Ahmed H. Badawi

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 “bulls 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 TH1 to Treg and/or TH2 phenotypes, leading to tolerance.

Immune modulating peptides for the treatment and suppression of multiple sclerosis

Multiple sclerosis (MS) is a neurodegenerative disease in which the immune system recognizes proteins of the myelin sheath as antigenic, thus initiating an inflammatory reaction in the central nervous system. This leads to demyelination of the axons, breakdown of the blood-brain barrier, and lesion formation. Current therapies for the treatment of MS are generally non-specific and weaken the global immune system, thus making the individual susceptible to opportunistic infections. Antigenic peptides and their derivatives are becoming more prevalent for investigation as therapeutic agents for MS because they possess immune-specific characteristics. In addition, other peptides that target vital components of the inflammatory immune response have also been developed. Therefore, the objectives of this review are to (a) summarize the immunological basis for the development of MS, (b) discuss specific and non-specific peptides tested in EAE and in humans, and (c) briefly address some problems and potential solutions with these novel therapies.

Structure, Size, and Solubility of Antigen Arrays Determines Efficacy in Experimental Autoimmune Encephalomyelitis

ABSTRACTPresentation of antigen with immune stimulating “signal” has been a cornerstone of vaccine design for decades. Here, the antigen plus immune “signal” of vaccines is modified to produce antigen-specific immunotherapies (antigen-SITs) that can potentially reprogram the immune response toward tolerance of an autoantigen. The codelivery of antigen with a cell adhesion inhibitor using Soluble Antigen Arrays (SAgAs) was previously shown to slow or halt experimental autoimmune encephalomyelitis (EAE), a murine form of multiple sclerosis (MS). SAgAs are comprised of a hyaluronic acid backbone with cografted intercellular cell adhesion molecule-1 ligand derived from αL-integrin (CD11a237–246, “LABL”) and an encephalitogenic epitope peptide of proteolipid protein (PLP139–151, “PLP”). Here, the physical characteristics of the carrier were investigated to evaluate how structure, size, and solubility drive the immune response when treating EAE. A bifunctional peptide (small, soluble), SAgAs (large, soluble), and PLGA nanoparticles (large, insoluble) all displaying PLP and LABL in equimolar ratios were compared. Maximum EAE suppression was achieved with coincident display of both peptides on a soluble construct.

Suppression of MOG- and PLP-induced experimental autoimmune encephalomyelitis using a novel multivalent bifunctional peptide inhibitor

Previously, bifunctional peptide inhibitors (BPI) with a single antigenic peptide have been shown to suppress experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner. In this study, a multivalent BPI (MVBMOG/PLP) with two antigenic peptides derived from myelin oligodendrocyte glycoprotein (MOG38-50) and myelin proteolipid protein (PLP139-151) was evaluated in suppressing MOG38-50- and PLP139-151-induced EAE. MVBMOG/PLP significantly suppressed both models of EAE even when there was some evidence of epitope spreading in the MOG38-50-induced EAE model. In addition, MVBMOG/PLP was found to be more effective than PLP-BPI and MOG-BPI in suppressing MOG38-50-induced EAE. Thus, the development of MVB molecules with broader antigenic targets can lead to suppression of epitope spreading in EAE.

Effect of modification of the physicochemical properties of ICAM-1-derived peptides on internalization and intracellular distribution in the human leukemic cell line HL-60.

The objective of this work is to test the hypothesis that increasing the hydrophilicity of DOX-peptide conjugates may modify their entry mechanisms into HL-60 cells from passive diffusion to receptor-mediated uptake. To test this hypothesis, the entry mechanisms and the intracellular disposition of DOX-cIBR7, DOX-PEGcIBR7, FITC-cIBR, and FITC-cIBR7 were evaluated in HL-60 cells. To increase the hydrophilicity of the peptide, the cIBR peptide (cyclo(1,12)Pen-PRGGSVLVTGC) was modified to cIBR7 peptide (cyclo(1,8)CPRGGSVC) by removing the hydrophobic residues at the C-terminus. DOX-cIBR7 conjugate, which has higher hydrophilicity than DOX-cIBR, was synthesized. Second, a hydrophilic linker (11-amino-3,6,9-trioxaundecanate linker) was incorporated between DOX and cIBR7 to generate DOX-PEGcIBR7 with higher hydrophilicity than DOX-cIBR7. As controls, FITC-cIBR and FITC-cIBR7 were used to check for any endocytic uptake process of the peptide. As previously found with DOX-cIBR, DOX-cIBR7, and DOX-PEGcIBR7, conjugates enter the cells via passive diffusion and not via the energy-dependent endocytic process. This result suggests that an increase in hydrophilicity does not influence the entry mechanism of the DOX-peptide conjugates. In contrast to the DOX-cIBR7 conjugate, the FITC-cIBR7 conjugate showed energy-dependent cellular entry into the cells and followed an endocytic pathway similar to that for dextran. Finally, the entry of DOX-cIBR7 and DOX-PEGcIBR into the cell cytosol was shown to be due to the properties of DOX and not to those of the peptide.

Immune Tolerance Induction against Experimental Autoimmune Encephalomyelitis (EAE) Using A New PLP-B7AP Conjugate that Simultaneously Targets B7/CD28 Costimulatory Signal and TCR/MHC-II Signal

Most of the current therapies used in the treatment of multiple sclerosis (MS) are either ineffective or have adverse side effects. As such, there is a need to develop better therapies that specifically target myelin-specific aberrant immune cells involved in CNS inflammation without compromising the general immune system. In the present study, we developed a new bifunctional peptide inhibitor (BPI) that is effective and specific. Our BPI (PLP-B7AP) is composed of an antigenic peptide from myelin proteolipid protein (PLP139–151) and a B7 antisense peptide (B7AP) derived from CD28 receptor. The main hypothesis is that PLP-B7AP simultaneously targets MHC-II and B7-costimulatory molecules on the surface of antigen presenting cells (APC) and possibly alters the differentiation of naïve T cells from inflammatory to regulatory phenotypes. Results showed that PLP-B7AP was very effective in suppressing experimental autoimmune encephalomyelitis (EAE) compared to various controls in a mouse model. PLP-B7AP was effective when administered both before and after disease induction. Secreted cytokines from splenocytes isolated during periods of high disease severity and remission indicated that PLP-B7AP treatment induced an increased production of anti-inflammatory cytokines and inhibited the production of pro-inflammatory cytokines. Further, analysis of cortical brain tissue sections showed that PLP-B7AP treated mice had significantly lower demyelination compared to the control group. All these taken together indicate that the T cell receptor (TCR) and the CD28 receptor can be targeted simultaneously to improve efficacy and specificity of potential MS therapeutics.

Controlling immune response and demyelination using highly potent bifunctional peptide inhibitors in the suppression of experimental autoimmune encephalomyelitis

In this study, we investigated the efficacy of new bifunctional peptide inhibitors (BPIs) in suppressing experimental autoimmune encephalomyelitis (EAE) in an animal model. BPI [e.g. proteolipid protein–cyclo(1,8)‐CPRGGSVC‐NH2 (PLP‐cIBR)] is a conjugate between the PLP139–151 peptide derived from proteolipid protein (PLP) and the cIBR7 peptide derived from domain‐1 (D1) of intercellular adhesion molecule‐1 (ICAM‐1). PLP–cIBR is designed to bind to major histocompatibility complex (MHC)‐II and leucocyte function‐associated antigen‐1 (LFA‐1) simultaneously to inhibit the formation of the immunological synapse and alter the differentiation and activation of a subpopulation of T cells, thus inducing immunotolerance. The results show that PLP–cIBR is highly potent in ameliorating EAE, even at low concentrations and less frequent injections. Mice treated with PLP–cIBR had a higher secretion of cytokines related to regulatory and/or suppressor cells compared to phosphate‐buffered saline (PBS)‐treated mice. In contrast, T helper type 1 (Th1) cytokines were higher in mice treated with PBS compared to PLP–cIBR, suggesting that it suppressed Th1 proliferation. Also, we observed significantly less demyelination in PLP‐cIBR‐treated mice compared to the control, further indicating that PLP–cIBR promoted protection against demyelination.

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.

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.

Peptides and Proteins for Treatment and Suppression of Type 1 Diabetes

Type-1 diabetes (T1D) is an autoimmune disease in which self-reactive immune cells infiltrate the islets in the pancreas to destroy -cells. One of many possible causes is that selfreactive T cells that are normally eliminated can escape from the thymus along with normal T cells. The escaped T cells can be activated in response to a low level of secondary selfantigens, which can lead to a major step for tissue self-recognition. For activation, T cells interact with antigen-presenting cells (APC) via formation of the immunological synapse, which has a “bull’s eye” structure at the membrane interface between both cells (Grakoui et al., 1999). The immunological synapse is composed of two segregated clusters of Signal-1 and Signal-2 molecular complexes. Signal-1 is generated by interaction between T-cell receptors (TCR) and antigen/multi histocompatibility complex-II (Ag/MHC-II). Signal-2 (costimulatory signal) can be delivered by a positive signal via B7/CD28 interactions or a negative signal via B7/CTLA-4 interactions. In addition, the CD40/CD154 costimulatory interaction between APC and T cells was found to induce an inflammatory immune response (Baker et al., 2008; Munro et al., 2007). Cell adhesion molecule interactions such as ICAM-1/LFA-1 interactions have also been categorized as a positive signal (Bromley et al., 2001; Grakoui et al., 1999). The positive costimulatory signal assists the induction of T-cell activation while the negative costimulatory signal suppresses T-cell activation (Bour-Jordan et al., 2011; Manikwar et al., 2011). The formation of the immunological synapse involves translocation of Signal-1 and Signal-2. Prior to the translocation process, Signal-1 is clustered at the periphery and Signal-2 is clustered at the center. Then, Signal-1 and Signal-2 switch places to establish the immunological synapse where Signal-1 is at the center (called central zone supramolecular activation complex or cSMAC) and Signal-2 is at the periphery (peripheral zone supramolecular activation complex or pSMAC) (Bromley et al., 2001; Grakoui et al., 1999). TCR on T cells recognize self-antigens presented on MHC-II molecules on the surface of APC for activation of self-reactive T cells to initiate autoimmune diseases. In T1D, glutamic acid decarboxylase-65 (GAD65), is one of the important self-antigens in humans, and is a reliable marker in overt diabetes (Tisch et al., 1998). Administration of GAD peptides in complete Freund’s adjuvant (CFA) into non-obese diabetes (NOD) mice triggers insulitis and destruction of -cells to cause diabetes (Liu et al., 1999; Tisch et al., 1999; Yoon et al., 1999). There is a correlation between islet expression of GAD enzymes and the development of T1D. Different types of MHC-II molecules such as I-Ag7 and I-Ag7.PD recognize different