Jan G. J. van de Winkel

Impaired IgG-dependent anaphylaxis and Arthus reaction in FcγRIII (CD16) deficient mice.

Abstract The family of receptors for IgG (FcγR) plays an essential role in antibody-mediated effector functions of the immune system. However, the specific contribution of each of the FcγR classes to in vivo immune reactions is still unclear. Here, we demonstrate that mice deficient for the ligand-binding α chain of FcγRIII lack NK cell–mediated antibody-dependent cytotoxicity and phagocytosis of IgG1-coated particles by macrophages. Strikingly, these mice lack IgG-mediated mast cell degranulation, are resistant to IgG-dependent passive cutaneous anaphylaxis, and exhibit an impaired Arthus reaction. These results indicate a prominent role for FcγRIII in inflammatory and anaphylactic responses, making this receptor a potential target in immunotherapy.

Clinical significance of IgG Fc receptors and FcγR-directed immunotherapies

Abstract Fc receptors for IgG (FcγRs) can trigger the inflammatory, cytotoxic and hypersensitivity functions of immune effector cells. Activation or deactivation of effector cells via FcγRs can be exploited to develop novel therapies for cancer, infectious diseases and autoimmune disorders. Initial results of clinical trials for several FcγR-directed immunotherapies show the potential promise of this approach.

Binding of Submaximal C1q Promotes Complement-Dependent Cytotoxicity (CDC) of B Cells Opsonized with Anti-CD20 mAbs Ofatumumab (OFA) or Rituximab (RTX): Considerably Higher Levels of CDC Are Induced by OFA than by RTX

The CD20 mAb ofatumumab (OFA) is more effective than rituximab (RTX) in promoting complement-dependent cytotoxicity (CDC) of B cells via the classical pathway (CP) of complement. CP activation is initiated by C1q binding to cell-bound IgG. Therefore, we examined the role of C1q in the dynamics of complement activation and CDC of B cell lines and primary cells from patients with chronic lymphocytic leukemia, reacted with OFA or RTX. C1q binding, complement activation, and colocalization of C1q with cell-bound mAbs were determined by flow cytometry and high-resolution digital imaging. C1q binds avidly to OFA-opsonized Raji and Daudi cells (KD = 12–16 nM) and colocalizes substantially with cell-bound OFA. Cells opsonized with OFA undergo high levels of complement activation and CDC in C1q-depleted serum supplemented with low concentrations of C1q. Under comparable conditions, RTX-opsonized cells bind less C1q; in addition, even when higher concentrations of C1q are used to achieve comparable C1q binding to RTX-opsonized cells, less complement activation and CDC are observed. Greater CDC induced by OFA may occur because C1q is bound in close proximity and with high avidity to OFA, resulting in effective CP activation. Moreover, OFA binds to the small, extracellular CD20 loop, placing the mAb considerably closer to the cell membrane than does RTX. This may facilitate effective capture and concentration of activated complement components closer to the cell membrane, potentially shielding them from inactivation by fluid phase agents and promoting efficient generation of the membrane attack complex.

FUNCTIONAL ASSOCIATION BETWEEN THE HUMAN MYELOID IMMUNOGLOBULIN A FC RECEPTOR (CD89) AND FCR GAMMA CHAIN : MOLECULAR BASIS FOR CD89/FCR GAMMA CHAIN ASSOCIATION

FcR γ chain has previously been shown to interact with the TCR-CD3 complex, the IgE Fc receptor I (FcεRI), and the class I and IIIA IgG receptors (FcγRI and FcγRIIIa). Here, we demonstrate that the Fc receptor γchain associates with FcαR in transfected IIA1.6 B lymphocytes. FcαR could be expressed at the surface of IIA1.6 B cells by itself, but was devoid of signaling capacity. Upon co-expression of FcR γchain, a physical interaction with FcαR could be demonstrated. This association proved crucial for the triggering of both proximal (intracellular calcium increase and tyrosine phosphorylation), as well as distal (IL-2 release), signal transduction responses. We next tested the hypothesis that a positively charged arginine residue (Arg209) within the transmembrane domain of FcαR promotes association with FcR γchain. We therefore constructed FcαR molecules where Arg209 was mutated to either a positively charged histidine, a negatively charged aspartic acid, or an uncharged leucine. A functional association between FcαR and FcR γchain was observed only with a positively charged residue (Arg209 or His209) present within the FcαR transmembrane domain. These data show that transmembrane signal transduction by the FcαR is mediated via FcR γchain, and that FcαR requires a positively charged residue within the transmembrane domain to promote functional association.

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.

Human IgG Fc receptors.

Human IgG receptors constitute a family of glycoprotein complexes consisting of ligand-binding, and associated signaling chains. Three leukocyte classes (Fc gamma RI, II, and III) and one separate endothelial Fc gamma R class (FcRB) are defined which are expressed on hematopoietic and endothelial cells. Upon interaction with IgG, Fc gamma R initiate a plethora of signaling cascades involving receptor signaling motifs, and protein tyrosine kinases and phosphatases. These cascades ultimately culminate in activation or deactivation of effector cells, resulting in initiation or down-modulation of cellular processes. Recent evidence points to a crucial in vivo role of Fc gamma R in both initiation and regulation of inflammatory and cytotoxic responses. These Fc gamma R-mediated immune responses can be exploited to develop novel immunotherapies.

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.

Fcγ receptor IIa polymorphism in caucasian patients with systemic lupus erythematosus: Association with clinical symptoms

OBJECTIVE The class II human leukocyte Fcy receptor for IgG (FcgammaRIIa) occurs in 2 codominantly expressed allelic forms (R131 and H131). Cells expressing IIa-H131 interact much more effectively with complexed IgG2 and IgG3 than do cells with IIa-R131. This might be linked to variability in immune complex handling, and therefore related to disease pathogenesis. The present study examines these possibilities in a cohort of Caucasian patients with systemic lupus erythematosus (SLE). METHODS One hundred eight Caucasian patients were diagnosed with SLE according to the American College of Rheumatology criteria. The SLE patients and 187 Caucasian controls were genotyped for the FcgammaRIIa polymorphism, and associations between FcgammaRIIa genotypes, selected HLA haplotypes, and clinical as well as laboratory features were analyzed. RESULTS No significant skewing of the FcgammaRIIa polymorphism was observed in the SLE cohort. Various clinical and serologic parameters were found more frequently or at a younger age in patients homozygous for the genotype IIa-R/R131 compared with those with the genotype IIa-H/H131. In patients with the genotype IIa-R/R131, significantly higher frequencies of proteinuria, hemolytic anemia, anti-nuclear RNP antibodies, and hypocomplementemia were found. The only clinical symptom observed more frequently in patients homozygous for IIa-H/H131 was livedo. Patients with the IIa-R/R131 genotype were significantly younger at disease onset and had an earlier incidence of arthritis, sicca syndrome, nephritis, lymphadenitis, hematologic abnormalities, immunologic abnormalities, lupus anticoagulant, cryoglobulinemia, and hypocomplementemia. HLA-DR3 was found in 41.7% of SLE patients, but was not associated with clinical symptoms, serologic abnormalities, or the homozygous genotypes of the FcgammaRIIa, although an association with a significantly later onset of SLE was found. CONCLUSION The FcgammaRIIa polymorphism constitutes an additional factor that might influence the clinical manifestations and course of SLE, but does not represent a genetic risk factor for the occurrence of SLE.

In vivo Cytotoxicity of Type I CD20 Antibodies Critically Depends on Fc Receptor ITAM Signaling

Antibody-Fc receptor (FcR) interactions play an important role in the mechanism of action of most therapeutic antibodies against cancer. Effector cell activation through FcR triggering may induce tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC). Reciprocally, FcR cross-linking of antibody may lead to the induction of apoptotic signaling in tumor cells. The relative importance of these bisecting pathways to in vivo antibody activity is unknown. To unravel these roles, we developed a novel mouse model with normal FcR expression but in which FcR signaling was inactivated by mutation of the associated gamma-chain. Transgenic mice showed similar immune complex binding compared with wild-type mice. In contrast, ADCC of cells expressing frequently used cancer targets, such as CD20, epidermal growth factor receptor, Her2, and gp75, was abrogated. Using the therapeutic CD20 antibodies ofatumumab and rituximab, we show that FcR cross-linking of antibody-antigen immune complexes in the absence of gamma-chain signaling is insufficient for their therapeutic activity in vivo. ADCC therefore represents an essential mechanism of action for immunotherapy of lymphoid tumors.