. Differences between allotypes within subclasses were minor for IgG1, IgG3, and IgG4 allotypes, and more substantial for IgG2. Allelic variant IGHG2*06, containing a unique serine at position 378 in the CH3 domain, showed less efficient complement activation and CDC compared with other IgG2 polymorphisms. We also observed variable cell lysis between IgG1 and IgG3, with IgG3 being superior in lysis of human RBCs and Ramos cells, and IgG1 being superior in lysis of Raji and Wien133 cells, demonstrating that a long-standing conundrum in the literature depends on cellular context. Furthermore, we compared IgG1 and IgG3 under different circumstances, showing that Ag density and Ab hinge length, but not complement regulators, define the context dependency of Ab-mediated CDC activity. Our results point toward a variation in the capacity of IgG subclasses to activate complement due to single amino acid changes and hinge length differences of allotypes to activate complement, which might give new insights on susceptibility to infectious, alloimmune, or autoimmune diseases and aid the design of Ab-based therapeutics.


Subject(s)
Complement Activation , Immunoglobulin G , Humans , Glycosylation
2.
Immunol Cell Biol ; 101(7): 657-662, 2023 08.
Article in English | MEDLINE | ID: mdl-36997299

ABSTRACT

The agonistic action of several immunomodulatory monoclonal antibodies (mAbs) requires both target antigen binding and clustering of this mAb:target complex by the Fcs interacting with Fcγ receptors (FcγRs), in particular FcγRIIb, on neighboring bystander cells. Fc mutations were made in the immunoglobulin G4 (IgG4)-based TGN1412 anti-CD28 mAb to define the role of FcγR interactions in its "super-agonist" activity. The dual mutation, IgG4-ED269,270 AA, ablated interaction with all human FcγRs and agonistic action was consequentially lost, confirming the FcγR dependence on the action of TGN1412. The IgG4 lower hinge region (F234 L235 G236 G237 ) was modified by L235 E mutation (F234 E235 G236 G237 ), a mutation commonly used to ablate FcγR binding, including in approved therapeutic mAbs. However, rather than ablating all FcγR binding, IgG4-L235 E conferred specific binding to FcγRIIb, the inhibitory Fc receptor. Furthermore, in combination with the core hinge-stabilizing mutation (IgG4-S228 P, L235 E), this mutation increased affinity for FcγRIIb compared with wild-type IgG4. In addition to having FcγRIIb specificity, these engineered TGN1412 antibodies retained their super-agonistic ability, demonstrating that CD28- and FcγRIIb-specific binding are together sufficient for agonistic function. The FcγRIIb-specific nature of IgG4-L235 E has utility for mAb-mediated immune agonism therapies that are dependent on FcγRIIb interaction and of anti-inflammatory mAbs in allergy and autoimmunity that harness FcγRIIb inhibitory signaling.


Subject(s)
Immunoglobulin G , Receptors, IgG , Humans , Receptors, IgG/metabolism , Antibodies, Monoclonal/therapeutic use , Mutation/genetics
3.
Front Immunol ; 14: 1304365, 2023.
Article in English | MEDLINE | ID: mdl-38259472

ABSTRACT

Immunoglobulin G (IgG) antibodies are a critical component of the adaptive immune system, binding to and neutralizing pathogens and other foreign substances. Recent advances in molecular antibody biology and structural protein engineering enabled the modification of IgG antibodies to enhance their therapeutic potential. This review summarizes recent progress in both natural and engineered structural modifications of IgG antibodies, including allotypic variation, glycosylation, Fc engineering, and Fc gamma receptor binding optimization. We discuss the functional consequences of these modifications to highlight their potential for therapeutical applications.


Subject(s)
Immunoglobulin G , Receptors, IgG , Gamma Rays , Glycosylation , Molecular Biology
4.
J Immunother Cancer ; 10(9)2022 09.
Article in English | MEDLINE | ID: mdl-36096528

ABSTRACT

BACKGROUND: Immunosuppressive extracellular adenosine is generated by the enzymatic activity of CD73. In preclinical models, antibodies (Abs) targeting different epitopes on CD73 exert anticancer activity through distinct mechanisms such as inhibition of enzymatic activity, engagement of Fc receptors, and spatial redistribution of CD73. METHODS: Using controlled Fab arm exchange, we generated biparatopic bispecific antibodies (bsAbs) from parental anti-CD73 Abs with distinct anticancer activities. The resulting anticancer activity was evaluated using in vitro and in vivo models. RESULTS: We demonstrate that different anticancer activities can be combined in a biparatopic bsAb. Remarkably, the bsAb significantly improved the enzyme inhibitory activity compared with the parental Abs, which led to neutralization of adenosine-mediated T-cell suppression as demonstrated by proliferation and interferon gamma (IFN-γ) production and prolonged survival of tumor-bearing mice. Additionally, the bsAb caused more efficient internalization of cell surface CD73 and stimulated potent Fc-mediated engagement of human immune effector cells in vitro and in vivo. CONCLUSIONS: Our data collectively demonstrate that complementary anticancer mechanisms of action of distinct anti-CD73 Abs can be combined and enhanced in a biparatopic bsAb. The multiple mechanisms of action and superior activity compared with the monospecific parental Abs make the bsAb a promising candidate for therapeutic targeting of CD73 in cancer. This concept may greatly improve future Ab design.


Subject(s)
Antibodies, Bispecific , Neoplasms , Adenosine , Animals , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Epitopes , Humans , Mice
5.
Cancers (Basel) ; 13(2)2021 Jan 14.
Article in English | MEDLINE | ID: mdl-33466732

ABSTRACT

Immunotherapy of cancer with CD3-bispecific antibodies is an approved therapeutic option for some hematological malignancies and is under clinical investigation for solid cancers. However, the treatment of solid tumors faces more pronounced hurdles, such as increased on-target off-tumor toxicities, sparse T-cell infiltration and impaired T-cell quality due to the presence of an immunosuppressive tumor microenvironment, which affect the safety and limit efficacy of CD3-bispecific antibody therapy. In this review, we provide a brief status update of the CD3-bispecific antibody therapy field and identify intrinsic hurdles in solid cancers. Furthermore, we describe potential combinatorial approaches to overcome these challenges in order to generate selective and more effective responses.

6.
Front Immunol ; 11: 740, 2020.
Article in English | MEDLINE | ID: mdl-32435243

ABSTRACT

Antibody dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. NK-cell mediated ADCC is mainly triggered by IgG-subclasses IgG1 and IgG3 through the IgG-Fc-receptor (FcγR) IIIa. Polymorphisms in the immunoglobulin gamma heavy chain gene likely form a layer of variation in the strength of the ADCC-response, but this has never been studied in detail. We produced all 27 known IgG allotypes and assessed FcγRIIIa binding and ADCC activity. While all IgG1, IgG2, and IgG4 allotypes behaved similarly within subclass, large allotype-specific variation was found for IgG3. ADCC capacity was affected by residues 291, 292, and 296 in the CH2 domain through altered affinity or avidity for FcγRIIIa. Furthermore, allotypic variation in hinge length affected ADCC, likely through altered proximity at the immunological synapse. Thus, these functional differences between IgG allotypes have important implications for therapeutic applications and susceptibility to infectious-, allo- or auto-immune diseases.


Subject(s)
Immunoglobulin Allotypes/metabolism , Immunoglobulin G/metabolism , Immunological Synapses/metabolism , Killer Cells, Natural/immunology , Receptors, IgG/metabolism , Antibody-Dependent Cell Cytotoxicity , Cells, Cultured , Glycosylation , Humans , Immunoglobulin Heavy Chains/genetics , Polymorphism, Genetic , Protein Binding , Receptors, IgG/genetics
7.
EBioMedicine ; 52: 102625, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31981978

ABSTRACT

BACKGROUND: DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A. METHODS: T-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys. FINDINGS: DuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies. FUNDING: Genmab.


Subject(s)
Antibodies, Bispecific/immunology , Antigens, CD20/metabolism , CD3 Complex/metabolism , Cytotoxicity, Immunologic , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Antibodies, Bispecific/genetics , Antibodies, Bispecific/pharmacology , Antibody Specificity/immunology , Antibody-Dependent Cell Cytotoxicity , Antineoplastic Agents, Immunological/pharmacology , Cell Line, Tumor , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Humans , Leukemia, B-Cell/drug therapy , Leukemia, B-Cell/etiology , Leukemia, B-Cell/pathology , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/etiology , Lymphoma, B-Cell/pathology , Macaca fascicularis , Mice , Mutation , Recombinant Proteins , Xenograft Model Antitumor Assays
8.
Nat Rev Drug Discov ; 18(8): 585-608, 2019 08.
Article in English | MEDLINE | ID: mdl-31175342

ABSTRACT

The term bispecific antibody (bsAb) is used to describe a large family of molecules designed to recognize two different epitopes or antigens. BsAbs come in many formats, ranging from relatively small proteins, merely consisting of two linked antigen-binding fragments, to large immunoglobulin G (IgG)-like molecules with additional domains attached. An attractive bsAb feature is their potential for novel functionalities - that is, activities that do not exist in mixtures of the parental or reference antibodies. In these so-called obligate bsAbs, the physical linkage of the two binding specificities creates a dependency that can be temporal, with binding events occurring sequentially, or spatial, with binding events occurring simultaneously, such as in linking an effector to a target cell. To date, more than 20 different commercialized technology platforms are available for bsAb creation and development, 2 bsAbs are marketed and over 85 are in clinical development. Here, we review the current bsAb landscape from a mechanistic perspective, including a comprehensive overview of the pipeline.


Subject(s)
Antibodies, Bispecific , Drug Design , Neoplasms , Animals , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/immunology , Antibodies, Bispecific/therapeutic use , Binding Sites, Antibody , Clinical Trials as Topic , Drug Evaluation, Preclinical , Humans , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplasms/immunology
9.
Mol Cancer Ther ; 18(2): 312-322, 2019 02.
Article in English | MEDLINE | ID: mdl-30381448

ABSTRACT

Immunotherapy of cancer with CD3-targeting bispecific antibodies (CD3 bsAb) is a fast developing field, and multiple tumor-associated antigens (TAA) are evaluated for hematologic and solid malignancies. The efficacy of these CD3 bsAb is usually examined in xenograft mouse tumor models with human T cells or in genetically engineered mouse models, where human TAA are introduced. These models often fail to fully recapitulate the natural tumor environment, especially for solid cancers, because of interspecies differences. Here, we investigated the systemic and intratumoral effects of a mouse CD3 bsAb in a fully immune-competent mouse melanoma model. Systemic administration of 0.5 mg/kg antibody induced a brief overall T-cell activation that was selectively sustained in the tumor microenvironment for several days. A fast subsequent influx of inflammatory macrophages into the tumor microenvironment was observed, followed by an increase in the number of CD4+ and CD8+ T cells. Although the capacity to directly kill melanoma cells in vitro was very modest, optimal tumor elimination was observed in vivo, even in the absence of CD8+ T cells, implying a redundancy in T-cell subsets for therapeutic efficacy. Finally, we took advantage of the full immune competence of our mouse model and tested immune memory induction. Despite a strong initial immunity against melanoma, treatment with the CD3 bsAb did not install protective memory responses. The observed mechanisms of action revealed in this immune-competent mouse model might form a rational basis for combinatorial approaches.


Subject(s)
Antibodies, Bispecific/administration & dosage , CD3 Complex/antagonists & inhibitors , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Melanoma/drug therapy , Animals , Antibodies, Bispecific/pharmacology , Antigens, Neoplasm/genetics , CD4-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/drug effects , Cell Line, Tumor , Female , Humans , Immunologic Memory , Lymphocyte Activation , Melanoma/immunology , Mice , Tumor Microenvironment/drug effects , Xenograft Model Antitumor Assays
10.
J Biol Chem ; 293(2): 651-661, 2018 01 12.
Article in English | MEDLINE | ID: mdl-29150443

ABSTRACT

Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined. Knowledge of the mechanism could lead to improved yields of bsAb at faster rates as well as foster adoption of process control. In this work, a combination of Förster resonance energy transfer (FRET), nonreducing SDS-PAGE, and strategic mutation of the Ab hinge region was employed to identify and characterize the individual steps of cFAE. Fluorescence correlation spectroscopy (FCS) was used to determine the affinity of parental (homodimer) and bispecific (heterodimer) interactions within the CH3 domain, further clarifying the thermodynamic basis for bsAb formation. The result is a clear sequence of events with rate constants that vary with experimental conditions, where dissociation of the K409R parental Ab into half-Ab controls the rate of the reaction.


Subject(s)
Antibodies, Bispecific/metabolism , Immunoglobulin Fab Fragments/metabolism , Animals , Humans , Kinetics , Spectrometry, Fluorescence
11.
Anal Chem ; 89(20): 10873-10882, 2017 10 17.
Article in English | MEDLINE | ID: mdl-28922593

ABSTRACT

Bispecific antibodies (bsAbs) are one of the most versatile and promising pharmaceutical innovations for countering heterogeneous and refractory disease by virtue of their ability to bind two distinct antigens. One critical quality attribute of bsAb formation requiring investigation is the potential randomization of cognate heavy (H) chain/light (L) chain pairing, which could occur to a varying extent dependent on bsAb format and the production platform. To assess the content of such HL-chain swapped reaction products with high sensitivity, we developed cysteine-stable isotope labeling using amino acids in cell culture (SILAC), a method that facilitates the detailed characterization of disulfide-bridged peptides by mass spectrometry. For this analysis, an antibody was metabolically labeled with 13C3,15N-cysteine and incorporated into a comprehensive panel of distinct bispecific molecules by controlled Fab-arm exchange (DuoBody technology). This technology is a postproduction method for the generation of bispecific therapeutic IgGs of which several have progressed into the clinic. Herein, two parental antibodies, each containing a single heavy chain domain mutation, are mixed and subjected to controlled reducing conditions during which they exchange heavy-light (HL) chain pairs to form bsAbs. Subsequently, reductant is removed and all disulfide bridges are reoxidized to reform covalent inter- and intrachain bonds. We conducted a multilevel (Top-Middle-Bottom-Up) approach focusing on the characterization of both "left-arm" and "right-arm" HL interchain disulfide peptides and observed that native HL pairing was preserved in the whole panel of bsAbs produced by controlled Fab-arm exchange.


Subject(s)
Antibodies, Bispecific/chemistry , Cysteine/chemistry , Disulfides/analysis , Immunoglobulin G/chemistry , Tandem Mass Spectrometry , Antibodies, Bispecific/metabolism , Antigens, CD20/immunology , Carbon Isotopes/chemistry , Chromatography, High Pressure Liquid , ErbB Receptors/immunology , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/metabolism , Isotope Labeling , Nitrogen Isotopes/chemistry
12.
Sci Rep ; 7(1): 2476, 2017 05 30.
Article in English | MEDLINE | ID: mdl-28559564

ABSTRACT

Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.


Subject(s)
Antibodies, Bispecific/biosynthesis , Immunoglobulin G/immunology , Neoplasms/therapy , Animals , Antibodies, Bispecific/immunology , Humans , Immunoglobulin G/genetics , Immunoglobulin G/therapeutic use , Mice , Models, Animal , Neoplasms/genetics , Neoplasms/immunology , Point Mutation/genetics , Point Mutation/immunology , Rats , Xenograft Model Antitumor Assays
13.
Sci Rep ; 7(1): 992, 2017 04 20.
Article in English | MEDLINE | ID: mdl-28428630

ABSTRACT

Autoantibodies against ion channels are the cause of numerous neurologic autoimmune disorders. Frequently, such pathogenic autoantibodies have a restricted epitope-specificity. In such cases, competing antibody formats devoid of pathogenic effector functions (blocker antibodies) have the potential to treat disease by displacing autoantibodies from their target. Here, we have used a model of the neuromuscular autoimmune disease myasthenia gravis in rhesus monkeys (Macaca mulatta) to test the therapeutic potential of a new blocker antibody: MG was induced by passive transfer of pathogenic acetylcholine receptor-specific monoclonal antibody IgG1-637. The effect of the blocker antibody (IgG4Δhinge-637, the hinge-deleted IgG4 version of IgG1-637) was assessed using decrement measurements and single-fiber electromyography. Three daily doses of 1.7 mg/kg IgG1-637 (cumulative dose 5 mg/kg) induced impairment of neuromuscular transmission, as demonstrated by significantly increased jitter, synaptic transmission failures (blockings) and a decrease in the amplitude of the compound muscle action potentials during repeated stimulations (decrement), without showing overt symptoms of muscle weakness. Treatment with three daily doses of 10 mg/kg IgG4Δhinge-637 significantly reduced the IgG1-637-induced increase in jitter, blockings and decrement. Together, these results represent proof-of principle data for therapy of acetylcholine receptor-myasthenia gravis with a monovalent antibody format that blocks binding of pathogenic autoantibodies.


Subject(s)
Autoantibodies/metabolism , Immunoglobulin G/administration & dosage , Myasthenia Gravis/drug therapy , Receptors, Cholinergic/metabolism , Animals , CHO Cells , Cholinergic Antagonists , Cricetulus , Disease Models, Animal , Gene Expression Regulation/drug effects , HEK293 Cells , Hinge Exons , Humans , Immunoglobulin G/pharmacology , Macaca mulatta , Myasthenia Gravis/immunology , Myasthenia Gravis/metabolism , Synaptic Transmission/drug effects , Treatment Outcome
14.
Science ; 354(6310): 284-285, 2016 10 21.
Article in English | MEDLINE | ID: mdl-27846516
15.
Mol Cell ; 63(1): 135-45, 2016 07 07.
Article in English | MEDLINE | ID: mdl-27320199

ABSTRACT

The classical complement pathway contributes to the natural immune defense against pathogens and tumors. IgG antibodies can assemble at the cell surface into hexamers via Fc:Fc interactions, which recruit complement component C1q and induce complement activation. Biophysical characterization of the C1:IgG complex has remained elusive primarily due to the low affinity of IgG-C1q binding. Using IgG variants that dynamically form hexamers efficient in C1q binding and complement activation, we could assess C1q binding in solution by native mass spectrometry and size-exclusion chromatography. Fc-domain deglycosylation, described to abrogate complement activation, affected IgG hexamerization and C1q binding. Strikingly, antigen binding by IgG hexamers or deletion of the Fab arms substantially potentiated complement initiation, suggesting that Fab-mediated effects impact downstream Fc-mediated events. Finally, we characterized a reconstituted 2,045.3 ± 0.4-kDa complex of intact C1 bound to antigen-saturated IgG hexamer by native mass spectrometry, providing a clear visualization of a complete complement initiation complex.


Subject(s)
Antigens/metabolism , Complement Activation , Complement C1q/metabolism , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/metabolism , Antigen-Antibody Reactions , Antigens/chemistry , Antigens/immunology , Binding Sites, Antibody , Cell Line, Tumor , Chromatography, Gel , Complement C1q/chemistry , Complement C1q/immunology , Glycosylation , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Mutation , Protein Binding , Protein Stability , Tandem Mass Spectrometry
16.
Nat Protoc ; 9(10): 2450-63, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25255089

ABSTRACT

The generation of bispecific antibodies (bsAbs) with natural IgG architecture in a practical and efficient manner has been a longstanding challenge. Here we describe controlled Fab-arm exchange (cFAE), which is an easy-to-use method to generate bispecific IgG1 (bsIgG1). The protocol involves the following: (i) separate expression of two parental IgG1s containing single matching point mutations in the CH3 domain; (ii) mixing of parental IgG1s under permissive redox conditions in vitro to enable recombination of half-molecules; (iii) removal of the reductant to allow reoxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or mass spectrometry (MS)-based methods. The protocol generates bsAbs with regular IgG architecture, characteristics and quality attributes both at bench scale (micrograms to milligrams) and at a mini-bioreactor scale (milligrams to grams) that is designed to model large-scale manufacturing (kilograms). Starting from good-quality purified proteins, exchange efficiencies of ≥95% can routinely be obtained within 2-3 d (including quality control).


Subject(s)
Antibodies, Bispecific/metabolism , Immunoglobulin G/metabolism , Protein Engineering/methods , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/genetics , Bioreactors , Genetic Vectors/genetics , Genetic Vectors/metabolism , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/genetics , Mass Spectrometry/methods , Oxidation-Reduction , Point Mutation , Protein Engineering/instrumentation , Quality Control
17.
MAbs ; 6(4): 812-9, 2014.
Article in English | MEDLINE | ID: mdl-24784437

ABSTRACT

Open innovation is the new buzz, with initiatives popping up left and right. Here, we give a personal perspective on a very successful, knowledge-driven innovation initiated in an academia- industry alliance, which culminated in technology platforms that enable the generation of therapeutic antibodies with novel properties. To start, we provide a general background on open innovation in the drug development field.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Drug Approval , Drug Design , Animals , Humans
18.
MAbs ; 5(6): 962-73, 2013.
Article in English | MEDLINE | ID: mdl-23995617

ABSTRACT

The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of>95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques.


Subject(s)
Antibodies, Bispecific/biosynthesis , Protein Engineering , Animals , Antibodies, Bispecific/genetics , Bioreactors , CHO Cells , Cricetinae , Cricetulus , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Point Mutation , Protein Stability , Spectrometry, Mass, Electrospray Ionization
19.
Proc Natl Acad Sci U S A ; 110(13): 5145-50, 2013 Mar 26.
Article in English | MEDLINE | ID: mdl-23479652

ABSTRACT

The promise of bispecific antibodies (bsAbs) to yield more effective therapeutics is well recognized; however, the generation of bsAbs in a practical and cost-effective manner has been a formidable challenge. Here we present a technology for the efficient generation of bsAbs with normal IgG structures that is amenable to both antibody drug discovery and development. The process involves separate expression of two parental antibodies, each containing single matched point mutations in the CH3 domains. The parental antibodies are mixed and subjected to controlled reducing conditions in vitro that separate the antibodies into HL half-molecules and allow reassembly and reoxidation to form highly pure bsAbs. The technology is compatible with standard large-scale antibody manufacturing and ensures bsAbs with Fc-mediated effector functions and in vivo stability typical of IgG1 antibodies. Proof-of-concept studies with HER2×CD3 (T-cell recruitment) and HER2×HER2 (dual epitope targeting) bsAbs demonstrate superior in vivo activity compared with parental antibody pairs.


Subject(s)
Antibodies, Bispecific/biosynthesis , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fc Fragments/biosynthesis , Immunoglobulin G/biosynthesis , Animals , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/genetics , CHO Cells , Cricetinae , Cricetulus , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/chemistry , Immunoglobulin G/genetics , Jurkat Cells , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
20.
MAbs ; 5(2): 219-28, 2013.
Article in English | MEDLINE | ID: mdl-23406897

ABSTRACT

Antibody engineering is increasingly being used to influence the properties of monoclonal antibodies to improve their biotherapeutic potential. One important aspect of this is the modulation of glycosylation as a strategy to improve efficacy. Here, we describe mutations of Y407 in the CH3 domain of IgG1 and IgG4 that significantly increase sialylation, galactosylation, and branching of the N-linked glycans in the CH2 domain. These mutations also promote the formation of monomeric assemblies (one heavy-light chain pair). Hydrogen-deuterium exchange mass spectrometry was used to probe conformational changes in IgG1-Y407E, revealing, as expected, a more exposed CH3-CH3 dimerization interface. Additionally, allosteric structural effects in the CH2 domain and in the CH2-CH3 interface were identified, providing a possible explanation for the dramatic change in glycosylation. Thus, the mutation of Y407 in the CH3 domain remarkably affects both antibody conformation and glycosylation, which not only alters our understanding of antibody structure, but also reveals possibilities for obtaining recombinant IgG with glycosylation tailored for clinical applications.


Subject(s)
Antibodies, Monoclonal/genetics , Immunoglobulin G/genetics , Mutation , Protein Engineering/methods , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Deuterium Exchange Measurement , Glycosylation , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Mass Spectrometry , Models, Molecular , N-Acetylneuraminic Acid , Protein Conformation , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Structure-Activity Relationship
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