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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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Related in: MedlinePlus

Dose-dependent binding of anti-CCR4 IgG antibodies to CCR4+ and CCR4− cell lines.Different concentrations of 17G, 9E and KM3060var antibodies were tested in flow cytometry for binding to avian DT40 cells stably transfected with human CCR4 (a), to human HEK-293 cells transiently transfected with human CCR4 (b) or to human T-cell leukemia line CCRF-CEM naturally expressing CCR4 (c). Binding to CCR4-negative non-transfected cells is also shown in panels (a) and (b). The results of representative experiments from three repeats are shown.
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pone-0103776-g002: Dose-dependent binding of anti-CCR4 IgG antibodies to CCR4+ and CCR4− cell lines.Different concentrations of 17G, 9E and KM3060var antibodies were tested in flow cytometry for binding to avian DT40 cells stably transfected with human CCR4 (a), to human HEK-293 cells transiently transfected with human CCR4 (b) or to human T-cell leukemia line CCRF-CEM naturally expressing CCR4 (c). Binding to CCR4-negative non-transfected cells is also shown in panels (a) and (b). The results of representative experiments from three repeats are shown.

Mentions: The selected scFv candidates 17G and 9E were reformatted as full length human IgG1 for further characterization. The variant of the KM2160-derived chimeric (mouse variable domains/human constant domains) IgG1 antibody KM3060 [30] made in-house (KM3060var) was used as a positive control. The anti-CCR4 IgG antibodies 17G, 9E and KM3060var demonstrated specific binding to an avian cell line stably transfected with human CCR4 gene (DT40, receptor density ∼92,000 CCR4 molecules per cell), to a human cell line transiently transfected with human CCR4 gene (HEK-293, receptor density ∼46,000 CCR4 molecules per cell) and to a human T-lymphoblastic leukemia cell line that endogenously expresses CCR4 (CCRF-CEM, receptor density ∼6,200 CCR4 molecules per cell), as determined by flow cytometry (Figure 2). No significant binding was observed to the tested non-transfected host cells.


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)

Dose-dependent binding of anti-CCR4 IgG antibodies to CCR4+ and CCR4− cell lines.Different concentrations of 17G, 9E and KM3060var antibodies were tested in flow cytometry for binding to avian DT40 cells stably transfected with human CCR4 (a), to human HEK-293 cells transiently transfected with human CCR4 (b) or to human T-cell leukemia line CCRF-CEM naturally expressing CCR4 (c). Binding to CCR4-negative non-transfected cells is also shown in panels (a) and (b). The results of representative experiments from three repeats are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103776-g002: Dose-dependent binding of anti-CCR4 IgG antibodies to CCR4+ and CCR4− cell lines.Different concentrations of 17G, 9E and KM3060var antibodies were tested in flow cytometry for binding to avian DT40 cells stably transfected with human CCR4 (a), to human HEK-293 cells transiently transfected with human CCR4 (b) or to human T-cell leukemia line CCRF-CEM naturally expressing CCR4 (c). Binding to CCR4-negative non-transfected cells is also shown in panels (a) and (b). The results of representative experiments from three repeats are shown.
Mentions: The selected scFv candidates 17G and 9E were reformatted as full length human IgG1 for further characterization. The variant of the KM2160-derived chimeric (mouse variable domains/human constant domains) IgG1 antibody KM3060 [30] made in-house (KM3060var) was used as a positive control. The anti-CCR4 IgG antibodies 17G, 9E and KM3060var demonstrated specific binding to an avian cell line stably transfected with human CCR4 gene (DT40, receptor density ∼92,000 CCR4 molecules per cell), to a human cell line transiently transfected with human CCR4 gene (HEK-293, receptor density ∼46,000 CCR4 molecules per cell) and to a human T-lymphoblastic leukemia cell line that endogenously expresses CCR4 (CCRF-CEM, receptor density ∼6,200 CCR4 molecules per cell), as determined by flow cytometry (Figure 2). No significant binding was observed to the tested non-transfected host cells.

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