Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis

Background: CC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis. Methodology: Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies. Significance: For the first time, successful generation of anti-G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer

Binding of anti-CCR4 scFv fragments to avian DT40 cells transfected with human CCR4.

<p>The results of titration on DT40/CCR4 and non-transfected DT40 cells are shown for scFv 17G (<b>a</b>), 9E (<b>b</b>), 1O (<b>c</b>) and 11F (<b>d</b>). Binding of scFv fragments in the presence of increasing concentrations of CCR4 ligands CCL22 and CCL17 is shown in panels (<b>e</b>) and (<b>f</b>), respectively. As a control, a scFv fragment derived from hybridoma KM2160 was used. Cell binding was analyzed by flow cytometry; bound scFv fragments were detected with anti-cMyc antibody followed by anti-human-PE immunoconjugates. Median fluorescence intensity is plotted against the scFv concentration (µg/mL); the results of representative experiments from three repeats are shown.</p

Cell surface retention of anti-CCR4 antibodies.

<p>Labeled anti-CCR4 antibodies were incubated with CCR4-positive DT40-cells and the decrease of fluorescent signals during the time course in the presence of excessive amounts of the corresponding unlabeled antibodies were recorded using flow cytometry. To prevent rebinding of labeled molecules, the unlabeled 9E10J IgG1 was added to the cells in all experiments. The off-rate (<i>k</i>_off) and half-life (<i>t</i>_1/2) values for dissociation of the antibody-antigen complexes were interpolated from the standard curves generated by one-phase exponential decay fit of the experimental data as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103776#pone-0103776-g005" target="_blank">Figure 5a, b</a>.</p

Apparent affinities of anti-CCR4 antibodies (human IgG1 format) as determined in cell binding experiments.

<p>Binding of diluted anti-CCR4 antibodies to different cell lines was measured using flow cytometry. Recorded median fluorescence intensity values were plotted against antibody concentrations and the affinities were calculated using the ‘one-site-total binding’ equation of the software PRISM (GraphPad). n.d., not determined; <b>-</b>, not detectable.</p

Dose-dependent binding of anti-CCR4 IgG antibodies to CCR4⁺ and CCR4⁻ cell lines.

<p>Different concentrations of 17G, 9E and KM3060var antibodies were tested in flow cytometry for binding to avian DT40 cells stably transfected with human CCR4 (<b>a</b>), to human HEK-293 cells transiently transfected with human CCR4 (<b>b</b>) or to human T-cell leukemia line CCRF-CEM naturally expressing CCR4 (<b>c</b>). Binding to CCR4-negative non-transfected cells is also shown in panels (<b>a</b>) and (<b>b</b>). The results of representative experiments from three repeats are shown.</p

Dose-dependent effect of anti-CCR4 antibodies on CCL17 or CCL22-induced chemotaxis of CCR4⁺ CCRF-CEM cells.

<p>Human T-cell leukemia CCRF-CEM cells were induced to migrate in the transwell plates, where the CCR4 ligands were placed in the lower chamber and the cells were co-incubated with either CCR4-specific antibodies or isotype control antibody in the upper chambers. The cells migrated to the lower chamber were detected and counted by flow cytometry. (<b>a</b>, <b>c</b>) Inhibition of CCL17-induced migration. (<b>b</b>, <b>d</b>) Inhibition of CCL22-induced migration. Mean and SD values of triplicates are plotted.</p

Effect of anti-CCR4 antibodies on calcium mobilization induced by CCR4-specific ligands CCL22 and CCL17 and by irrelevant ligand CXCL12.

<p>CCR4-positive CCRF-CEM cells, loaded with the Ca²⁺ sensing dye Fluo-4, were stimulated with either CCR4-specific ligands CCL22 and CCL17 or the irrelevant chemokine CXCL12, in presence or absence of antibodies. Samples were analyzed by FACS and the AUC value, corresponding to CCR4 receptor activation, was recorded over 2 min. Ratios of integrated AUCs expressed as percentage of AUC for stimulation with the ligand alone are shown.</p

Comparative ADCC activity of anti-CCR4 antibodies.

<p>CCR4-positive CCRF-CEM cells were labeled with calcein and incubated with isolated human PBMCs as effector cells in presence of different concentrations of anti-CCR4 antibodies. Induction of ADCC was expressed as percentage of cells lysed; the maximal lysis using detergent was set to 100%. Means and SDs from 7–11 independent experiments are shown.</p

Effect of human anti-CCR4 antibody 9E10J on platelet aggregation.

<p>(<b>a</b>) Binding of 9E10J IgG1 at concentration 10 µg/mL to human platelets isolated from the fresh donor blood. As negative controls, binding histograms for an isotype control antibody (Isotype) and of a secondary antibody alone (anti-human-PE) are shown. (<b>b</b>) Effect of anti-CCR4 antibody 9E10 and of the isotype control antibody on platelet aggregation in comparison with ADP. (<b>c</b>) Platelet aggregation induced by CCR4 ligands, CCL17 and CCL22, in comparison with ADP. (<b>d, e</b>) Ligand-induced aggregation of platelets pre-incubated with either the isotype control antibody (<b>d</b>) or anti-CCR4 antibody 9E10J (<b>e</b>).</p