A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to ...targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.
Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies ...activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG subclasses. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.
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.
IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic ...surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.
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.
IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell ...killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.
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.
Activation of membrane receptors through clustering is a common mechanism found in various biological systems. Spatial proximity of receptors may be transduced across the membrane to initiate ...signaling pathways or alternatively be recognized by peripheral proteins or immune cells to trigger external effector functions. Here we show how specific immunoglobulin G (IgG) binding induces clustering of monomeric target molecules in lipid membranes through Fc–Fc interactions. We visualize and characterize the dynamic IgG oligomerization process and the molecular interactions involved using high-speed atomic force microscopy, single-molecule force spectroscopy, and quartz crystal microbalance experiments. We found that the Fc–Fc interaction strength is precisely tuned to be weak enough to prevent IgG oligomerization in solution at physiological titers, but enabling IgG oligomerization when Fabs additionally bind to their cognate surface epitopes, a mechanism that ultimately targets IgG-mediated effector functions such as classical complement activation to antigenic membranes.
Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates ...and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 IgG1) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with "Fc-unmodified" chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 μg intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) "complement-inactive" Fc modifications that engaged Fc gamma receptor (FcγR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q-/- mice, when C5 function was blocked, or in C9-/- mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics.
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