Development of Novel Promiscuous Anti-Chemokine Peptibodies for Treating Autoimmunity and Inflammation

Abstract

Chemokines and their receptors play critical roles in the progression of autoimmunity and inflammation. Typically, multiple chemokines are involved in the development of these pathologies. Indeed, targeting single chemokines or chemokine receptors has failed to achieve significant clinical benefits in treating autoimmunity and inflammation. Moreover, the binding of host atypical chemokine receptors to multiple chemokines as well as the binding of chemokine-binding proteins secreted by various pathogens can serve as a strategy for controlling inflammation. In this work, promiscuous chemokine-binding peptides that could bind and inhibit multiple inflammatory chemokines, such as CCL2, CCL5, and CXCL9/10/11, were selected from phage display libraries. These peptides were cloned into human mutated immunoglobulin Fc-protein fusions (peptibodies). The peptibodies BKT120Fc and BKT130Fc inhibited the ability of inflammatory chemokines to induce the adhesion and migration of immune cells. Furthermore, BKT120Fc and BKT130Fc also showed a significant inhibition of disease progression in a variety of animal models for autoimmunity and inflammation. Developing a novel class of antagonists that can control the courses of diseases by selectively blocking multiple chemokines could be a novel way of generating effective therapeutics.

Keywords: autoimmunity; chemokines; inflammation; peptibodies; phage display.

Figure 1

ELISA of chemokine-binding peptides (CBPs) with CCL2 and CXCL12. ELISA was performed on biotinylated peptides to detect their interaction with (A) CCL2 or (B) CXCL12. The data are presented as the fold binding that was obtained using two concentrations of the various peptides (10 and 100 ng/ml).

Figure 2

Effect of various peptides on the chemokine-induced immune cell-dependent adhesion to VCAM-1. Adhesion was measured using the laminar flow assay. The number of adherent cells resisting detachment by elevated shear forces (dyn/cm²) is expressed as the percentage of originally settled cells. The effects of the peptides BKT130, BKT120, and peptide 23 (P23) on the (A) CCL2-, (B) CCL5-, and (C)CXCL12-induced immune cell-dependent adhesion to VCAM-1 were measured. All the adhesion experiments were performed at least three times on multiple test fields.

Figure 3

Effect of the peptibodies BKT130Fc and BKT120Fc on the chemokine-induced immune cell-dependent adhesion to VCAM-1. Adhesion was measured using the laminar flow assay. The number of adherent cells resisting detachment by elevated shear forces (dyn/cm²) is expressed as the percentage of originally settled cells. The effects of the peptibodies BKT130Fc and BKT120Fc on the (A) CCL2-, (B) CCL5-, (C) CCL11-, (D) CXCL8-, (E) CXCL9-, (F) CXCL10-, (G) CXCL11-, and (H) CXCL12-induced immune cell-dependent adhesion to VCAM-1 were measured. All the adhesion experiments were performed at least three times on multiple test fields.

Figure 4

ELISA for the binding of BKT130Fc to different chemokines. ELISA was performed with several dilutions of BKT130Fc (1, 10, and 50 μg/ml) to detect the interaction to different chemokines that were bound to the plates. The data are presented as the optical density (O.D.) obtained at wavelength of 450 nm. (A) presents the chemokines that demonstrate binding to BKT130Fc and (B) presents the chemokine with no binding to BKT130Fc.

Figure 5

The peptibodies BKT130Fc and BKT120Fc inhibit the in vitro migration of different chemokines. A transmigration assay was performed to study the effects of the BKT130Fc, BKT120Fc and BKT110Fc peptibodies on in vitro migration. (A) Migration toward CCL2 was assessed using THP-1 cells. (B) Migration toward CCL5 was assessed using THP-1 cells. (C) Migration toward CXCL10 was assessed using Jurkat cells overexpressing CXCR3. (D) Migration toward CXCL11 was assessed using Jurkat cells overexpressing CXCR3. (E) Migration toward CXCL12 was assessed using CD4+ T cells. BKT130Fc, BKT120Fc, and BKT110Fc were added at 10 µg/ml. The results, analyzed using Student’s t-tests (*p ≤ 0.05), are expressed as the mean percentage of migration ±SD. (F) Dosing of BKT130Fc pharmacokinetics (PK). C57BL/6 mice (n = 3) were intravenously (i.v.) injected with 25, 50, or 100 μg of BKT130Fc. The serum peptibody levels were tested after 2 h, 24 h, and 6 days. (G) PK of BKT130Fc and BKT120c in mice. C57BL/6 mice (n = 5) were i.v. injected with BKT120Fc or BKT130Fc at 50 µg/mouse. The serum peptibody levels were tested after 2 h, 24 h, and 6, 11, and 18 days. (H) BKT130Fc and BKT120Fc inhibited delayed-type hypersensitivity (DTH) in vivo. DTH model was established in female BALB/c mice (n = 12). BKT120Fc, BKT130Fc, or BKT110Fc were intravenously injected (50 μg/mouse) on day 0 and 24 h before the challenge (day 5) (total of 100 μg/mouse). Dexamethasone (100 µg/muse) served as the positive control. The two dotted lines indicate the normal range of ear swelling. The results, analyzed using Student’s t-tests (*p ≤ 0.05), are expressed as the mean ± SE of ear swelling (mm). All the DTH experiments were performed at least four times.

Figure 6

BKT130Fc and BKT120Fc inhibited the infiltration of inflammatory cells into the synovial cavities of mice of the antigen-induced arthritis (AIA) model. Male C57BL/6 mice (n = 12) were immunized with methylated bovine serum albumin. BKT130Fc or BKT120Fc were administered via i.v. once at 50 µg/per a mouse, 24 h before the challenge. The control group represents normal mice, the AIA group represents untreated AIA mice and the BKT130Fc or BKT120Fc groups represent AIA mice treated with BKT130Fc or BKT120Fc, respectively. The number of infiltrated cells into the knee cavity was evaluated by FACScaliber: (A) number of neutrophils, (B) number of MNCs. Representative FACS analysis of cells within the knee cavity is presented in (D). The R1 gate shows the neutrophil population and the R2 gate shows the MNCs. The level of myeloperoxidase (MPO) activity is presented in (C) as the relative number of neutrophils per mg of wet periarticular tissue. (E) Histology analysis of the knee joint shows the infiltration of inflammatory cells following hematoxylin and eosin (H&E) staining.

Figure 7

BKT130Fc and BKT120Fc reduced the severity of experimental autoimmune encephalomyelitis (EAE) by inhibiting the infiltration of immune cells into the central nervous system (CNS). (A) Female SJL mice (n = 12) were immunized with the proteolipid protein (PLP) peptide to induce relapsing-remitting EAE. BKT130Fc or BKT120Fc were intravenously administered once (50 μg/mouse) on day 9 post-disease induction. (B) Female C57BL/6 mice (n = 12) were immunized with a myelin oligodendrocyte glycoprotein (MOG) peptide to induce chronic EAE. BKT130Fc or BKT120Fc were intravenously administered once (50 μg/mouse) on day 9 post-disease induction. The data are presented as the EAE mean clinical score measured for 47 days post-immunization. (C) Female C57BL/6 mice (n = 8) were immunized with a MOG peptide to induce chronic EAE. BKT110Fc was intravenously administered once (50 μg/mouse) on day 9 post-disease induction. (D) Accumulating score of MOG induced EAE after treating with BKT130Fc, BKT120Fc, or BKT110Fc. (E) Histology analysis of the spinal cords from SJL mice shows the infiltration of immune cells into the CNS following hematoxylin and eosin (H&E) staining and the area of demyelination marked with luxol fast blue (light blue staining = demyelination area). (F–G) Cytokine levels in the sera of mice on day 30 post-immunization as measured by the Mouse Th1/Th2 Cytokine Kit Array in (F) SJL mice immunized with PLP and (G) C57BL/6 mice immunized with MOG. The results, analyzed using Student’s t-tests (*p ≤ 0.05), are expressed as the mean ± SE. All the EAE experiments were performed at least three times.