Neutralizing nanobodies targeting diverse chemokines effectively inhibit chemokine function

Abstract

Chemokine receptors and their ligands play a prominent role in immune regulation but many have also been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, allograft rejection after transplantation, and also in cancer metastasis. Most approaches to therapeutically target the chemokine system involve targeting of chemokine receptors with low molecular weight antagonists. Here we describe the selection and characterization of an unprecedented large and diverse panel of neutralizing Nanobodies (single domain camelid antibodies fragment) directed against several chemokines. We show that the Nanobodies directed against CCL2 (MCP-1), CCL5 (RANTES), CXCL11 (I-TAC), and CXCL12 (SDF-1α) bind the chemokines with high affinity (at nanomolar concentration), thereby blocking receptor binding, inhibiting chemokine-induced receptor activation as well as chemotaxis. Together, we show that neutralizing Nanobodies can be selected efficiently for effective and specific therapeutic treatment against a wide range of immune and inflammatory diseases.

Keywords: Antibodies; Chemokines; Chemotaxis; G Protein-coupled Receptors (GPCR); Nanobodies; Radioreceptor Assays.

FIGURE 1.

Screening and specificity of Nanobody libraries. A, screening of Nanobodies directed against CXCL11. Periplasm of anti-CXCL11 Nanobody-producing bacteria was diluted (1:10), incubated with ¹²⁵I-CXCL11 (50 pm), and used for a binding experiment on CXCR3-expressing HEK293T. As positive control an anti-CXCL11 antibody (2 μg/ml) was used. B, screening of Nanobodies directed against CCL2. Periplasm of anti-CCL2 Nanobody-producing bacteria was diluted (1:10), incubated with ¹²⁵I-CCL2 (50 pm), and used for a binding experiment on HCMV-US28-expressing HEK293T cells. As positive control unlabeled CCL2 was used. C, specificity of Nanobodies directed against CCL2. Periplasm of anti-CCL2 Nanobody-producing bacteria was diluted (1:10), incubated with ¹²⁵I-CXCL11 (50 pm) and used for a binding experiment on CXCR3-expressing HEK293T. Experiments were performed in triplicate for the controls and single concentrations for the Nanobodies.

FIGURE 2.

Inhibition of chemokine binding. A, inhibition of CXCL11 binding to CXCR3. Purified Nanobodies were incubated at the indicated concentrations with ¹²⁵I-CXCL11 (50 pm). Subsequently, binding was analyzed on CXCR3-expressing HEK293T cells. The Nanobodies inhibited ¹²⁵I-CXCL11 binding with the following pIC₅₀ ± S.E. values: 11B7 (○), 9.4 ± 0.1 (n = 3); 11B1 (●), 9.3 ± 0.1 (n = 4); 11B2 (■), 8.8 ± 0.1 (n = 3); 11A4 (□), 8.6 ± 0.0 (n = 3); 11H2 (▴), 8.3 ± 0.1 (n = 3); 11F2 (▵), 7.7 ± 0.0 (n = 3). Unlabeled CXCL11 (♢, dashed line) inhibited ¹²⁵I-CXCL11 binding with pIC₅₀ ± S.E. of 8.8 ± 0.1. B, inhibition of CXCL12 binding to CXCR4. Purified Nanobody 12A4 was incubated at the indicated concentrations with ¹²⁵I-CXCL12 (50 pm). Subsequently, binding was analyzed on CXCR4-expressing HEK293T cells. The Nanobody 12A4 inhibited ¹²⁵I-CXCL12 binding with a pIC₅₀ ± S.E. value of 8.8 ± 0.1 (n = 3). C, inhibition of CCL2 binding to CCR2. Purified Nanobodies were incubated at the indicated concentrations with ¹²⁵I-CCL2 (50 pm). Subsequently, binding was performed on CCR2-expressing HEK293T cells. The Nanobodies inhibited ¹²⁵I-CCL2 binding with the following pIC₅₀ ± S.E. values: 8E3 (●), 9.0 ± 0.0 (n = 3); 8E10 (○), 8.8 ± 0.1 (n = 3). D, inhibition of CCL5 binding to CCR1. Purified Nanobodies were incubated at the indicated concentrations with ¹²⁵I-CCL5 (50 pm). Subsequently, binding was performed on CCR1-expressing HEK293T cells. The Nanobodies inhibited ¹²⁵I-CCL5 binding with the following pIC₅₀ ± S.E. values: 17B11 (●), 8.8 ± 0.1 (n = 3); 10C8 (○), 9.2 ± 0.1 (n = 3). Experiments were performed in duplicate and repeated the indicated amount of times. E, inhibition of CXCL12 and CXCL11 binding to CXCR7. CXCL12 (100 nm) and CXCL11 (100 nm) effectively displace ¹²⁵I-CXCL12 (50 pm). Purified Nanobody 12A4 (1 μm) was incubated with ¹²⁵I-CXCL12 (50 pm) and CXCL11 (100 nm) with CXCL11 Nanobodies 11B1, 11B7, and 11B2 (1 μm) and binding was analyzed on CXCR7-expressing NIH-3T3 cells. Purified anti-CXCL12 Nanobody 12A4 prevented binding of ¹²⁵I-CXCL12 to CXCR7 and preincubation of CXCL11 with purified anti-CXCL11 Nanobodies 11B1, 11B7, and 11B2 neutralized CXCL11 allowing ¹²⁵I-CXCL12 to bind to CXCR7 (n = 4).

FIGURE 3.

Inhibition of chemokine receptor activation. A, inhibition of CXCR3 activation. Purified Nanobodies were incubated at the indicated concentrations with CXCL11 (5 nm). Subsequently, CXCR3-mediated PLC activation was determined in HEK293T cells co-expressing CXCR3 and Gα_qi5. The Nanobodies inhibited CXCL11-induced signaling with the following pIC₅₀ ± S.E. values: 11B1 (●), 7.9 ± 0.1 (n = 3); 11B7 (○), 7.7 ± 0.1 (n = 3). B, inhibition of CXCR4 activation. Purified 12A4 Nanobody was incubated at the indicated concentrations with CXCL12 (5 nm). Subsequently, CXCR4-mediated PLC activation was determined in HEK293T cells co-expressing CXCR4 and Gα_qi5. Nanobody 12A4 inhibited CXCL12-induced signaling with a pIC₅₀ ± S.E. value of 7.1 ± 0.1 (n = 5).

FIGURE 4.

Inhibition of chemotaxis. A, migration of CXCR3-expressing L1.2 cells. A migration assay with increasing concentrations of CXCL11 was performed using L1.2 cells transfected with cDNA encoding CXCR3. Data are shown as percentage of migrated cells and obtained in three experiments. B, inhibition of CXCL11-induced chemotaxis. Purified Nanobodies were preincubated at the indicated concentrations with CXCL11 (1 nm). Subsequently, CXCL11-induced migration of CXCR3-expressing L1.2 cells was determined. The Nanobodies inhibited CXCL11-induced chemotaxis with the following pIC₅₀ ± S.E. values: 11B1 (●), 9.0 ± 0.1 (n = 4); 11B7 (○), 7.8 ± 0.2 (n = 4). C, migration of L1.2 cells. A migration assay with increasing concentrations of CXCL12 was performed using L1.2 cells. The CXCR4 antagonist AMD3100 (10 μm) inhibited migration toward CXCL12 (1 nm). Data are shown as percentage of migrated cells and was obtained in three experiments. D, inhibition of CXCL12-induced chemotaxis. Purified 12A4 Nanobody was incubated at the indicated concentrations with CXCL12 (1 nm) for 1 h at RT while shaking. Subsequently, CXCL12-induced migration of L1.2 cells was determined. The 12A4 Nanobody inhibited CXCL12-induced chemotaxis with a pIC₅₀ ± S.E. value of 7.9 ± 0.1 (n = 5). Experiments were performed in triplicate.