Stefanie Derer

G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation.

Molecular danger signals attract neutrophilic granulocytes (polymorphonuclear leukocytes (PMNs)) to sites of infection. The G protein-coupled receptor (GPR) 43 recognizes propionate and butyrate and is abundantly expressed on PMNs. The functional role of GPR43 activation for in vivo orchestration of immune response is unclear. We examined dextrane sodium sulfate (DSS)-induced acute and chronic intestinal inflammatory response in wild-type and Gpr43-deficient mice. The severity of colonic inflammation was assessed by clinical signs, histological scoring, and cytokine production. Chemotaxis of wild-type and Gpr43-deficient PMNs was assessed through transwell cell chemotactic assay. A reduced invasion of PMNs and increased mortality due to septic complications were observed in acute DSS colitis. In chronic DSS colitis, Gpr43−/− animals showed diminished PMN intestinal migration, but protection against inflammatory tissue destruction. No significant difference in PMN migration and cytokine secretion was detected in a sterile inflammatory model. Ex vivo experiments show that GPR43-induced migration is dependent on activation of the protein kinase p38α, and that this signal acts in cooperation with the chemotactic cytokine keratinocyte chemoattractant. Interestingly, shedding of L-selectin in response to propionate and butyrate was compromised in Gpr43−/− mice. These results indicate a critical role for GPR43-mediated recruitment of PMNs in containing intestinal bacterial translocation, yet also emphasize the bipotential role of PMNs in mediating tissue destruction in chronic intestinal inflammation.

Human IgG2 Antibodies against Epidermal Growth Factor Receptor Effectively Trigger Antibody-Dependent Cellular Cytotoxicity but, in Contrast to IgG1, Only by Cells of Myeloid Lineage

Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcγRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcγRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcγRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R–directed immunotherapy.

Recombinant Dimeric IgA Antibodies against the Epidermal Growth Factor Receptor Mediate Effective Tumor Cell Killing

Dimeric IgA Abs contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. In this article, we describe the production, purification, and functional evaluation of recombinant dimeric IgA against the epidermal growth factor receptor. Human joining chain-containing IgA was produced by nonadherent Chinese hamster ovarian (CHO)-K1 cells under serum-free conditions. Purification by anti-human κ and anti–His-tag affinity, as well as size exclusion chromatography, resulted in a homogenous preparation of highly pure IgA dimers. Functional studies demonstrated dimeric IgA to be at least as effective as monomeric IgA in triggering Ab-dependent cellular cytotoxicity by isolated monocytes or polymorphonuclear cell and in human whole-blood assays. Importantly, dimeric IgA was more effective in F(ab)-mediated killing mechanisms, such as inhibition of ligand binding, receptor downmodulation, and growth inhibition. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric Ig receptor through an epithelial cell monolayer. Together, these studies demonstrate that recombinant dimeric IgA Abs recruit a distinct repertoire of effector functions compared with monomeric IgA or IgG1 Abs.

Fc‐engineered EGF‐R antibodies mediate improved antibody‐dependent cellular cytotoxicity (ADCC) against KRAS‐mutated tumor cells

Oncogenic mutations of the KRAS gene have emerged as a common mechanism of resistance against epidermal growth factor receptor (EGF‐R)‐directed tumor therapy. Mutated KRAS leads to ligand‐independent activation of signaling pathways downstream of EGF‐R. Thereby, direct effector mechanisms of EGF‐R antibodies, such as blockade of ligand binding and inhibition of signaling, are bypassed. Thus, a humanized variant of the approved EGF‐R antibody Cetuximab inhibited growth of wild‐type KRAS‐expressing A431 cells, but did not inhibit KRAS‐mutated A549 tumor cells. We then investigated whether killing of tumor cells harboring mutated KRAS can be improved by enhancing antibody‐dependent cellular cytotoxicity (ADCC). Protein‐ and glyco‐engineering of antibodies’ Fc region are established technologies to enhance ADCC by increasing antibodies’ affinity to activating Fcγ receptors. Thus, EGF‐R antibody variants with increased affinity for the natural killer (NK) cell‐expressed FcγRIIIa (CD16) were generated and analyzed. These variants triggered significantly enhanced mononuclear cell (MNC)‐mediated killing of KRAS‐mutated tumor cells compared to wild‐type antibodies. Additionally, cells transfected with mutated KRAS were killed as effectively by ADCC as vector‐transfected control cells. Together, these data demonstrate that KRAS mutations are not sufficient to render tumor cells resistant to ADCC. Consequently Fc‐engineered EGF‐R antibodies may prove effective against KRAS‐mutated tumors, which are not susceptible to signaling inhibition by EGF‐R antibodies.

Impact of Epidermal Growth Factor Receptor (EGFR) Cell Surface Expression Levels on Effector Mechanisms of EGFR Antibodies

The epidermal growth factor receptor (EGFR) is a widely expressed Ag that is successfully targeted in tumor patients by mAbs or tyrosine kinase inhibitors. A clinical study in non-small cell lung cancer patients demonstrated a positive correlation between EGFR expression levels and the therapeutic efficacy of the EGFR mAb cetuximab. However, the impact of EGFR expression on the different mechanisms of action (MoAs) triggered by the EGFR mAb has not been defined. In this study, BHK-21 cells were stably transfected to express different EGFR levels, which were quantified by immunofluorescence and immunohistochemistry and compared with EGFR levels of clinical non-small cell lung cancer samples. These cells were used to systematically investigate the impact of target Ag expression levels on Fab- or Fc-mediated MoAs of EGFR mAb. A negative correlation between EGFR levels and potency of Fab-mediated MoA was observed. Interestingly, Ab-dependent cell-mediated cytotoxicity (ADCC) by NK cells, monocytes, or polymorphonuclear cells as well as complement-dependent cytotoxicity positively correlated with the number of EGFR molecules. In comparison with ADCC by mononuclear cells, polymorphonuclear cell-mediated ADCC and complement-dependent cytotoxicity required higher EGFR expression levels and higher mAb concentrations to trigger significant tumor cell killing. This correlation between EGFR expression levels and Fc-mediated MoA was confirmed in an independent panel of human tumor cell lines carrying diverse genetic alterations. Furthermore, RNA interference-induced knockdown experiments reinforced the impact of EGFR expression on tumor cell killing by EGFR mAb. In conclusion, these results suggest that EGFR expression levels may determine distinct patterns of MoAs that contribute to the therapeutic efficacy of EGFR mAb.

mTNF reverse signalling induced by TNFα antagonists involves a GDF-1 dependent pathway: implications for Crohn's disease

Objective Mechanisms of action (MoA) of anti-tumour necrosis factor α (TNFα) therapies in Crohns disease (CD) may critically involve induction of immune cell apoptosis via membrane-bound TNFα (mTNFα) binding. Certolizumab pegol (CZP), which is effective in induction and maintenance of remission in CD lacks the ability to induce apoptosis. The aim of this study was to analyse transcriptomal responses of reverse signalling induced by the TNFα binding agents infliximab (IFX) and CZP in myelomonocytic cells. Design Induction of transcriptional patterns upon anti-TNFα stimulation was assessed using oligonucleotide microarrays. mRNA expression of GDF-1/ LASS1, which was identified as a shared target, was studied in inflammatory bowel disease by real-time PCR, while signalling pathways induced by growth and differentiation factor 1 (GDF-1) were investigated using western blots and ELISA. Results IFX and CZP induced a common signature of 20 transcripts that could be categorised into control of cell cycle, transcription activation and pre-mRNA processing. We selected GDF-1/LASS1 for functional follow-up, which was found to be upregulated in inflamed CD tissues. We show that downregulation of GDF-1/LASS1 depends on autocrine release of transforming growth factor β after mTNFα ligation. We demonstrate that GDF-1 itself acts as a novel proinflammatory factor via induction of interleukin 6 and signal transducer and activator of transcription 3 and is downregulated after IFX treatment. Conclusion Commonalities in the MoA of IFX and CZP comprise modulation of non-apoptotic pathways through downregulation of proinflammatory GDF-1. Further characterisation of the molecular role of GDF-1 in complex inflammatory processes in vivo is warranted to decide whether this proinflammatory molecule is a promising therapeutic target in patients with CD.

Systematic expression profiling of innate immune genes defines a complex pattern of immunosenescence in peripheral and intestinal leukocytes

Immunosenescence is characterized by a quantitative decline of adequate immune responses, which renders the elderly individual particularly susceptible to bacterial, viral and fungal pathogens. Whereas changes of the aging adaptive immune system (for example, reduced immunoglobulin secretion) have been extensively characterized, alterations of the innate immune system are still poorly understood. The aim of the present study was to systematically examine mRNA expression levels of innate immune genes and proinflammatory cytokines in peripheral and intestinal leukocytes of subjects of different ages. In both, whole blood samples and in colonic biopsies most of the Toll-like receptors (TLRs) and nucleotide-binding and oligomerization domain-like receptors (NLRs) transcript levels were significantly downregulated in elderly subjects (90–99 years). Older individuals, when compared to the younger, exhibited an increased expression and/or secretion of proinflammatory cytokines by peripheral and intestinal leukocytes as well as an increased level of nuclear factor-κB activation in colonic biopsies. The observed downregulation of TLRs and NLRs during the aging process may contribute to the lack of effective recognition of invading pathogens or the commensal flora. This effect results in aberrant secondary immune cell activation and could significantly contribute to morbidity and mortality at advanced age.

Increasing FcγRIIa affinity of an FcγRIII-optimized anti-EGFR antibody restores neutrophil-mediated cytotoxicity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested as an essential mechanism for the in vivo activity of cetuximab, an epidermal growth factor receptor (EGFR)-targeting therapeutic antibody. Thus, enhancing the affinity of human IgG1 antibodies to natural killer (NK) cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc portion has been demonstrated to improve NK cell-mediated ADCC and to represent a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) effector cells express the highly homologous FcγRIIIb isoform, which is described to be ineffective in triggering ADCC. Here, non-fucosylated or protein-engineered anti-EGFR antibodies with optimized FcγRIIIa affinities demonstrated the expected benefit in NK cell-mediated ADCC, but did not mediate ADCC by PMN, which could be restored by FcγRIIIb blockade. Furthermore, eosinophils and PMN from paroxysmal nocturnal hemoglobinuria patients that expressed no or low levels of FcγRIIIb mediated effective ADCC with FcγRIII-optimized anti-EGFR antibody. Additional experiments with double FcγRIIa/FcγRIII-optimized constructs demonstrated enhanced PMN-mediated ADCC compared with single FcγRIII-optimized antibody. In conclusion, our data demonstrate that FcγRIIIb engagement impairs PMN-mediated ADCC activity of FcγRIII-optimized anti-EGFR antibodies, while further optimization of FcγRIIa binding significantly restores PMN recruitment.

Characterization of a mutated IgA2 antibody of the m(1) allotype against the epidermal growth factor receptor for the recruitment of monocytes and macrophages.

Background: IgA constitutes a promising antibody isotype, which requires optimization before immunotherapeutic application. Results: P221R-mutated and wild type IgA2m(1) antibodies were similarly effective in killing tumor cells and in recruiting myeloid effector cells. Conclusion: Improved IgA antibodies constitute promising next generation antibodies for tumor therapy. Significance: These studies support the clinical development of therapeutic IgA antibodies. IgA antibodies constitute an important part of the mucosal immune system, but their immunotherapeutic potential remains rather unexplored, in part due to biotechnological issues. For example, the IgA2m(1) allotype carries an unusual heavy and light chain pairing, which may confer production and stability concerns. Here, we report the generation and the biochemical and functional characterization of a P221R-mutated IgA2m(1) antibody against the epidermal growth factor receptor (EGFR). Compared with wild type, the mutated antibody demonstrated heavy chains covalently linked to light chains in monomeric as well as in joining (J)-chain containing dimeric IgA. Functional studies with wild type and mutated IgA2m(1) revealed similar binding to EGFR and direct effector functions such as EGFR down-modulation and growth inhibition. Furthermore, both IgA molecules triggered similar levels of indirect tumor cell killing such as antibody-dependent cell-mediated cytotoxicity (ADCC) by isolated monocytes, activated polymorphonuclear cells, and human whole blood. Interestingly, the dimeric IgA antibodies demonstrated higher efficiency in direct as well as in indirect effector mechanisms compared with their respective monomeric forms. Both wild type and mutated antibody triggered effective FcαRI-mediated tumor cell killing by macrophages already at low effector to target cell ratios. Interestingly, also polarized macrophages mediated significant IgA2-mediated ADCC. M2 macrophages, which have been described as promoting tumor growth and progression, may convert to ADCC-mediating effector cells in the presence of EGFR-directed antibodies. In conclusion, these results provide further insight into the immunotherapeutic potential of recombinant IgA antibodies for tumor immunotherapy and suggest macrophages as an additional effector cell population.

Complement‐mediated tumor‐specific cell lysis by antibody combinations targeting epidermal growth factor receptor (EGFR) and its variant III (EGFRvIII)

Monoclonal antibodies (mAb) against variant III of epidermal growth factor receptor (EGFRvIII) hold promise for improving tumor selectivity of EGFR‐targeted therapy. Here, we compared Fc‐mediated effector functions of three mAb against EGFRvIII (MR1‐1, ch806, 13.1.2) with those of zalutumumab, a high affinity EGFR mAb in advanced clinical trials. MR1‐1 and ch806 demonstrated preferential and 13.1.2 exclusive binding to EGFRvIII, in contrast to zalutumumab, which bound both wild‐type and EGFRvIII. All four human IgG1κ mAb mediated antibody‐dependent cellular cytotoxicity (ADCC) of EGFRvIII‐expressing cells with mononuclear cells and isolated monocytes, while only zalutumumab in addition triggered ADCC by polymorphonuclear cells. Interestingly, combinations of zalutumumab and EGFRvIII mAb specifically mediated complement‐dependent cytotoxicity (CDC) of EGFRvIII‐transfected but not wild‐type cells. Moreover, EGFRvIII‐specific CDC was significantly enhanced when zalutumumab was combined with a Fc‐engineered variant of MR1‐1 (K326A/E333A). These observations confirm the immunotherapeutic potential of antibody combinations against EGFR, and demonstrate that tumor selectivity can be improved by combining therapeutic EGFR mAb with an antibody against EGFRvIII. (Cancer Sci 2011; 102: 1761–1768)