Janine Schuurman

Anti-Inflammatory Activity of Human IgG4 Antibodies by Dynamic Fab Arm Exchange

Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.

Immunoglobulin G4: an odd antibody

Despite its well‐known association with IgE‐mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half‐molecules (i.e. a heavy and attached light‐chain) exchange. This process, also referred to as ‘Fab‐arm exchange’, results usually in asymmetric antibodies with two different antigen‐combining sites. While these antibodies are hetero‐ bivalent, they will behave as monovalent antibodies in most situations. Another aspect of IgG4, still poorly understood, is its tendency to mimic IgG rheumatoid factor (RF) activity by interacting with IgG on a solid support. In contrast to conventional RF, which binds via its variable domains, the activity of IgG4 is located in its constant domains. This is potentially a source of false positives in IgG4 antibody assay results. Because regulation of IgG4 production is dependent on help by T‐helper type 2 (Th2) cells, the IgG4 response is largely restricted to non‐microbial antigens. This Th2‐dependency associates the IgG4 and IgE responses. Another typical feature in the immune regulation of IgG4 is its tendency to appear only after prolonged immunization. In the context of IgE‐mediated allergy, the appearance of IgG4 antibodies is usually associated with a decrease in symptoms. This is likely to be due, at least in part, to an allergen‐blocking effect at the mast cell level and/or at the level of the antigen‐presenting cell (preventing IgE‐facilitated activation of T cells). In addition, the favourable association reflects the enhanced production of IL‐10 and other anti‐inflammatory cytokines, which drive the production of IgG4. While in general, IgG4 is being associated with non‐activating characteristics, in some situations IgG4 antibodies have an association with pathology. Two striking examples are pemphigoid diseases and sclerosing diseases such as autoimmune pancreatitis. The mechanistic basis for the association of IgG4 with these diseases is still enigmatic. However, the association with sclerosing diseases may reflect an excessive production of anti‐inflammatory cytokines triggering an overwhelming expansion of IgG4‐producing plasma cells. The bottom line for allergy diagnosis: IgG4 by itself is unlikely to be a cause of allergic symptoms. In general, the presence of allergen‐specific IgG4 indicates that anti‐inflammatory, tolerance‐inducing mechanisms have been activated. The existence of the IgG4 subclass, its up‐regulation by anti‐inflammatory factors and its own anti‐inflammatory characteristics may help the immune system to dampen inappropriate inflammatory reactions.

Complement Is Activated by IgG Hexamers Assembled at the Cell Surface

Hexing Complement Complement activation is an immediate and potent immune defense mechanism, but how immunoglobulin G (IgG) antibodies activate complement at the molecular level is poorly understood. Using high-resolution crystallography, Diebolder et al. (p. 1260) show that human IgGs form hexameric structures by interacting with neighboring IgG molecules, and the complex then activates complement. Thus, IgG molecules and the complement system can coexist in the blood because complement activation will only be triggered after IgG senses a surface antigen and starts to aggregate. Hexameric platforms of antibodies on the cell surface trigger the complement cascade. 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.

Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo

Two humanized IgG4 antibodies, natalizumab and gemtuzumab, are approved for human use, and several others, like TGN1412, are or have been in clinical development. Although IgG4 antibodies can dynamically exchange half-molecules, Fab-arm exchange with therapeutic antibodies has not been demonstrated in humans. Here, we show that natalizumab exchanges Fab arms with endogenous human IgG4 in natalizumab-treated individuals. Gemtuzumab, in contrast, contains an IgG4 core-hinge mutation that blocks Fab-arm exchange to undetectable levels both in vitro and in a mouse model. The ability of IgG4 therapeutics to recombine with endogenous IgG4 may affect their pharmacokinetics and pharmacodynamics. Although pharmacokinetic modeling lessens concerns about undesired cross-linking under normal conditions, unpredictability remains and mutations that completely prevent Fab-arm exchange in vivo should be considered when designing therapeutic IgG4 antibodies.

Dual Mode of Action of a Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Cancer Therapy

Epidermal growth factor receptor (EGF-R) overexpression is common in a large number of solid tumors and represents a negative prognostic indicator. Overexpression of EGF-R is strongly tumor associated, and this tyrosine kinase type receptor is considered an attractive target for Ab therapy. In this study, we describe the evaluation of mAb 2F8, a high avidity human mAb (IgG1κ) directed against EGF-R, developed using human Ig transgenic mice. mAb 2F8 effectively blocked binding of EGF and TGF-α to the EGF-R. At saturating concentrations, 2F8 completely blocked EGF-R signaling and inhibited the in vitro proliferation of EGF-R-overexpressing A431 cells. At much lower concentrations, associated with low receptor occupancy, 2F8 induced efficient Ab-dependent cell-mediated cytotoxicity (ADCC) in vitro. In vivo studies showed potent antitumor effects in models with A431 tumor xenografts in athymic mice. Ex vivo analysis of the EGF-R status in tumor xenografts in 2F8-treated mice revealed that there are two therapeutic mechanisms. First, blocking of EGF-R signaling, which is most effective at complete receptor saturation and therefore requires a relatively high Ab dose. Second, at very low 2F8 receptor occupancy, we observed potent antitumor effects in mice, which are likely based on the engagement of immune effector mechanisms, in particular ADCC. Taken together, our findings indicate that ADCC represents an important effector mechanism of this Ab, which is effective at relatively low dose.

Resolution of psoriasis upon blockade of IL-15 biological activity in a xenograft mouse model.

Psoriasis is a chronic inflammatory disease of the skin characterized by epidermal hyperplasia, dermal angiogenesis, infiltration of activated T cells, and increased cytokine levels. One of these cytokines, IL-15, triggers inflammatory cell recruitment, angiogenesis, and production of other inflammatory cytokines, including IFN-gamma, TNF-alpha, and IL-17, which are all upregulated in psoriatic lesions. To investigate the role of IL-15 in psoriasis, we generated mAbs using human immunoglobulin-transgenic mice. One of the IL-15-specific antibodies we generated, 146B7, did not compete with IL-15 for binding to its receptor but potently interfered with the assembly of the IL-15 receptor alpha, beta, gamma complex. This antibody effectively blocked IL-15-induced T cell proliferation and monocyte TNF-alpha release in vitro. In a human psoriasis xenograft model, antibody 146B7 reduced the severity of psoriasis, as measured by epidermal thickness, grade of parakeratosis, and numbers of inflammatory cells and cycling keratinocytes. These results obtained with this IL-15-specific mAb support an important role for IL-15 in the pathogenesis of psoriasis.

Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

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.

Effect of Target Dynamics on Pharmacokinetics of a Novel Therapeutic Antibody against the Epidermal Growth Factor Receptor: Implications for the Mechanisms of Action

The epidermal growth factor receptor (EGFR) is overexpressed on many solid tumors and represents an attractive target for antibody therapy. Here, we describe the effect of receptor-mediated antibody internalization on the pharmacokinetics and dose-effect relationship of a therapeutic monoclonal antibody (mAb) against EGFR (2F8). This mAb was previously found therapeutically active in mouse tumor models by two dose-dependent mechanisms of action: blockade of ligand binding and induction of antibody-dependent cell-mediated cytotoxicity. In vitro studies showed 2F8 to be rapidly internalized by EGFR-overexpressing cells. In vivo, accelerated 2F8 clearance was observed in cynomolgus monkeys at low doses but not at high doses. This enhanced clearance seemed to be receptor dependent and was included in a pharmacokinetic model designed to explain its nonlinearity. Receptor-mediated clearance was also found to affect in situ antibody concentrations in tumor tissue. Ex vivo analyses of xenograft tumors of 2F8-treated nude mice revealed that relatively high antibody plasma concentrations were required for maximum EGFR saturation in high-EGFR-expressing human A431 tumors, in contrast to lower-EGFR-expressing human xenograft tumors. In summary, receptor-mediated antibody internalization and degradation provides a saturable route of clearance that significantly affects pharmacokinetics, particularly at low antibody doses. EGFR saturation in normal tissues does not predict saturation in tumor tissue as local antibody concentrations in EGFR-overexpressing tumors may be more rapidly reduced by antibody internalization. Consequently, antibody saturation of the receptor may be affected, thereby affecting the local mechanism of action.

Production of a mouse/human chimeric IgE monoclonal antibody to the house dust mite allergen Der p 2 and its use for the absolute quantification of allergen-specific IgE☆☆☆★★★

A chimeric human IgE monoclonal antibody was developed against the house dust mite allergen Der p 2. This chimeric antibody (hIgE-Dp2A) was composed of the heavy-chain variable domains and light chains of the original murine monoclonal antibody retaining its binding characteristics, whereas the heavy-chain constant domains were exchanged with the human IgE heavy chain. The chimeric IgE expression level was IgE 600 IU/ml (1 IU = 2.4 ng/ml). The binding of the chimeric hIgE-Dp2A to mite extract was indistinguishable from that of the original mouse monoclonal antibody. Parallel dose-response curves were found when the binding of hIgE-Dp2A to mite extract and anti-IgE coupled to sepharose were compared. Binding levels were not identical; however, hIgE-Dp2A bound significantly better to the mite-extract sepharose. This result indicates that the commonly used anti-IgE on solid phase calibration systems may lead to an overestimation of the amount of allergen-specific IgE present in the serum sample. The less efficient binding of the detector anti-IgE in case of the anti-IgE sepharose is likely to be because of the occupation of epitopes of the IgE by the sepharose-bound anti-IgE. Dose-response curves of serial dilutions of patient samples were parallel with the hIgE-Dp2A dose-response curve, which indicates that hIgE-Dp2A behaves like natural IgE antibodies in binding to allergen coupled to solid phase. This antibody is well suited for use as a reference reagent in the RAST and enables the expression of the amount of allergen-specific IgE present in a patient sample in absolute amounts.

Human IgG4 Binds to IgG4 and Conformationally Altered IgG1 via Fc-Fc Interactions

The Fc fragment of IgG4 can interact with the Fc fragment of another IgG molecule. This interaction is a confounding factor when measuring IgG4 rheumatoid factor levels. Recently, we demonstrated that half-molecules of IgG4 can exchange to form a bispecific Ab. We expected these two phenomena to be related and investigated the physicochemical aspects of IgG4 Fc-Fc interactions. We found that IgG4 is >99% monomeric by size-exclusion chromatography; therefore, IgG4 Fc-Fc interactions in the fluid phase (if any) would be short-lived. However, 125I-labeled IgG4 does bind to IgG1 and IgG4 coupled to a solid phase. By contrast, IgG1 does not bind to coupled IgG4. Furthermore, conditions that induce partial unfolding/dissociation of the CH3 domains enhance IgG4 Fc binding, suggesting that Fc binding is primarily CH3 mediated. IgG4 slowly associates with both IgG4 and IgG1 coupled to a biosensor chip. Remarkably, subsequent dissociation was much faster for IgG4 than for IgG1. Moreover, after binding of an IgG4 mAb to Sepharose-coupled Ag, we observed additional binding of IgG4 with irrelevant specificity, whereas similar binding was not observed with Ag-bound IgG1. We propose that the IgG4-IgG4 Fc interaction resembles an intermediate of the Fab-arm (half-molecule) exchange reaction that is stabilized because one of the IgG4 molecules is coupled to a solid phase. By contrast, IgG4 Fc recognizes IgG1 only after a conformational change that renders CH3(IgG1) accessible to an interaction with the CH3(IgG4). Such Fc interactions may enhance Ag binding of IgG4 in vivo.