Bifunctional Peptide Inhibitors Suppress Interleukin-6 Proliferation and Ameliorates Murine Collagen-Induced Arthritis.

The objective of this study is to evaluate the efficacy and potential mechanism of action of type-II collagen bifunctional peptide inhibitor (CII-BPI) molecules in suppressing rheumatoid arthritis in the collagen-induced arthritis (CIA) mouse model. CII-BPI molecules (CII-BPI-1, CII-BPI-2, and CII-BPI-3) were formed through conjugation between an antigenic peptide derived from type-II collagen and a cell adhesion peptide LABL (CD11a237-246) from the I-domain of LFA-1 via a linker molecule. The hypothesis is that the CII-BPI molecules simultaneously bind to MHC-II and ICAM-1 on the surface of APC and block maturation of the immunological synapse. As a result, the differentiation of naïve T cells is altered from inflammatory to regulatory and/or suppressor T cells. The efficacies of CII-BPI molecules were evaluated upon intravenous injections in CIA mice. Results showed that CII-BPI-1 and CIIBPI-2 suppressed the joint inflammations in CIA mice in a dose-dependent manner and were more potent than the respective antigenic peptides alone. CII-BPI-3 was not as efficacious as CII-BPI-1 and CII-BPI-2. Significantly less joint damage was observed in CII-BPI-2 and CII-2 treated mice than in the control. The production of IL-6 was significantly lower at the peak of disease in mice treated with CII-BPI-2 compared to those treated with CII-2 and control. In conclusion, this is the first proof-of-concept study showing that BPI molecules can be used to suppress RA and may be a potential therapeutic strategy for the treatment of rheumatoid arthritis.

Vaccinelike and prophylactic treatments of EAE with novel I-domain antigen conjugates (IDAC): targeting multiple antigenic peptides to APC.

The objective of this work is to utilize novel I-domain antigenic-peptide conjugates (IDAC) for targeting antigenic peptides to antigen-presenting cells (APC) to simulate tolerance in experimental autoimmune encephalomyelitis (EAE). IDAC-1 and IDAC-3 molecules are conjugates between the I-domain protein and PLP-Cys and Ac-PLP-Cys-NH(2) peptides, respectively, tethered to N-terminus and Lys residues on the I-domain. The hypothesis is that the I-domain protein binds to ICAM-1 and PLP peptide binds to MHC-II on the surface of APC; this binding event inhibits the formation of the immunological synapse at the APC-T-cell interface to alter T-cell differentiation from inflammatory to regulatory phenotypes. Conjugation of peptides to the I-domain did not change the secondary structure of IDAC molecules as determined by circular dichroism spectroscopy. The efficacies of IDAC-1 and -3 were evaluated in EAE mice by administering iv or sc injections of IDAC in a prophylactic or a vaccinelike dosing schedule. IDAC-3 was better than IDAC-1 in suppressing and delaying the onset of EAE when delivered in prophylactic and vaccinelike manners. IDAC-3 also suppressed subsequent relapse of the disease. The production of IL-17 was lowered in the IDAC-3-treated mice compared to those treated with PBS. In contrast, the production of IL-10 was increased, suggesting that there is a shift from inflammatory to regulatory T-cell populations in IDAC-3-treated mice. In conclusion, the I-domain can effectively deliver antigenic peptides in a vaccinelike or prophylactic manner for inducing immunotolerance in the EAE mouse model.

Vaccine-like controlled-release delivery of an immunomodulating peptide to treat experimental autoimmune encephalomyelitis.

The objective of this work is to use colloidal gel from alginate-chitosan-PLGA complex to deliver Ac-PLP-BPI-NH2-2 peptide in a controlled-release manner as a vaccine-like therapeutic to suppress experimental autoimmune encephalomyelitis (EAE) in the mouse model. Oppositely charged PLGA nanoparticles were prepared by a solvent diffusion method. The carboxyl group of the alginate and the amine group of the chitosan coated the nanoparticles with negative and positive charges, respectively. The peptide (Ac-PLP-BPI-NH2-2), designed to bind to MHC-II and ICAM-1 simultaneously, was formulated into the colloidal gel by physical mixture. Vaccine-like administration of the peptide-loaded colloidal gel (Ac-PLP-BPI-NH2-2-NP) was achieved by subcutaneous (sc) injection to EAE mice. Disease severity was measured using clinical scoring and percent change in body weight. Cytokine production was determined using the splenocytes from Ac-PLP-BPI-NH2-2-NP-treated mice and compared to that of controls. Ac-PLP-BPI-NH2-2-NP suppressed and delayed the onset of EAE as well as Ac-PLP-BPI-NH2-2 when delivered in a vaccine-like manner. IL-6 and IL-17 levels were significantly lower in the Ac-PLP-BPI-NH2-2-NP-treated mice compared to the mouse group treated with blank colloidal gel, suggesting that the mechanism of suppression of EAE is due to a shift in the immune response away from Th17 production. The results of this study suggest that a one-time sc administration of Ac-PLP-BPI-NH2-2 formulated in a colloidal gel can produce long-term suppression of EAE by reducing Th17 proliferation.

I-domain-antigen conjugate (IDAC) for delivering antigenic peptides to APC: synthesis, characterization, and in vivo EAE suppression.

The objectives of this work are to characterize the identity of I-domain-antigen conjugate (IDAC) and to evaluate the in vivo efficacy of IDAC in suppressing experimental autoimmune encephalomyelitis (EAE) in mouse model. The hypothesis is that the I-domain delivers PLP(139-151) peptides to antigen-presenting cells (APC) and alters the immune system by simultaneously binding to ICAM-1 and MHC-II, blocking immunological synapse formation. IDAC was synthesized by derivatizing the lysine residues with maleimide groups followed by conjugation with PLP-Cys-OH peptide. Conjugation with PLP peptide does not alter the secondary structure of the protein as determined by CD. IDAC suppresses the progression of EAE, while I-domain and GMB-I-domain could only delay the onset of EAE. As a positive control, Ac-PLP-BPI-NH(2)-2 can effectively suppress the progress of EAE. The number of conjugation sites and the sites of conjugations in IDAC were determined using tryptic digest followed by LC-MS analysis. In conclusion, conjugation of I-domain with an antigenic peptide (PLP) resulted in an active molecule to suppress EAE in vivo.

Antigen-specific blocking of CD4-specific immunological synapse formation using BPI and current therapies for autoimmune diseases.

In this review, we discuss T-cell activation, etiology, and the current therapies of autoimmune diseases (i.e., MS, T1D, and RA). T-cells are activated upon interaction with antigen-presenting cells (APC) followed by a "bull's eye"-like formation of the immunological synapse (IS) at the T-cell-APC interface. Although the various disease-modifying therapies developed so far have been shown to modulate the IS and thus help in the management of these diseases, they are also known to present some undesirable side effects. In this study, we describe a novel and selective way to suppress autoimmunity by using a bifunctional peptide inhibitor (BPI). BPI uses an intercellular adhesion molecule-1 (ICAM-1)-binding peptide to target antigenic peptides (e.g., proteolipid peptide, glutamic acid decarboxylase, and type II collagen) to the APC and therefore modulate the immune response. The central hypothesis is that BPI blocks the IS formation by simultaneously binding to major histocompatibility complex-II and ICAM-1 on the APC and selectively alters the activation of T cells from T(H)1 to T(reg) and/or T(H)2 phenotypes, leading to tolerance.

A peptide from the beta-strand region of CD2 protein that inhibits cell adhesion and suppresses arthritis in a mouse model.

Cell adhesion molecules play a central role at every step of the immune response. The function of leukocytes can be regulated by modulating adhesion interactions between cell adhesion molecules to develop therapeutic agents against autoimmune diseases. Among the different cell adhesion molecules that participate in the immunologic response, CD2 and its ligand CD58 (LFA-3) are two of the best-characterized adhesion molecules mediating the immune response. To modulate the cell adhesion interaction, peptides were designed from the discontinuous epitopes of the beta-strand region of CD2 protein. The two strands were linked by a peptide bond. beta-Strands in the peptides were nucleated by inserting a beta-sheet-inducing Pro-Gly sequence with key amino acid sequences from CD2 protein that binds to CD58. Using a fluorescence assay, peptides that exhibited potential inhibitory activity in cell adhesion were evaluated for their ability to bind to CD58 protein. A model for peptide binding to CD58 protein was proposed based on docking studies. Administration of one of the peptides, P3 in collagen-induced arthritis in the mouse model, indicated that peptide P3 was able to suppress rheumatoid arthritis in mice.