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Fully human antagonistic antibodies against CCR4 potently inhibit cell signaling and chemotaxis.

Hagemann UB, Gunnarsson L, Géraudie S, Scheffler U, Griep RA, Reiersen H, Duncan AR, Kiprijanov SM - PLoS ONE (2014)

Bottom Line: In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies.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 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.

View Article: PubMed Central - PubMed

Affiliation: Affitech Research AS, Oslo, Norway.

ABSTRACT

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.

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Binding of anti-CCR4 scFv fragments to avian DT40 cells transfected with human CCR4.The results of titration on DT40/CCR4 and non-transfected DT40 cells are shown for scFv 17G (a), 9E (b), 1O (c) and 11F (d). Binding of scFv fragments in the presence of increasing concentrations of CCR4 ligands CCL22 and CCL17 is shown in panels (e) and (f), 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.
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pone-0103776-g001: Binding of anti-CCR4 scFv fragments to avian DT40 cells transfected with human CCR4.The results of titration on DT40/CCR4 and non-transfected DT40 cells are shown for scFv 17G (a), 9E (b), 1O (c) and 11F (d). Binding of scFv fragments in the presence of increasing concentrations of CCR4 ligands CCL22 and CCL17 is shown in panels (e) and (f), 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.

Mentions: In order to generate initial antibodies against CCR4, phage display selection was performed using a scFv antibody library derived from a naïve human IgM/IgD repertoire [33]. To identify antagonistic antibodies against CCR4, the phages binding to the CCR4+ cells and not to CCR4− cells were competitively eluted in presence of CCR4-specific ligands CCL17 or CCL22. The second strategy included elution using trypsin or citric acid to generate antibodies that could potentially have either higher affinities than the CCR4 ligands, CCL17 and CCL22, or bound to epitopes outside of the ligand-binding pocket on the receptor. After completion of three rounds of panning, the selected phage pools were subjected to screening in cell binding assays using a cell reporter-assisted high-throughput screening system. Screening of 12,240 clones resulted in identification of 132 scFv candidates binding CCR4+ cells and showing no binding to CCR4− cells. Sequencing led to identification of four different CCR4-specific antibody variants, 17G, 9E, 1O and 11F. These scFv fragments demonstrated specific binding to cells transfected with the gene encoding human CCR4 (Figure 1a–d) and to cell lines naturally expressing CCR4 (not shown). In contrast, none of the four selected candidates showed significant binding to CCR4-negative cells (Figure 1a–d). Analysis of CCR4+ cell binding in presence of increasing concentrations of CCR4 ligands CCL22 and CCL17 demonstrated a clear decrease in staining signals for all scFv variants with both CCL22 and CCL17 (Figure 1e, f). In contrast, no effect of CCL17 or CCL22 was observed on binding of a control scFv fragment derived from the murine hybridoma KM2160 recognizing the N-terminal part of human CCR4 [30]. The results indicated that the antibodies 17G, 9E, 1O and 11F interfered with ligand binding and thus might have CCR4-blocking activity.


Fully human antagonistic antibodies against CCR4 potently inhibit cell signaling and chemotaxis.

Hagemann UB, Gunnarsson L, Géraudie S, Scheffler U, Griep RA, Reiersen H, Duncan AR, Kiprijanov SM - PLoS ONE (2014)

Binding of anti-CCR4 scFv fragments to avian DT40 cells transfected with human CCR4.The results of titration on DT40/CCR4 and non-transfected DT40 cells are shown for scFv 17G (a), 9E (b), 1O (c) and 11F (d). Binding of scFv fragments in the presence of increasing concentrations of CCR4 ligands CCL22 and CCL17 is shown in panels (e) and (f), 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4117600&req=5

pone-0103776-g001: Binding of anti-CCR4 scFv fragments to avian DT40 cells transfected with human CCR4.The results of titration on DT40/CCR4 and non-transfected DT40 cells are shown for scFv 17G (a), 9E (b), 1O (c) and 11F (d). Binding of scFv fragments in the presence of increasing concentrations of CCR4 ligands CCL22 and CCL17 is shown in panels (e) and (f), 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.
Mentions: In order to generate initial antibodies against CCR4, phage display selection was performed using a scFv antibody library derived from a naïve human IgM/IgD repertoire [33]. To identify antagonistic antibodies against CCR4, the phages binding to the CCR4+ cells and not to CCR4− cells were competitively eluted in presence of CCR4-specific ligands CCL17 or CCL22. The second strategy included elution using trypsin or citric acid to generate antibodies that could potentially have either higher affinities than the CCR4 ligands, CCL17 and CCL22, or bound to epitopes outside of the ligand-binding pocket on the receptor. After completion of three rounds of panning, the selected phage pools were subjected to screening in cell binding assays using a cell reporter-assisted high-throughput screening system. Screening of 12,240 clones resulted in identification of 132 scFv candidates binding CCR4+ cells and showing no binding to CCR4− cells. Sequencing led to identification of four different CCR4-specific antibody variants, 17G, 9E, 1O and 11F. These scFv fragments demonstrated specific binding to cells transfected with the gene encoding human CCR4 (Figure 1a–d) and to cell lines naturally expressing CCR4 (not shown). In contrast, none of the four selected candidates showed significant binding to CCR4-negative cells (Figure 1a–d). Analysis of CCR4+ cell binding in presence of increasing concentrations of CCR4 ligands CCL22 and CCL17 demonstrated a clear decrease in staining signals for all scFv variants with both CCL22 and CCL17 (Figure 1e, f). In contrast, no effect of CCL17 or CCL22 was observed on binding of a control scFv fragment derived from the murine hybridoma KM2160 recognizing the N-terminal part of human CCR4 [30]. The results indicated that the antibodies 17G, 9E, 1O and 11F interfered with ligand binding and thus might have CCR4-blocking activity.

Bottom Line: In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies.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 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.

View Article: PubMed Central - PubMed

Affiliation: Affitech Research AS, Oslo, Norway.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus