Tanja Schneider-Merck

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.

Effector Mechanisms of Recombinant IgA Antibodies against Epidermal Growth Factor Receptor

IgA is the most abundantly produced Ab isotype in humans, but its potential as immunotherapeutic reagent has hardly been explored. In this study, we describe anti-tumor mechanisms of mouse/human chimeric IgA Abs against the epidermal growth factor receptor (EGF-R). EGF-R Abs of IgG isotype are currently approved for the treatment of colon or head and neck cancers. As expected, the human IgG1, IgA1, and IgA2 variants of the 225 Ab demonstrated similar binding to EGF-R. Furthermore, IgA Abs were as effective as IgG in mediating direct effector mechanisms such as blockade of EGF binding, inhibition of EGF-R phosphorylation, and induction of growth inhibition. None of the three variants induced complement-mediated lysis. Human IgG1 effectively recruited MNC for ADCC, but activated PMN only weakly, whereas both IgA isoforms proved to be effective in triggering neutrophils. Interestingly, the IgA2 isoform was significantly superior to its IgA1 counterpart in recruiting PMN as effector cells. Because neutrophils constitute the most abundant effector cell population in human blood, this enhanced neutrophil recruitment lead to increased killing of EGF-R expressing tumor cells in whole blood assays. This killing was further enhanced when blood from G-CSF-primed donors was compared with healthy donor blood. Together, these data suggest EGF-R Abs of human IgA isotype to bear promise for therapeutic use in cancer.

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.

Physical Interaction and Mutual Transrepression between CCAAT/Enhancer-binding Protein β and the p53 Tumor Suppressor

The tumor suppressor protein p53 is not only involved in defending cells against genotoxic insults but is also implicated in differentiation processes, a function that it shares with the CCAAT/enhancer-binding protein β (C/EBPβ). We previously reported an up-regulation of both factors in the cycle-dependent differentiation process of human endometrial stromal cells, termed decidualization. C/EBPβ-mediated activation of a decidualization marker, the decidual prolactin promoter, was antagonized by p53. Here we report that C/EBPβ in turn represses the transcriptional activity of p53. Competition for limiting amounts of coactivator CREB-binding protein/p300 was ruled out as the underlying mechanism of transrepression. Physical interaction between p53 and C/EBPβ was demonstrated in vitro and in vivo and shown to depend on the C-terminal domains of both proteins. In gel shift experiments, C/EBPβ reduced complex formation between p53 and its response element. Conversely, p53 strongly inhibited binding of endogenous C/EBPβ from endometrial stromal cells to the C/EBP-responsive region in the decidual prolactin promoter. The observed negative cross-talk between p53 and C/EBPβ is likely to impact expression of their respective target genes.

Anti‐tumor activity of a B‐cell receptor‐targeted peptide in a novel disseminated lymphoma xenograft model

Receptor‐targeted therapies have become standard in the treatment of various lymphomas. In view of its unparalleled specificity for the malignant B‐cell clone, the B‐cell receptor (BCR) on B cell lymphoma cells is a potential therapeutic target. We have used two BCR epitope mimicking peptides binding to the Burkitts lymphoma cell lines CA46 and SUP‐B8. We proved their functionality by demonstrating calcium flux and BCR‐mediated endocytosis upon peptide receptor binding. Toxicity experiments in vitro via cross‐linking of the BCR with tetramerized epitope mimics lead to apoptosis in both cell lines but was far more effective in SUP‐B8 cells. We established a SUP‐B8‐based disseminated Burkitts lymphoma model in NOD/SCID mice. Treatment of tumor‐bearing mice with tetramerized epitope mimics had significant anti‐tumor effects in vivo. We conclude that peptide‐mediated, BCR‐targeted therapy is a promising approach which may be explored and further developed for application in highly aggressive lymphoma.