In tumor biopsies, macrophages constitute a major component of the leukocyte infiltrate, where monocyte-derived M0 macrophages are thought to polarize into the tumor-inhibiting M1 or the tumor-promoting M2 phenotypes (27, 28). 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 FcRI-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. Keywords: Antibodies, Epidermal Growth Factor Receptor (EGFR), FC Receptors, Immunotherapy, Macrophages, IgA, Cytotoxicity Introduction Monoclonal antibodies and their derivatives constitute a rapidly growing class of biotherapeutics for an increasing number of clinical indications (1, 2). In tumor therapy, all therapeutic antibodies being approved or in clinical trials, are of the human IgG isotype, with clinical experience being available for IgG1, IgG2, and IgG4. At present, many arguments favor human IgG1 as the preferred isotype: first, human IgG1 effectively recruits NK cells and complement for tumor cell killing (3, 4). Second, IgG antibodies display an extended serum half-life, which is mediated by binding to neonatal Fc receptor (FcRn) (5). In addition, production technologies and purification protocols are well established, and regulatory agencies are acquainted with relevant safety issues (6). The Rabbit polyclonal to ABCA3 importance of antibody stability is underlined by recent observations with human IgG4 antibodies because IgG4 antibodies undergo fragment antibody binding (Fab) arm exchange under experimental and physiological conditions (7, 8). Recent data demonstrated that myeloid effector cells were effectively recruited for ADCC by human IgG2 antibodies (9). However, granulocytes and monocytes were particularly cytotoxic for tumor cells with antibodies of the IgA isotype (4, 10C12), which has not been explored clinically. IgA antibodies contribute significantly to the humoral arm of the mucosal immune system, which provides important barrier functions to protect the large area of serosal surfaces (13). Three forms of IgA (monomeric, dimeric, and secretory IgA) are distinguished, which have distinct molecular characteristics and serve different functions in the immune system (14). Secretory IgA constitutes a first line of serosal defense against invading pathogens. Secretory IgA is a heavily glysoylated multimeric protein consisting of two monomeric IgA molecules, covalently linked by the joining J-chain and the secretory component (15). (26). In tumor biopsies, macrophages constitute a major component of the leukocyte infiltrate, where monocyte-derived M0 macrophages are thought to polarize into the tumor-inhibiting M1 or ALW-II-41-27 the tumor-promoting M2 ALW-II-41-27 phenotypes (27, 28). lectin/lectin 1/concanavalin A/lectin, or agglutinin I (all from Vector Laboratories). After washing three times with buffer B (50 mm Tris/HCl, pH 8, 90 mm NaCl, 2 mm CaCl2, 5 mg/ml BSA, 0.05% Tween 20) for 10 min, deposition of lectin was detected using HRP-labeled NeutrAvidin (Vector Labs). Membranes were developed using enhanced chemiluminescent reaction reagent (Pierce). Flow Cytometry and EGFR Down-modulation Binding to EGFR and to FcRI was analyzed by flow cytometry as described previously (12). Down-modulation of EGFR was analyzed on murine BaF3 cells transfected with human EGFR, which were incubated with 225-IgA and control IgA (6 nm) for 4 or 24 h. Residual surface EGFR was detected with Alexa Fluor 488-labeled murine 425 antibody using the DyLight Fluor antibody labeling kit (Pierce). Results were ALW-II-41-27 calculated as % EGFR down-modulation = 100 ? (relative fluorescence intensity (RFI) m425-FITC/RFI sample) 100. All samples were analyzed on a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter), collecting 1 104 events for each experimental value. Data were analyzed using XL-System II software (version 3.0, Beckman Coulter). Relative fluorescence intensities were calculated as the ratio of mean linear fluorescence intensity of relevant to irrelevant isotype-matched antibodies. Growth Inhibition Assay Growth inhibition of DiFi colon carcinoma cells was analyzed using 3-(3,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2tests. EC50 values were calculated from dose-response curves, reported as.
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