Isotype selection for antibody-based cancer therapy

The clinical application of monoclonal antibodies (mAbs) has revolutionized the field of cancer therapy, as it has enabled the successful treatment of previously untreatable types of cancer. Different mechanisms play a role in the anti‐tumour effect of mAbs. These include blocking of tumour‐specific growth factor receptors or of immune modulatory molecules as well as complement and cell‐mediated tumour cell lysis. Thus, for many mAbs, Fc‐mediated effector functions critically contribute to the efficacy of treatment. As immunoglobulin (Ig) isotypes differ in their ability to bind to Fc receptors on immune cells as well as in their ability to activate complement, they differ in the immune responses they activate. Therefore, the choice of antibody isotype for therapeutic mAbs is dictated by its intended mechanism of action. Considering that clinical efficacy of many mAbs is currently achieved only in subsets of patients, optimal isotype selection and Fc optimization during antibody development may represent an important step towards improved patient outcome. Here, we discuss the current knowledge of the therapeutic effector functions of different isotypes and Fc‐engineering strategies to improve mAbs application

FcγRIII and FcγRIV are indispensable for acute glomerular inflammation induced by switch variant monoclonal antibodies

The relative ability of IgG subclasses to cause acute inflammation, and the roles of specific effector mechanisms in this process is not clear. We explored this in an in vivo model of glomerular inflammation in the mouse. TNP was planted on the glomerular basement membrane after conjugation to nephrotoxic antibody. The relative nephritogenicity of anti-TNP switch-variant monoclonal antibodies was then explored and shown to be IgG2a>IgG2b, with no disease caused by IgG1. Using knockout mice, we showed that FcγRIII was necessary for both neutrophil influx and glomerular damage induced by IgG2a and IgG2b. Surprisingly IgG1 did not cause disease although it binds to FcγRIII. Using blocking antibodies, we showed that this was explained by an additional requirement for FcγRIV which does not bind to IgG1. IgG2a or IgG2b induced neutrophil influx was not affected by deficiency of either FcγRI or C3. Bone marrow chimeras were constructed to test the effect of combined deficiency of FcγRI and C3, and there was no effect on IgG2a or IgG2b mediated neutrophil influx. However, IgG2b-induced albuminuria and thrombosis was reduced in C3 deficient mice, showing an additional role for complement in IgG2b-mediated glomerular damage. The results show that IgG2a and IgG2b are the pathogenic subclasses in acute neutrophil-mediated glomerular inflammation, with an indispensible role for both FcγRIII and FcγRIV. In addition complement contributes to IgG2b induced glomerular injury

Complement Activation Selectively Potentiates the Pathogenicity of the IgG2b and IgG3 Isotypes of a High Affinity Anti-Erythrocyte Autoantibody

By generating four IgG isotype-switch variants of the high affinity 34–3C anti-erythrocyte autoantibody, and comparing them to the IgG variants of the low affinity 4C8 anti-erythrocyte autoantibody that we have previously studied, we evaluated in this study how high affinity binding to erythrocytes influences the pathogenicity of each IgG isotype in relation to the respective contributions of Fcγ receptor (FcγR) and complement. The 34–3C autoantibody opsonizing extensively circulating erythrocytes efficiently activated complement in vivo (IgG2a = IgG2b > IgG3), except for the IgG1 isotype, while the 4C8 IgG autoantibody failed to activate complement. The pathogenicity of the 34–3C autoantibody of IgG2b and IgG3 isotypes was dramatically higher (>200-fold) than that of the corresponding isotypes of the 4C8 antibody. This enhanced activity was highly (IgG2b) or totally (IgG3) dependent on complement. In contrast, erythrocyte-binding affinities only played a minor role in in vivo hemolytic activities of the IgG1 and IgG2a isotypes of 34–3C and 4C8 antibodies, where complement was not or only partially involved, respectively. The remarkably different capacities of four different IgG isotypes of low and high affinity anti-erythrocyte autoantibodies to activate FcγR-bearing effector cells and complement in vivo demonstrate the role of autoantibody affinity maturation and of IgG isotype switching in autoantibody-mediated pathology

A Critical Role for Syk Protein Tyrosine Kinase in Fc Receptor-Mediated Antigen Presentation and Induction of Dendritic Cell Maturation

AbstractDendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation

Immune Antibodies and Helminth Products Drive CXCR2-Dependent Macrophage-Myofibroblast Crosstalk to Promote Intestinal Repair

Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing

A Dual-Color Bioluminescence Reporter Mouse for Simultaneous in vivo Imaging of T Cell Localization and Function

Non-invasive imaging technologies to visualize the location and functionality of T cells are of great value in immunology. Here, we describe the design and generation of a transgenic mouse in which all T cells constitutively express green-emitting click-beetle luciferase (CBG99) while expression of the red-emitting firefly luciferase (PpyRE9) is induced by Nuclear Factor of Activated T cells (NFAT) such as during T cell activation, which allows multicolor bioluminescence imaging of T cell location and function. This dual-luciferase mouse, which we named TbiLuc, showed high constitutive luciferase expression in lymphoid organs such as lymph nodes and the spleen. Ex vivo purified CD8+ and CD4+ T cells both constitutively expressed luciferase, whereas B cells showed no detectable signal. We cross-bred TbiLuc mice to T cell receptor-transgenic OT-I mice to obtain luciferase-expressing naïve CD8+ T cells with defined antigen-specificity. TbiLuc*OT-I T cells showed a fully antigen-specific induction of the T cell activation-dependent luciferase. In vaccinated mice, we visualized T cell localization and activation in vaccine-draining lymph nodes with high sensitivity using two distinct luciferase substrates, D-luciferin and CycLuc1, of which the latter specifically reacts with the PpyRE9 enzyme. This dual-luciferase T cell reporter mouse can be applied in many experimental models studying the location and functional state of T cells

A Restricted Role for FcγR in the Regulation of Adaptive Immunity.

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV-/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV-/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity

Markedly Different Pathogenicity of Four Immunoglobulin G Isotype-Switch Variants of an Antierythrocyte Autoantibody Is Based on Their Capacity to Interact in Vivo with the Low-Affinity Fcγ Receptor III

Using three different Fcγ receptor (FcγR)-deficient mouse strains, we examined the induction of autoimmune hemolytic anemia by each of the four immunoglobulin (Ig)G isotype-switch variants of a 4C8 IgM antierythrocyte autoantibody and its relation to the contributions of the two FcγR, FcγRI, and FcγRIII, operative in the phagocytosis of opsonized particles. We found that the four IgG isotypes of this antibody displayed striking differences in pathogenicity, which were related to their respective capacity to interact in vivo with the two phagocytic FcγRs, defined as follows: IgG2a > IgG2b > IgG3/IgG1 for FcγRI, and IgG2a > IgG1 > IgG2b > IgG3 for FcγRIII. Accordingly, the IgG2a autoantibody exhibited the highest pathogenicity, ∼20–100-fold more potent than its IgG1 and IgG2b variants, respectively, while the IgG3 variant, which displays little interaction with these FcγRs, was not pathogenic at all. An unexpected critical role of the low-affinity FcγRIII was revealed by the use of two different IgG2a anti–red blood cell autoantibodies, which displayed a striking preferential utilization of FcγRIII, compared with the high-affinity FcγRI. This demonstration of the respective roles in vivo of four different IgG isotypes, and of two phagocytic FcγRs, in autoimmune hemolytic anemia highlights the major importance of the regulation of IgG isotype responses in autoantibody-mediated pathology and humoral immunity

Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Th17 responses to fungal infection

Innate immune cells detect pathogens via pattern recognition receptors (PRRs), which signal for initiation of immune responses to infection. Studies with Dectin-1, a PRR for fungi, have defined a novel innate signaling pathway involving Syk kinase and the adaptor CARD9, which is critical for inducing Th17 responses to fungal infection. We show that another C-type lectin, Dectin-2, also signals via Syk and CARD9, and contributes to dendritic cell (DC) activation by fungal particles. Unlike Dectin-1, Dectin-2 couples to Syk indirectly, through association with the FcRγ chain. In a model of Candida albicans infection, blockade of Dectin-2 did not affect innate immune resistance but abrogated Candida-specific T cell production of IL-17 and, in combination with the absence of Dectin-1, decreased Th1 responses to the organism. Thus, Dectin-2 constitutes a major fungal PRR that can couple to the Syk–CARD9 innate signaling pathway to activate DCs and regulate adaptive immune responses to fungal infection