Antigen-specific suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor: structure optimization and pharmacokinetics

Abstract

The objective of this study was to optimize the in vivo activity of proteolipid protein (PLP)-bifunctional peptide inhibitor (BPI) molecule to suppress experimental autoimmune encephalomyelitis (EAE) in SJL/J mice and evaluate pharmacokinetic profiles of PLP-BPI. PLP-BPI is constructed via conjugation of myelin PLP(139-151) with CD11a(237-246)-derived peptide (LABL) via a spacer. The hypothesis is that PLP-BPI binds simultaneously to major histocompatibility complex-II and intercellular adhesion molecule-1 on the antigen-presenting cell (APC) and inhibits the formation of the immunological synapse during T-cell and APC interactions. In this study, the structure of BPI was modified by varying the spacer and was evaluated in the EAE model. Intravenous injections of BPI derivatives inhibited the onset, severity, and incidence of EAE more effectively and induced a lower incidence of anaphylaxis than that produced by unmodified PLP-BPI. As anticipated, production of interleukin-17, a proinflammatory cytokine commonly found in elevated levels among multiple sclerosis (MS) patients, was significantly lower in Ac-PLP-BPI-PEG6- or Ac-PLP-BPI-NH(2)-2-treated mice than in phosphate-buffered saline-treated mice. These results suggest that BPI-type molecules can be modified to achieve more efficient and better tolerated BPI-based derivatives for the treatment of MS.

Fig. 1.

The therapeutic efficacy of BPI derivatives in reversing EAE in the mouse model. The mice were immunized with PLP_139-151/CFA followed by intravenous treatments up to a maximum of three injections with the BPI molecules starting on the day of disease onset as described in Materials and Methods. A, clinical EAE disease score. B, change in body weight. Results are expressed as the mean ± S.D. (n = 10). There are significant differences between peptide-treated and PBS-treated groups in clinical disease scores: Ac-PLP-BPI-NH₂-2, p < 0.001; Ac-PLP-BPI-PEG6, p < 0.001; and Ac-PLP-BPI-PEG3, p < 0.001. Statistical values for the loss of body weight compared with PBS were as follows: Ac-PLP-BPI-NH₂-2, p < 0.001; Ac-PLP-BPI-PEG6, p < 0.001; and Ac-PLP-BPI-PEG3, p > 0.05.

Fig. 2.

In vivo EAE-suppressive activity of BPI derivatives in the mouse EAE model. PLP_139-151/CFA-immunized mice received intravenous injections of the indicated peptide (100 nmol/mouse/day) on days 4 and 7. A, clinical EAE disease score. B, change in body weight. C, incidence of disease. Results are expressed as the mean ± S.D. (n = 10). There are significant differences between BPI-treated and PBS-treated groups in clinical disease scores and loss of body weight: Ac-PLP-BPI-NH₂-2, p < 0.001 and Ac-PLP-BPI-EG6, p < 0.001. There was no significant difference (p > 0.05) in EAE clinical scores and loss of body weight between scrambled peptide (Ac-PLP-BPI-PEG_SC) and PBS groups. In addition, no significant difference (p > 0.05) between Ac-PLP-BPI-NH₂-2 and Ac-PLP-BPI-PEG6 was observed.

Fig. 3.

Production of IL-17 in serum in vivo. SJL/J female mice were immunized subcutaneouslywith PLP_139-151/CFA and injected intraperitoneally with pertussis toxin on days 0 and 2. Then, the mice received intravenous injections of the indicated peptide (100 nmol/mouse/day) or PBS on days 4, 7, and 10. A, serum concentration of IL-17 determined on days 12 and 35 in Ac-PLP-BPI-NH₂-2-treated, Ac-PLP-BPI-PEG6-treated, PBS-treated, and unprimed mice by using enzyme-linked immunosorbent assays. Data are represented as mean ± S.D. (six mice per group), and experiments were done in duplicate. B to D, disease progression was evaluated by EAE clinical disease score (B), percentage change in body weight (C), and incidence of disease (D). There are significant differences between BPI-treated and PBS-treated groups in clinical disease scores and loss of weight: Ac-PLP-BPI-NH₂-2, p < 0.001 and Ac-PLP-BPI-PEG6, p < 0.001. There was no significant difference (p > 0.05) in EAE clinical scores and loss of body weight between Ac-PLP-BPI-NH₂-2 and Ac-PLP-BPI-PEG6.

Fig. 4.

Plasma concentration profiles of Ac-PLP-BPI-NH₂-2 after intravenous administration in Spraque-Dawley rats in vivo. Data are represented as mean ± S.D. with (n = 3) and in triplicate. Point marks represent actual plasma levels, and the solid lines represent predicted plasma levels after fitting the raw data into compartmental models using commercial pharmacokinetic software.

Fig. 5.

Potential mechanisms of action of antigenic peptide or BPI molecule in the suppression of EAE. Mature dendritic cells present processed antigenic peptides to naive T cells to generate Th1 and/or Th17 cells. BPI molecule binds simultaneously to MHC-II and ICAM-1 on mature dendritic cells and prevents the formation of the immunological synapse. As a result, BPI molecule inhibits the formation of Th1 and generates Th2 cells. Immature dendritic cells can also present a soluble antigenic and BPI molecule to naive T cells to produce IL-10-producing T-regs. Produced IL-10 suppresses the generation of Th1 and Th17 cells, resulting in the suppression of the disease.