Development of a human antibody that exhibits antagonistic activity toward CC chemokine receptor 7

Abstract

Background: CC chemokine receptor 7 (CCR7) is a member of G-protein-coupled receptor family and mediates chemotactic migration of immune cells and different cancer cells induced via chemokine (C-C motif) ligand 19 (CCL19) or chemokine (C-C motif) ligand 21 (CCL21). Hence, the identification of blockade antibodies against CCR7 could lead to the development of therapeutics targeting metastatic cancer.

Methods: CCR7 was purified and stabilized in its active conformation, and antibodies specific to purified CCR7 were screened from the synthetic M13 phage library displaying humanized scFvs. The in vitro characterization of selected scFvs identified two scFvs that exhibited CCL19-competitive binding to CCR7. IgG₄'s harboring selected scFv sequences were characterized for binding activity in CCR7⁺ cells, inhibitory activity toward CCR7-dependent cAMP attenuation, and the CCL19 or CCL21-dependent migration of CCR7⁺ cells.

Results: Antibodies specifically binding to purified CCR7 and CCR7⁺ cells were isolated and characterized. Two antibodies, IgG₄(6RG11) and IgG₄(72C7), showed ligand-dependent competitive binding to CCR7 with K_D values of 40 nM and 50 nM, respectively. Particularly, IgG₄(6RG11) showed antagonistic activity against CCR7, whereas both antibodies significantly blocked the ligand-induced migration and invasion activity of CCR7⁺ cancer cells.

Conclusions: Two antibody clones were successfully identified from a synthetic scFv-displaying phage library using purified recombinant CCR7 as an antigen. Antibodies specifically bound to the surface of CCR7⁺ cells and blocked CCR7⁺ cell migration. Particularly, 6RG11 showed antagonist activity against CCR7-dependent cAMP attenuation.

Keywords: CC chemokine receptor 7; GPCR; antibody; metastasis; phage display.

Figure 1

Purification of functional P9-CCR7. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of P9-CCR7 after Ni-NTA chromatography. M = size marker, MP = soluble membrane fraction after solubilization in 1% sarkosyl, FT = flow-through from Ni-NTA chromatography, W1, W2 = wash fraction with each 0, 5 mM imidazole, respectively, E = elution fraction with each 20 mM imidazole from Ni-NTA chromatography. Purified P9-CCR7 is indicated by triangles. (B) Size exclusion chromatography of purified P9-CCR7 stabilized with APG. P9-CCR7 was eluted as a monodisperse peak (Peak 1) followed by free APG (Peak 2). (C) A binding curve of P9-CCR7 to CCL19-H₆-Flag (blue dots) or CCL21-H₆-Flag (orange squares). Different concentration of P9-CCR7 was added to plates containing 10 μg/mL CCL19-H₆-Flag (blue dots) or CCL21-H₆-Flag (orange squares) and P9-CCR7 bound to wells was measured via an anti-P9 antibody and HRP-conjugated antimouse antibody.

Figure 2

scFv clone specificity toward CCR7. (A) Specificity of the purified scFvs toward CCR7. Sixteen scFv clones with unique sequences were purified to homogeneity and the purified scFvs (500 nM) were incubated in 96-well plates containing immobilized P9-CCR7 or P9-GLP1R, with bound scFvs detected using an HRP-conjugated anti-HA antibody. The ratio of the absorption value P9-CCR7 to P9-GLP1R values was plotted. (B) Inhibition of the interactions between CCL19 and CCR7 by two scFvs (6RG11 and 72C7). CCL19-H₆-Flag (100 nM) was incubated with different scFv concentrations in a 96-well plate containing immobilized P9-CCR7, and bound CCL19-H₆-Flag was measured using a monoclonal anti-Flag®M2 antibody and HRP-conjugated antimouse Fc antibody. (C) Concentration-dependent binding of scFv(6RG11) and scFv(72C7) to CCR7. Different scFv(6RG11) (blue dots) and scFv(72C7) (green squares) concentrations were added to immobilized P9-CCR7, and bound scFvs were detected using an HRP-conjugated anti-HA antibody.

Figure 3

Specific interactions between CCR7 and IgG₄(6RG11) or IgG₄(72C7). (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified IgG₄(6RG11) and IgG₄(72C7). M = Size marker, NR = 6 μg of IgG₄ in non-reducing conditions, R = 6 μg of IgG₄ in reducing conditions plus 1 mM DTT. (B) Concentration-dependent binding of IgG₄ (6RG11) and IgG₄(72C7). Different purified IgG₄ concentrations were added to a 96-well plate containing immobilized P9 or P9-CCR7, and bound IgG₄(6RG11) (blue dots) or IgG₄(72C7) (green dots) to P9-CCR7 or P9 (black squares) was measured using an HRP-conjugated antihuman Fc. (C) Cytometric analysis of IgG₄(6RG11) and IgG₄(72C7) using CCR7⁺ cells. HEK293 or CCR7-transformed HEK293 cells (HEK293/CCR7) were incubated with mock (red), 20 nM IgG₄(Opdivo)(blue) as an isotype control, or 20 nM IgG₄(6RG11) or IgG₄(72C7) (orange), and then treated with an Alexa488-conjugated antihuman Fc antibody.

Figure 4

The antagonist activity of IgG₄(6RG11) and IgG₄(72C7). (A) CCL19 or CCL21-dependent cAMP attenuation in HEK293 and MDA-MB-231 cells overexpressing CCR7 in the presence of forskolin. Different concentrations of CCL19 (left panel) or CCL21 (right panel) were added to HEK293 (upper panel, black square) or HEK293/CCR7 cells (upper panel, blue or orange dots) and cAMP levels were measured. Ligand effects in MDA-MB-231 (lower panel, black squares) or MDA-MB-231/CCR7 cells (lower panel, blue or orange dots) were also measured. (B) The effects of IgG₄(6RG11) and IgG₄(72C7) on ligand-dependent cAMP attenuation in HEK293 and MDA-MB-231 cells overexpressing CCR7 in the presence of forskolin. Different IgG₄(6RG11) (blue circles) and IgG₄(72C7) (green triangles) concentrations were added to HEK293/CCR7 (upper panel) or MDA-MB-231/CCR7 cells (lower panel) in the presence of 20 nM CCL19 (left panel) or CCL21 (right panel) and cAMP levels measured.

Figure 5

Inhibition of IgG₄(6RG11) and IgG₄(72C7) on CCL19 or CCL21-dependent migration of MDA-MB-231/CCR7 cells. (A) Image of migrated MDA-MB-231/CCR7 cells after treatment of 30 nM CCL19 in the presence of 0, 10, 50, or 200 nM IgG₄(6RG11) (upper panel) or IgG₄(72C7) (lower panel). (B) The number of migrated cells in lower chambers was plotted against CCL19 and IgG₄(6RG11) or IgG₄(72C7) concentrations. (C) Image of migrated cells after treatment with 30 nM CCL21 in the presence of 0, 10, 50, or 200 nM IgG₄(6RG11) (upper panel) or IgG₄(72C7) (lower panel). (D) The number of migrated cells in lower chambers was plotted against CCL21 and IgG₄(6RG11) or IgG₄(72C7) concentrations (P > 0.05, no significance; ^*P ≤ 0.05; ^**P ≤ 0.01; ^***P ≤ 0.001).