ArticleNovember 21, 2025

Overcoming Synthetic Challenges and Evaluating in Vivo Efficacy of the MOG-Fc-Bifunctional Peptide Inhibitor for EAE in Mice
Click to copy article linkArticle link copied!

Rucha Mahadik
Rucha Mahadik
Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States
More by Rucha Mahadik
Andrea L. Villela-Nava
Andrea L. Villela-Nava
Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States
More by Andrea L. Villela-Nava
https://orcid.org/0009-0008-4299-6703
Lun Xin
Lun Xin
Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States
BioDev Department, WuXi Biologics USA, Cranbury, New Jersey 08512, United States
More by Lun Xin
Teruna J. Siahaan*
Teruna J. Siahaan
Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States
*Email: [email protected]. Tel.: +1 785-864-7327.
More by Teruna J. Siahaan
https://orcid.org/0000-0001-7250-0627
Thomas Tolbert*
Thomas Tolbert
Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States
*Email: [email protected]. Tel.: +1 785-864-1898.
More by Thomas Tolbert
https://orcid.org/0000-0001-7726-3456

Open PDF Supporting Information (1)

Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2025, 36, 12, 2700–2709

Click to copy citationCitation copied!

https://doi.org/10.1021/acs.bioconjchem.5c00518

Published November 21, 2025

Request reuse permissions

Abstract

Click to copy section linkSection link copied!

Multiple sclerosis (MS) is an autoimmune disease that causes neural degeneration as a result of the immune system launching an attack on the myelin sheath surrounding neurons. MS has multiple disease states; each one has been associated with a different onset pathway and requires a separate treatment. Primary progressive MS (PPMS) is a rare form of MS that affects 10–15% of MS patients, and Ocrelizumab is currently the only FDA-approved treatment on the market. While it can be effective in managing PPMS, Ocrelizumab can only delay the onset of the disease. In this study, MOG-Fc-BPI was designed as a potential therapeutic agent to suppress experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner, altering immune cells from an inflammatory to a regulatory phenotype. Here, MOG-Fc-BPI was successfully synthesized by conjugating the MOG-R₅ peptide using sortase A enzyme to the C-terminus of the Fc-domain with LABL peptide at the N-terminus. Purified MOG-Fc-BPI was formulated to reach a concentration of 15 mg/mL for the in vivo study. MOG-stimulated EAE in C57BL/6 mice (a model for PPMS) that were treated with MOG-Fc-BPI on days 4 and 7 at 35 nmol/dose showed complete disease suppression on day 19 (score = 0; without symptoms) compared to PBS. The MOG-Fc-BPI-treated mice showed increased body weights throughout the study, while PBS-treated mice lost around 10% bodyweight during the peak of the disease without recovery up to the end of the study. Overall, this study provided a proof-of-concept that MOG-Fc-BPI has the potential to suppress PPMS.

This publication is licensed for personal use by The American Chemical Society.

Subjects

Introduction

Click to copy section linkSection link copied!

Multiple sclerosis (MS) is an autoimmune disease characterized by neuronal degeneration in the central nervous system (CNS). (1−3) Immune cells infiltrate the CNS to attack the myelin sheath surrounding neurons, resulting in inflammation, demyelination, axonal loss, and gliosis. (3) The progression of neural degeneration leads to both physical and cognitive impairment in MS patients. The onset of the disease has been attributed to the activation of autoreactive T cells against various myelin sheath proteins such as proteolipid protein (PLP), myelin basic protein (MBP), and myelin oligodendrocyte glycoprotein (MOG). (2) Depending on which antigenic protein is responsible for the activation of autoreactive T cells, the patients can develop a different disease state of MS, such as relapse-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), and progressive relapsing MS (PRMS). (3) RRMS is the most common form of MS, affecting 87% of MS patients; these patients experience periods of disease progression, followed by periods of symptom improvement. A majority of RRMS patients will later develop SPMS, in which they reach a state of irreversible damage to the neurons and lose their remittance period. PPMS is experienced by 10–15% of MS patients; they have constant disease progression from the time of onset without any remittance periods. (3) PRMS is the least common form (approximately 5% of MS patients), in which patients experience constant disease progression with periods of increased disease symptoms.

MS is difficult to treat because the different disease states could be related to different myelin antigens; as a result, each disease state may require a different treatment strategy. (2,3) Current treatments of MS include anti-inflammatories and disease-modifying therapies (DMTs). (4) Anti-inflammatories are used to reduce disease symptoms without suppressing disease progression. DMTs aim at interfering with the disease mechanism that can damage neurons to reduce symptoms. While DMTs can be effective in treating MS, they are limited to treating only one disease state, usually RRMS. Because DMTs can suppress the global immune system, this treatment can also make patients vulnerable to pathogenic infections. To account for these limitations, our group has designed a class of potential therapeutics called the bifunctional peptide inhibitors (BPIs) to target only the subset of autoreactive T cells in an antigen-specific manner related to myelin sheath proteins (PLP, MBP, and MOG). (5,6) BPIs are composed of two peptides, an antigenic peptide such as a myelin antigenic peptide (PLP, MBP, or MOG) and a signal-2 blocking peptide (i.e., LABL peptide) that are connected by a linker. (5,6)

BPIs are designed to bind antigen-presenting cells (APC) to present the antigenic peptide via major histocompatibility complex II (MHC-II) to alter the balance of T cells from inflammatory to regulatory phenotypes. (2,5,6) Naïve T cells can interact with the antigen-MHC-II complex (Ag/MHC-II) on APCs via the T cell receptor (TCR), which is known as Signal-1. Simultaneously, costimulatory molecules on the surface of APCs and T cells can interact as Signal-2; in this case, intercellular adhesion molecule-1 (ICAM-1) on the surface of the APC can bind with LFA-1 on the surface of the T cell. (2,5,6) During the initial interaction of T cell and APC, TCR-Ag/MHC-II complexes (Signal-1) are formed and located at the periphery of the interface, while the costimulatory molecules (Signal-2) are clustered in the center of the two cells. During the translocation of signal clusters, the TCR-Ag/MHC-II complexes move to the center cluster of the cells, while the costimulatory molecules LFA-1 and ICAM-I move to the periphery cluster to finally form the immunological synapse (IS). The IS signals the naïve T cell to differentiate into inflammatory Th1 and Th17 cells that attack the myelin sheath in the CNS. To prevent the inflammatory T cell response, the BPI is designed to selectively target the myelin-specific T cells and switch the differentiation to a regulatory response (e.g., Treg cells). (2) The hypothesis is that the antigenic peptide portion of BPI can selectively influence subpopulations of autoreactive T cells that recognize a particular myelin sheath protein (i.e., PLP, MOG, or MBP). In addition, the LABL peptide derived from LFA-1 binds to ICAM-1 and inhibits IS and Signal-2 formation; in such a situation, the naïve T cell is then differentiated into regulatory T cells (Tregs) to promote tolerance. (6,7)

BPIs have been shown to suppress experimental autoimmune encephalomyelitis (EAE), (8−10) the mouse model of MS, as well as in Type-I diabetes (11) and rheumatoid arthritis (12) in animal models. Different antigenic peptides of MS are derived from PLP, MBP, and MOG, and these peptides can be used to induce different disease states of EAE as models for the different disease states of MS. PLP peptide in complete Freund’s Adjuvant (CFA) induces relapse-remitting EAE, while MOG peptide in CFA induces primary progressive EAE. Finally, the MBP peptide in CFA induces a mixed form of relapse-remitting and primary progressive EAE. The original BPI molecules used aminocaproic acid (8,9) or PEG (13) chains as linkers between the antigenic peptide and LABL peptide; however, these BPIs have a short plasma half-life (e.g., 2–3 h). (13) To improve half-life, new Fc-BPI molecules were designed in which the Fc region of an IgG1 antibody was used as a linker between the two peptides. (2,14) PLP-Fc-BPI and MBP-Fc-BPI have been successfully synthesized using sortase-mediated ligations (15−17) and have been found to have efficacy in suppressing PLP- and MBP-stimulated EAE, respectively. (14) The MBP-Fc-BPI fusion protein has a better half-life of 21.3 h than the parent peptide-based BPIs (2–3 h). (13) It was found that MBP-Fc-BPI suppresses EAE at a much lower dose (14) compared to that of the parent MBP-BPI; (8,9,13) thus, it may potentially reduce side effects such as anaphylaxis.

In this study, we synthesize MOG-Fc-BPI (Figure 1) and evaluate its efficacy in MOG-stimulated EAE in mice as an animal model for PPMS. Due to the hydrophobicity of the MOG peptide, it was shown previously that direct expression of MOG-Fc-BPI was very challenging. (14) Thus, it is difficult to test the efficacy of MOG-Fc-BPI in the EAE mouse model of PPMS, which lacks a remission period. Recently, we have successfully synthesized MOG-Fc-BPI by conjugating the MOG-R₅ peptide with the LABL-Fc-ST molecule using sortase A enzyme (Figure 1). Finally, the MOG-Fc-BPI was very effective in suppressing and reversing the disease symptoms of MOG-stimulated EAE.

Figure 1. Structure of MOG-Fc-BPI consists of an Fc-domain with the LABL peptide at the N-terminus and the MOG-R₅ peptide at the C-terminus.

Materials and Methods

Click to copy section linkSection link copied!

Expression and Purification of LABL-Fc-ST

LABL-Fc-ST was expressed in P. pastoris (Komagataella phaffi) cells using the protocol described in White et al., and the experimental detail was described in the Supplementary Method 1. (14,15,18) The LABL-Fc-ST identity was determined in reduced and deglycosylated form using electrospray Ionization ESI-TOF MS (Figure 2A) as well as reduced and various glycoforms using MALDI-TOF (Figure 2B).

Figure 2. MS spectra of the purified LABL-Fc-ST protein. (A) Intact-MS spectrum of the reduced and deglycosylated LABL-Fc-ST protein with [M + H]: 27,024 amu. (B) The MS spectrum of fully glycosylated LABL-Fc-ST with the Man8 glycan (expected [M + Na]: 28,749 amu; observed mass: 28,749 amu) as well as the Man9 glycan (expected [M + Na]: 28,910 amu; observed mass: 28,911 amu).

Mass Spectrometry Analysis

The molecular weights (MWs) of LABL-Fc-St and MOG-Fc-BPI were determined using two mass spectrometry methods using (a) the reduced and glycosylated form and (b) the reduced and deglycosylated form. The intact-MS analysis was performed in the Waters BioAccord LC-MS system with ESI ionization and TOF for detection. To reduce the protein, 40 μg of the protein sample (1.0 mg/mL) was treated with 25 mM dithiothreitol (DTT) in 1× Rapid PNGase F buffer (New England BioLabs, Ipswich, MA) and incubated at 75 °C for 8 min to achieve reduction. To deglycosylate the protein, 2.0 μL of Rapid PNGase F (New England BioLabs) was added to the reaction, and the mixture was incubated at 50 °C for 15 min. Then, the sample was diluted with Milli-Q water to a final protein concentration of 0.4 mg/mL. The Waters ACQUITY Protein BEH C4 column (300 Å, 1.7 μm, 2.1 × 50 mm) was used for protein separation with the mobile phases containing (A) 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. A 10 μL aliquot of the sample was injected for analysis, and the resulting spectra were deconvoluted to calculate the molecular weight.

Synthesis and Purification of the MOG Peptide

MOG peptide with N-terminal triglycine and C-terminal penta-arginine modifications (GGGWYRSPFSRVVHLGRRRRR-NH₂), G₃MOGR₅, was obtained from DG Peptides (Hangzhou City, China). The peptide has >96% purity as determined by HPLC. The expected MW of the peptide was 2555.9 amu with the observed masses of 512.18 m/z for [M + 5H], 639.98 m/z for [M + 4H], and 853.09 m/z for [M + 3H] (Figure 3).

Figure 3. MS spectrum of G₃MOGR₅ peptide showed peaks for [M + 5H] (512.18 m/z), [M + 4H] (639.98 m/z), and [M + 3H] (853.09 m/z) with an overall observed mass of 2555.9 amu, which was similar to the expected mass of 2555.94 amu.

Sortase-Mediated Ligation with LABL-Fc-ST and MOG Peptide

Initially, the MOG peptide was ligated to the C-terminus of LABL-Fc-ST using sortase-mediated ligation on a submilligram scale. In each ligation, 10 μM LABL-Fc-ST protein was combined with 6 mM CaCl₂, 1 mM G₃MOGR₅, and 5 μM sortase in TBS at pH 7.5 in a 150 μL reaction. The ligations were incubated at 35 °C for 24 h. After 24 h, they were quenched by the addition of 25 mM EDTA. The ligations were then observed by mass spectrometry.

On a larger scale (60 mg, 6 reactions, 5 mL reaction), the MOG peptide was ligated to the C-terminus of LABL-Fc-ST protein using sortase-mediated ligation. In total, the ligation reaction contained: 37 μM LABL-Fc-ST protein (60 mg), 6 mM CaCl₂, 1.0 mM G₃MOGR₅ (77.0 mg), and 7.0 μM sortase (3.7 mg). The pH was adjusted to pH 7.5 using a 1.0 M Tris base. The ligation was allowed to proceed for 24 h at 35 °C and then stored at −20 °C until purification. The sample was purified by using protein G affinity chromatography. The purified MOG-Fc-BPI was stored in the protein G elution buffer (90% (v/v) 0.1 M Glycine, pH 2.7, and 10% (v/v) 1 M Tris, pH 8–9) at −20 °C until formulation for in vivo dosing. The MOG-Fc-BPI was characterized using WES capillary cartridge analysis in reduced and intact forms (Supplementary Figure S1) and by mass spectrometry in reduced-deglycosylated (Figure 5A) and reduced-glycosylated forms (Figure 5B).

Efficacy of MOG-Fc-BPI in EAE Mice

The efficacy of MOG-Fc-BPI was determined in MOG-induced EAE mice. On day 0, a total of 20 female C57BL/6 mice (6-week old; 2 groups; 10 mice per group) were immunized subcutaneously with 560 μg of MOG in a 0.2 mL emulsion of equal volume phosphate-buffered saline (PBS) and complete Freund’s adjuvant (CFA) containing killed Mycobacterium tuberculosis strain H37RA (final concentration of 4 mg/mL; Becton, Dickinson and Company (BD), Sparks, MD). The MOG/CFA emulsion was administered above the shoulders and the flanks (total of 4 doses; 50 μL per dose) subcutaneously (s.q.). Each mouse received a 400 ng dose of pertussis toxin (List Biological Laboratories Inc., Campbell, CA) administered intraperitoneally (ip) on days 0 and 2. On days 4 and 7, the mice were given 150 μL/day of treatment compounds via intravenous (i.v.) injections: group 1 received 2 doses of MOG-Fc-BPI (at 35 nmol/dose), and group 2 received PBS. For the duration of the 25-day study, the mice were scored from 0 to 5 based on the physical symptoms with the following scoring scale: 0 = no symptoms, 1 = tail weakness or ataxia, 2 = incomplete paralysis of one to two hind limbs, 3 = complete paralysis of one or two hind limbs, 4 = complete paralysis of one or two hind limbs and paralysis of one or two fore limbs, and 5 = moribund or dead.

Results

Click to copy section linkSection link copied!

Synthesis of MOG-Fc-BPI

After expression of LABL-Fc-ST on a 10 L scale (2 × 5 L), the supernatant was separated from the cells, filtered, and purified using protein A chromatography to obtain purified LABL-Fc-ST. The identities of the purified glycosylated and deglycosylated LABL-Fc-ST were confirmed using several mass spectrometry methods (Figure 2). The reduced and deglycosylated form of LABL-Fc-ST protein showed a single peak at [M + H]: 27,024 amu. The sodium adduct for the Man8-LABL-Fc-ST glycoform was observed at 28,749 amu (expected [M + Na⁺] = 28,749 amu), and the Man9-LABL-Fc-ST glycoform sodium adduct was observed at 28,911 amu (expected [M + Na⁺] = 28910 amu), confirming the identity of the starting materials for the conjugation reaction.

G₃MOGR₅ peptide was used to improve solubility to a millimolar concentration that is necessary for the sortase A catalyzed reaction with LABL-Fc-ST. A large excess of the G₃MOGR₅ peptide is needed as a nucleophile to react with the sortase-protein intermediate. Without the addition of several arginine residues, the MOG peptide precipitated during the enzymatic reaction. (14) The expected MW of G₃MOGR₅ is 2555.91 amu, and the observed MW of 2555.9 amu was determined from the observed multiple charged states (m/z): [M + 5H] = 512.18 amu; [M + 4H] = 639.98 amu; and [M + 3H] = 853.09 amu (Figure 3).

Sortase-Mediated Ligation

Sortase enzyme was used to conjugate the N-terminal tri-Gly on the MOG peptide to the C-terminal sortase tag region (LPETGGG) of LABL-Fc-ST (Figure 4). During the sortase reaction, the active site cysteine in the sortase cleaves between the threonine and glycine of the sortase tag of LABL-Fc-ST to create a thioester with the threonine while removing the C-terminal triglycine (Figure 1). The N-terminal triglycine on the MOG peptide acts as a nucleophile to react with the thioester intermediate to make a peptide bond, generating the final MOG-Fc-BPI conjugate (Figure 4). A molar ratio of peptide to protein of 100:1 was used in small-scale reactions, and a molar ratio of 27:1 was used in large-scale reactions to favor the formation of the desired MOG-Fc-BPI product.

Figure 4. Synthetic scheme for the production of MOG-Fc-BPI. LABL-Fc-ST contains the C-terminal sortase recognition tag, LPETGGG. The G₃-MOG-R₅ peptide was conjugated to the C-terminus of LABL-Fc-ST using sortase A, which recognizes the LPETGGG sequence and cleaves between the threonine and glycine residues to form a thioester with threonine while releasing the C-terminal triglycine peptide. The N-terminal triglycine residues of G₃-MOG-R₅ act as a nucleophile in the sortase A reaction to attack the thioester and produce the desired product, MOG-Fc-BPI.

The mass spectrometry analysis of the reduced and deglycosylated form of MOG-Fc-BPI showed a major peak at [M + H]: 29,391 amu (Figure 5A). The glycosylated forms of MOG-Fc-BPI are shown in Figure 5B. The majority species was the Man8 glycoform of MOG-Fc-BPI (Figure 5B; Peak a). The nonglycosylated product of MOG-Fc-BPI was found in a low amount (Figure 5B; Peak b). There was a small amount of unligated LABL-Fc-ST found in the reaction product, suggesting that the reaction was not complete (Figure 5; Peak c).

Figure 5. MS spectra of MOG-Fc-BPI. (A) The intact-MS spectrum of the reduced and deglycosylated MOG-Fc-BPI protein with [M + H] = 29,391 amu. (B) MS spectrum of purified glycosylated MOG-Fc-BPI with major species such as (a) Man8 glycoform of MOG-Fc-BPI (expected = 31,093.8 amu, observed = 31,092.8 amu); (b) MOG-Fc-BPI with no glycan (expected = 29,372.3 amu, observed = 29,392.1 amu); and (c) LABL-Fc-ST (expected = 28,726.9 amu, observed = 28,726.7 amu).

MOG-Fc-BPI Formulation

Formulation design for the MOG-Fc-BPI was required to find a stable condition for in vivo dosing at ∼15 mg/mL. The buffer solution of the MOG-Fc-BPI protein was switched from purification buffer to the injection buffer using a 10K MWCO concentrator to achieve the desired injected dose. Various excipients were added to stabilize the protein formulation, and after buffer exchange, the formation of a cloudy solution and particulates was monitored by visual observation and optical density. The protein aggregates and precipitates were filtered using a syringe through 0.22 μm filters; then, the concentration of protein in the filtrate was determined by measuring the optical density at 280 nm. We found that MOG-Fc-BPI has limited solubility up to 3 mg/mL in PBS. In contrast, a buffer containing 0.1 M sodium phosphate, 0.15 M NaCl, and 50 mM sucrose at pH 7.2 can stabilize MOG-Fc-BPI at a concentration of ∼15 mg/mL for in vivo evaluation in the EAE animal model.

Animal Studies

The activity of MOG-Fc-BPI to suppress the MOG-stimulated EAE was evaluated in C57BL/6 mice. Two groups of mice were induced to stimulate the disease by dosing with an emulsion of MOG peptide in CFA along with Mycobacterium tuberculosis and pertussis toxin. The first group (n = 6; treated group) was administered with 150 μL of MOG-Fc-BPI on days 4 and 7 at 35 nmol/dose after disease induction on day 0. Similarly, the second group (n = 8) was injected with 150 μL of PBS on days 4 and 7 as control mice. The disease severity was determined using clinical scores that ranked their physical symptoms (Figure 6A, B). The changes in body weights were monitored throughout the study (Figure 6C, D).

Figure 6. Activity of MOG-Fc-BPI to suppress EAE was determined by comparing (A) daily clinical scores, (B) AUC of clinical scores, (C) daily body weights, and (D) AUC of body weights to those of PBS control. (A) The MOG-Fc-BPI-treated mice received 2 doses of 35 nmol/dose injections on days 4 and 7 and showed lower clinical scores than those of PBS-treated mice. (B) AUC of clinical scores for PBS-treated mice was significantly higher than that of MOG-Fc-BPI-treated mice over 25 days (p < 0.001). (C) The differences in the increase body weights over 25 day period for MOG-Fc-BPI- and PBS-treated mice. There is inhibition in the increase of body weights on days 14–25 in PBS-treated mice but not MOG-Fc-BPI-treated mice. (D) A significantly higher ACU of body weights of MOG-Fc-BPI-treated mice compared to PBS-treated mice (p = 0.0274).

In PBS-treated mice, the disease scores started to increase on day 13 and continued to increase the severity up to day 25, with the average score around 3, indicating complete paralysis of one or two hind limbs (Figure 6A). In contrast, mice treated with MOG-Fc-BPI experienced the onset of disease on day 12, with a peak of the disease on day 14, with an average score of 1.5, indicating a tail weakness and a sign of a limb weakness at the peak. Then, the scores reversed to 0 or normal on day 18, and these scores were maintained at 0 until day 25, indicating a complete disease suppression in all mice treated with MOG-Fc-BPI. To assess the difference in disease scores, the area under the curve (AUC) for disease scores for each group was determined (Figure 6B); the results indicated that the PBS-treated mice had significantly higher disease scores than MOG-Fc-BPI-treated mice (p < 0.001).

The body weights of the animals can also be used to measure the quantitative degree of sickness of the mice during the study. The MOG-Fc-BPI-treated mice continuously increased in body weights from days 0 to 13, and the body weights plateaued from days 13 to 25, indicating that the treatment kept the animals reasonably healthy (Figure 6C). In contrast, the PBS-treated mice decreased in body weights from days 13 to 17 and never completely regained this lost weight from days 17 to 25 in comparison to MOG-Fc-BPI-treated mice (Figure 6C). PBS-treated mice had significantly lower AUC body weights than those treated with MOG-Fc-BPI (Figure 6D), indicating that MOG-Fc-BPI maintained the growth of body weights in EAE mice.

Discussion

Click to copy section linkSection link copied!

The PPMS state of MS disease has been very challenging to treat, and it is characterized by constant disease progression without any period of improvement. When compared with the specific inflammatory lesions in RRMS, progressive forms of MS have been characterized by global inflammation. Furthermore, PPMS patients have specifically shown oligodendrocyte degeneration and loss. (19) These differences in disease characteristics, such as symptoms and forms of neurological degeneration, suggest differences in the mechanism of disease onset. Therefore, different treatments to treat the disease may be necessary depending on what triggers the disease onset. As DMT, Ocrelizumab is used for PPMS and RRMS treatments; however, it only delays the disease progression without completely suppressing PPMS. (20) Some patients treated with Ocrelizumab developed progressive multifocal leukoencephalopathy (PML). (21) Because of the limited treatment options, there is a need to develop alternative treatments for PPMS with lower side effects.

In this study, we focused on an antigen-specific approach to alter the balance of T cells from inflammatory to regulatory phenotypes in MS. MOG-Fc-BPI contains an MOG peptide antigen and an LABL peptide as a Signal-2 blocker that can selectively modulate T-cell activation against myelin sheath proteins. The MOG peptide is derived from an active epitope of the MOG protein associated with PPMS. Thus, MOG-Fc-BPI was investigated to suppress MOG-stimulated EAE, a model for PPMS. Two mechanisms of action have been proposed for MOG-Fc-BPI in suppressing EAE disease (Figure 7). The first mechanism is via the MOG-Fc-BPI interaction with immature dendritic cells (iDC) (Figure 7A). Here, the MOG and LABL peptides simultaneously bind to MHC-II and ICAM-1, respectively. Then, the naïve T cell through the T-cell receptor (TCR) recognizes MOG/MHC-II complex on iDC (Signal-1); however, the LFA-1 receptor is blocked from recognizing ICAM-1 as Signal-2 by LABL. The presence of Signal-1 without Signal-2 causes prevention of the IS formation during naïve T cell and iDC interaction. This process directs the differentiation of the naïve T cell to a regulatory T cell (Treg) to alter the balance of the immune system from inflammatory to regulatory conditions. The second mechanism involves the simultaneous binding of MOG-Fc-BPI to MHC-II and ICAM-1 on mDC, followed by an interaction with a naïve T cell (Figure 7B). TCR on the naïve T cell recognizes the MOG/MHC-II complex on mDC as Signal-1. As previously indicated, the LABL fragment of MOG-Fc-BPI blocks Signal-2 while Signal-1 is connected; therefore, the process prevents the naïve T cell differentiation to inflammatory Th1 and Th17 cells.

Figure 7. Proposed mechanisms of action for MOG-Fc-BPI suppression of EAE disease involving iDC and mDC. (A) MOG-Fc-BPI binds simultaneously to ICAM-1 and MHC-II using LABL and MOG peptides, respectively, on iDC to block Signal-2 in the presence of Signal-1. and this also prevents immunological synapse (IS) formation. The presence of Signal-1 in the absence of Signal-2 causes naïve T cells to become Treg cells. (B) MOG-Fc-BPI binds to ICAM-1 and MHC-II on the surface of mDC. The TCRs on naïve T cells recognize the MOG/MHC-II complexes (Signal-1) with blocked Signal-2; as a result, this prevents the differentiation of naïve T cells to inflammatory Th17 or Th1 cells.

Fc-BPIs were designed to improve the BPI plasma stability, efficacy, and safety. The Fc-BPI uses the Fc domain of IgG1 to link between MOG and LABL peptides (Figure 1). PLP-Fc-BPI and MBP-Fc-BPI have been shown to completely suppress PLP- and MBP-induced EAE in mouse models, respectively. (14) MOG-Fc-BPI completely suppressed EAE with a lower dose and number of injections (2 × 35 nmol) compared to MOG-BPI, which only reduced the disease scores with three larger doses (3 × 100 nmol). (10) In another study, MBP-Fc-BPI showed a better in vivo half-life (21.3 h) than the parent BPI peptide (t_1/2 = 2–3 h), suggesting that the Fc domain improved the systemic circulation of Fc-BPIs. (13) (Figure 6A, B). (10) The complete suppression of the disease by MOG-Fc-BPI in PPMS EAE mice is remarkable. Normally, this animal model does not show any remittance period as observed in the RRMS EAE model. (8,9) The increase in body weights of MOG-Fc-BPI-treated mice indicates a healthy group of animals (Figure 6C, D). In contrast, PBS-treated mice lost body weight from days 13 to 17 and never returned to normal (Figure 6C). These results suggest MOG-Fc-BPI has the potential to treat PPMS as an alternative to Ocrelizumab. (20)

Previously, the axon myelination of PLP-Fc-BPI-treated EAE mice was similar to that of healthy mice (no demyelination) and significantly better than PBS control mice. (14) A BPI molecule, Ac-PLP-cIBR-NH₂-1, also effectively suppressed EAE and prevented axon demyelination. (22) The BPI treatment also stopped the blood-brain barrier (BBB) leakiness as detected by magnetic resonance imaging (MRI); this was indicated by the low brain deposition of gadolinium diethylenetriaminepentaacetate (Gd-DTPA), which was similar to healthy mice. (23) In contrast, untreated EAE mice had a high Gd-DTPA brain deposition due to the BBB leakiness. (23)

Recently, PLP-Fc-BPI showed proliferation of the Treg FoxP3+ subpopulation compared to a PBS control group, suggesting that Tregs control proinflammatory cells (i.e., Th1 and Th17) to suppress the disease. (24−27) Tregs lowered the activity of Th17 cells. (26,28,29) PLP-Fc-BPI treatment also increased the population of Tr1 cells with FoxP3- but IL10+ CD4+ markers; Tr1 cells have been described as ex-Th17 cells that switched to Tr1 cells. (30−33,29) Finally, the PLP-Fc-BPI-treated group had increased IL10 and suppressed IL17 compared with the PBS group; this could be attributed to the trans-differentiation of Th17 to Tr1 cells.

The responses of Fc-BPI in EAE mice were similar to those of BPI peptides and I-domain-antigen conjugate (IDAC) molecules. PLP-BPI, MOG-BPI, MOG/PLP-BPI suppressed IL17 production while stimulating the production of IL10. (8,9,13,22) MOG-BPI and MOG/PLP-BPI suppressed the production of IL6 and IFNγ cytokines in EAE mice compared to a PBS control group. (10) Both EAE mice and MS patients produce high levels of IL-6 and IFNγ, including in the central nervous system (CNS). (34) In addition, IL6 induces BBB leakiness in MS and EAE. (35) IFNγ also inhibits axon remyelination in the CNS of EAE mice. (36) Furthermore, PLP-BPI and PLP-IDAC in EAE mice enhanced production of IL10, indicating the potential involvement of Tregs. (9,37) IDAC molecules induced the production of IL5 and suppressed IL2 production in EAE mice, suggesting the suppression of Th1 that shifts into the stimulation of Th2 cells. (37) The populations of TGFβ- and IL-4-producing CD4⁺CD25+ T cells were significantly upregulated in PLP-BPI-treated EAE mice to tip the balance from inflammatory to regulatory phenotypes. (9)

During formulation, MOG-Fc-BPI generated subvisible particulates that could potentially induce immunogenicity to MOG-Fc-BPI in vivo. To counter this problem, we have developed a stable formulation of MOG-Fc-BPI; however, its repeated administrations may induce a hypersensitivity reaction. This hypersensitivity reaction to BPI and Fc-BPI molecules could be due to the production of antibodies to these molecules during the initial burst of immune cell activation. (6) This event triggers the cross-linking of IgE antibodies that bind the high-affinity immunoglobulin E receptor (FcεRI) on mast cells. (38,39) This process could lead to bronchoconstriction due to excessive mucus production in the lungs in the EAE mice. (39) Therefore, altering the route of administration from i.v. to subcutaneous, intradermal, or oral can lower the incidence of hypersensitivity reactions. (40) Similar to treatment with allergens, Fc-BPI can be delivered in a gradual dose escalation to avoid adverse effects. (41)

The process to synthesize MOG-Fc-BPI was challenging; previous attempts to express it as a single protein failed because of MOG peptide proteolysis. (14) Therefore, a sortase ligation method was utilized to successfully produce PLP-Fc-BPI and MBP-Fc-BPI. In this method, the MOG and LABL-Fc-ST were ligated using sortase at 35 °C and pH 7.5 (Figure 4); however, the solubility of different components became the limiting condition. The high hydrophobicity of the MOG peptide at pH 7.5 was incompatible with the sortase reaction conditions. Initially, two Arg residues were added to the C-terminus of the MOG peptide (G₃MOGR₂); unfortunately, although the G₃MOGR₂ peptide was soluble in the sortase reaction conditions and generated a product in a small scale reaction, (14) a high level of precipitate was still observed after a 24 h period, suggesting that the peptide and/or MOG-Fc-BPI were not stable or aggregated in the reaction solution.

Other ligation approaches such as split intein ligation methods have been investigated to make MOG-Fc-BPI. (42) Inteins are naturally occurring sequences that self-splice themselves to ligate the two exteins that flank the sequence. (42) The ligations can occur under mild denaturing conditions and then be refolded to the final folded state. (43) In addition, two different expression systems can be used, for example, one where a correctly folded glycoprotein and insoluble peptide are produced, respectively; then, they are combined in the last step of a synthesis. Unfortunately, this method failed to conjugate LABL-Fc and MOG peptide to make MOG-Fc-BPI and generated a high level of precipitate in denaturing or nondenaturing conditions. The final product cannot be detected in solution by HPLC and mass spectrometry. Similarly, synthesizing MOG-IDAC had similar problems because it produced aggregates and precipitates upon standing at room temperature, while PLP-IDAC was successfully synthesized and had high suppressive activity against EAE. (44,45)

The aggregation and precipitation problems can be overcome with the G₃MOGR₅ peptide, which was successfully ligated to LABL-Fc-ST to produce the MOG-Fc-BPI (Figure 5). G₃MOGR₅ and MOG-Fc-BPI were physically stable in solution; this is due to the addition of five positive charges from the arginines. Addition of five Arg residues did not influence the efficacy of MOG-Fc-BPI in suppressing EAE (Figure 6), suggesting that the MOG epitope can still bind to MHC-II on APCs.

This study also overcomes the challenge of administering a sufficiently high dose of MOG-Fc-BPI for in vivo studies without the presence of protein aggregation and precipitation. Based on the target dose of 35 nmol in 150 μL (approximately 15 mg/mL), various formulation conditions were tested to see whether MOG-Fc-BPI was stable at that concentration. This was necessary because MOG-Fc-BPI has a limited solubility of approximately 3 mg/mL in PBS. MOG-Fc-BPI was stable at the target concentration for in vivo studies in a buffer containing 0.1 M sodium phosphate, 0.15 M NaCl, and 50 mM sucrose at pH 7.2. Recently, we carried out high-throughput formulation studies to stabilize the MOG-Fc-BPI by screening varying buffer types, pH, ionic strengths, and excipients. This molecule was found to be sensitive to pH and ionic strength changes. The presence of certain divalent anions, sugar, β-cyclodextrin, and polysorbate 80 as excipients prevented the aggregation of the MOG-Fc-BPI molecule in the formulation. The optimized formulation suppresses the formation of aggregates and subvisible particles, as well as the fragmentation of MOG-Fc-BPI molecules. Currently, we are developing methods to produce various Fc-BPI molecules in Cho cells to further develop Fc-BPI molecules in the future.

Conclusions

Click to copy section linkSection link copied!

There is a desperate need for treatment of PPMS, with only one FDA-approved treatment (i.e., Ocrelizumab) that can only delay the disease progression. (6) Here, MOG-Fc-BPI was successfully synthesized and formulated to overcome the aggregation problems. The effective production, formulation, and administration of MOG-Fc-BPI can suppress MOG-stimulated EAE to a disease score of zero in the PPMS EAE mouse model. Thus, MOG-Fc-BPI has a promising future as a therapeutic agent for PPMS.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.5c00518.

Procedure to express and purify the LABL-Fc-ST protein as a starting material; experimental method to produce sortase A enzyme; WES capillary cartridge analysis of the MOG-Fc-BPI molecule in reduced and intact forms (Figure S1) (PDF)

bc5c00518_si_001.pdf (90.62 kb)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!

Corresponding Authors
- Teruna J. Siahaan - Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States; https://orcid.org/0000-0001-7250-0627; Email: [email protected]
- Thomas Tolbert - Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States; https://orcid.org/0000-0001-7726-3456; Email: [email protected]
Authors
- Rucha Mahadik - Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States; Present Address: Department of Sterile Product Development, Bristol-Myers Squibb Company, One Squibb Drive, New Brunswick, New Jersey 08903, United States
- Andrea L. Villela-Nava - Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States; https://orcid.org/0009-0008-4299-6703
- Lun Xin - Department of Pharmaceutical Chemistry, The University of Kansas, 2093 Constant Ave., Lawrence, Kansas 66047, United States; BioDev Department, WuXi Biologics USA, Cranbury, New Jersey 08512, United States
Notes
There was no unexpected safety hazard observed in all experiments. The protocol number AUS 75-04 for the EAE animal studies had been approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Kansas.
The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

We acknowledge the support for RM from the National Institutes of Health (NIH) Graduate Training at the Biology-Chemistry Interface Grant T32 GM132061 from the National Institutes of General Medical Sciences. We also acknowledge grant supports to T.J.S. and T.T. from the National Institutes of Health, including R01-AG082273 (NIA), R01-AG071682 (NIA), and P20-GM113117 (Pilot Grant, COBRE Chemical Biology Infectious Disease, NIGMS).

Abbreviations

Click to copy section linkSection link copied!

CNS	central nervous system
EAE	experimental autoimmune encephalomyelitis
MS	multiple sclerosis (MS)
MBP	myelin basic protein
MOG	myelin oligodendrocyte glycoprotein
PLP	proteolipid protein (PLP)
PPMS	primary progressive MS
PRMS	progressive relapsing MS
RRMS	relapse-remitting MS
SPMS	secondary progressive MS

References

Click to copy section linkSection link copied!

This article references 45 other publications.

1
Brandstadter, R.; Katz Sand, I. The use of natalizumab for multiple sclerosis. Neuropsychiatr Dis Treat 2017, 13, 1691– 1702, DOI: 10.2147/NDT.S114636
Google Scholar
There is no corresponding record for this reference.
2
Mahadik, R.; Kiptoo, P.; Tolbert, T.; Siahaan, T. J. Immune Modulation by Antigenic Peptides and Antigenic Peptide Conjugates for Treatment of Multiple Sclerosis. Med. Res. Arch. 2022, 10 (5), 2804, DOI: 10.18103/mra.v10i5.2804
Google Scholar
There is no corresponding record for this reference.
3
Ghasemi, N.; Razavi, S.; Nikzad, E. Multiple Sclerosis: Pathogenesis, Symptoms, Diagnoses and Cell-Based Therapy. Cell J. 2017, 19 (1), 1– 10, DOI: 10.22074/cellj.2016.4867
Google Scholar
There is no corresponding record for this reference.
4
Kammona, O.; Kiparissides, C. Recent Advances in Antigen-Specific Immunotherapies for the Treatment of Multiple Sclerosis. Brain Sci. 2020, 10 (6), 333, DOI: 10.3390/brainsci10060333
Google Scholar
There is no corresponding record for this reference.
5
Manikwar, P.; Kiptoo, P.; Badawi, A. H.; Buyuktimkin, B.; Siahaan, T. J. Antigen-specific blocking of CD4-specific immunological synapse formation using BPI and current therapies for autoimmune diseases. Med. Res. Rev. 2012, 32 (4), 727– 764, DOI: 10.1002/med.20243
Google Scholar
There is no corresponding record for this reference.
6
Badawi, A. H.; Siahaan, T. J. Immune modulating peptides for the treatment and suppression of multiple sclerosis. Clin Immunol 2012, 144 (2), 127– 138, DOI: 10.1016/j.clim.2012.05.010
Google Scholar
There is no corresponding record for this reference.
7
Moral, M. E. G.; Siahaan, T. J. Conjugates of Cell Adhesion Peptides for Therapeutics and Diagnostics Against Cancer and Autoimmune Diseases. Curr. Top Med. Chem. 2018, 17 (32), 3425– 3443, DOI: 10.2174/1568026618666180118154514
Google Scholar
There is no corresponding record for this reference.
8
Kobayashi, N.; Kiptoo, P.; Kobayashi, H.; Ridwan, R.; Brocke, S.; Siahaan, T. J. Prophylactic and therapeutic suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor. Clin Immunol 2008, 129 (1), 69– 79, DOI: 10.1016/j.clim.2008.06.002
Google Scholar
There is no corresponding record for this reference.
9
Kobayashi, N.; Kobayashi, H.; Gu, L.; Malefyt, T.; Siahaan, T. J. Antigen-specific suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor. J. Pharmacol Exp Ther 2007, 322 (2), 879– 886, DOI: 10.1124/jpet.107.123257
Google Scholar
There is no corresponding record for this reference.
10
Badawi, A. H.; Siahaan, T. J. Suppression of MOG- and PLP-induced experimental autoimmune encephalomyelitis using a novel multivalent bifunctional peptide inhibitor. J. Neuroimmunol 2013, 263 (1–2), 20– 27, DOI: 10.1016/j.jneuroim.2013.07.009
Google Scholar
There is no corresponding record for this reference.
11
Murray, J. S.; Oney, S.; Page, J. E.; Kratochvil-Stava, A.; Hu, Y.; Makagiansar, I. T.; Brown, J. C.; Kobayashi, N.; Siahaan, T. J. Suppression of type 1 diabetes in NOD mice by bifunctional peptide inhibitor: modulation of the immunological synapse formation. Chem. Biol. Drug Des 2007, 70 (3), 227– 236, DOI: 10.1111/j.1747-0285.2007.00552.x
Google Scholar
There is no corresponding record for this reference.
12
Buyuktimkin, B.; Kiptoo, P.; Siahaan, T. J. Bifunctional Peptide Inhibitors Suppress Interleukin-6 Proliferation and Ameliorates Murine Collagen-Induced Arthritis. J. Clin. Cell. Immunol. 2014, 5 (6), 1000273 DOI: 10.4172/2155-9899.1000273
Google Scholar
There is no corresponding record for this reference.
13
Ridwan, R.; Kiptoo, P.; Kobayashi, N.; Weir, S.; Hughes, M.; Williams, T.; Soegianto, R.; Siahaan, T. J. Antigen-specific suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor: structure optimization and pharmacokinetics. J. Pharmacol Exp Ther 2010, 332 (3), 1136– 1145, DOI: 10.1124/jpet.109.161109
Google Scholar
There is no corresponding record for this reference.
14
White, D. R.; Khedri, Z.; Kiptoo, P.; Siahaan, T. J.; Tolbert, T. J. Synthesis of a Bifunctional Peptide Inhibitor-IgG1 Fc Fusion That Suppresses Experimental Autoimmune Encephalomyelitis. Bioconjug Chem. 2017, 28 (7), 1867– 1877, DOI: 10.1021/acs.bioconjchem.7b00175
Google Scholar
There is no corresponding record for this reference.
15
Xiao, J.; Chen, R.; Pawlicki, M. A.; Tolbert, T. J. Targeting a homogeneously glycosylated antibody Fc to bind cancer cells using a synthetic receptor ligand. J. Am. Chem. Soc. 2009, 131 (38), 13616– 13618, DOI: 10.1021/ja9045179
Google Scholar
There is no corresponding record for this reference.
16
Zou, Z.; Ji, Y.; Schwaneberg, U. Empowering Site-Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase-Mediated Ligation. Angew. Chem., Int. Ed. Engl. 2024, 63 (12), e202310910 DOI: 10.1002/anie.202310910
Google Scholar
There is no corresponding record for this reference.
17
Antos, J. M.; Truttmann, M. C.; Ploegh, H. L. Recent advances in sortase-catalyzed ligation methodology. Curr. Opin Struct Biol. 2016, 38, 111– 118, DOI: 10.1016/j.sbi.2016.05.021
Google Scholar
There is no corresponding record for this reference.
18
Heistinger, L.; Gasser, B.; Mattanovich, D. Microbe Profile: Komagataella phaffii: a methanol devouring biotech yeast formerly known as Pichia pastoris. Microbiology (Reading) 2020, 166 (7), 614– 616, DOI: 10.1099/mic.0.000958
Google Scholar
There is no corresponding record for this reference.
19
Miller, D. H.; Leary, S. M. Primary-progressive multiple sclerosis. Lancet Neurol 2007, 6 (10), 903– 912, DOI: 10.1016/S1474-4422(07)70243-0
Google Scholar
There is no corresponding record for this reference.
20
Lamb, Y. N. Ocrelizumab: A Review in Multiple Sclerosis. Drugs 2022, 82 (3), 323– 334, DOI: 10.1007/s40265-022-01672-9
Google Scholar
There is no corresponding record for this reference.
21
Hauser, S. L.; Kappos, L.; Montalban, X.; Craveiro, L.; Chognot, C.; Hughes, R.; Koendgen, H.; Pasquarelli, N.; Pradhan, A.; Prajapati, K. Safety of Ocrelizumab in Patients With Relapsing and Primary Progressive Multiple Sclerosis. Neurology 2021, 97 (16), e1546– e1559, DOI: 10.1212/WNL.0000000000012700
Google Scholar
There is no corresponding record for this reference.
22
Kiptoo, P.; Buyuktimkin, B.; Badawi, A. H.; Stewart, J.; Ridwan, R.; Siahaan, T. J. Controlling immune response and demyelination using highly potent bifunctional peptide inhibitors in the suppression of experimental autoimmune encephalomyelitis. Clin. Exp. Immunol. 2013, 172 (1), 23– 36, DOI: 10.1111/cei.12029
Google Scholar
There is no corresponding record for this reference.
23
Badawi, A. H.; Kiptoo, P.; Wang, W. T.; Choi, I. Y.; Lee, P.; Vines, C. M.; Siahaan, T. J. Suppression of EAE and prevention of blood-brain barrier breakdown after vaccination with novel bifunctional peptide inhibitor. Neuropharmacology 2012, 62 (4), 1874– 1881, DOI: 10.1016/j.neuropharm.2011.12.013
Google Scholar
There is no corresponding record for this reference.
24
Littman, D. R.; Rudensky, A. Y. Th17 and regulatory T cells in mediating and restraining inflammation. Cell 2010, 140 (6), 845– 858, DOI: 10.1016/j.cell.2010.02.021
Google Scholar
There is no corresponding record for this reference.
25
Moorman, C. D.; Bastian, A. G.; DeOca, K. B.; Mannie, M. D. A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4(+) CD25(+) FOXP3(+) Tregs that inhibit experimental autoimmune encephalomyelitis (EAE). J. Neuroinflammation 2020, 17 (1), 180, DOI: 10.1186/s12974-020-01856-8
Google Scholar
There is no corresponding record for this reference.
26
Gagliani, N.; Amezcua Vesely, M. C.; Iseppon, A.; Brockmann, L.; Xu, H.; Palm, N. W.; de Zoete, M. R.; Licona-Limon, P.; Paiva, R. S.; Ching, T. Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation. Nature 2015, 523 (7559), 221– 225, DOI: 10.1038/nature14452
Google Scholar
There is no corresponding record for this reference.
27
Mari, E. R.; Rasouli, J.; Ciric, B.; Moore, J. N.; Conejo-Garcia, J. R.; Rajasagi, N.; Zhang, G. X.; Rabinovich, G. A.; Rostami, A. Galectin-1 is essential for the induction of MOG35–55 -based intravenous tolerance in experimental autoimmune encephalomyelitis. Eur. J. Immunol. 2016, 46 (7), 1783– 1796, DOI: 10.1002/eji.201546212
Google Scholar
There is no corresponding record for this reference.
28
Leno-Duran, E.; Ng, S. L.; Strominger, J. L. Regulation of EAE by spontaneously generated IL-10-secreting regulatory T cells in HLA-DR15/TCR.Ob1A12 double transgenic mice. Immunology 2021, 163 (3), 338– 343, DOI: 10.1111/imm.13321
Google Scholar
There is no corresponding record for this reference.
29
Raverdeau, M.; Christofi, M.; Malara, A.; Wilk, M. M.; Misiak, A.; Kuffova, L.; Yu, T.; McGinley, A. M.; Quinn, S. M.; Massilamany, C. Retinoic acid-induced autoantigen-specific type 1 regulatory T cells suppress autoimmunity. EMBO Rep. 2019, 20 (5), e47121 DOI: 10.15252/embr.201847121
Google Scholar
There is no corresponding record for this reference.
30
Loos, J.; Schmaul, S.; Noll, T. M.; Paterka, M.; Schillner, M.; Loffel, J. T.; Zipp, F.; Bittner, S. Functional characteristics of Th1, Th17, and ex-Th17 cells in EAE revealed by intravital two-photon microscopy. J. Neuroinflammation 2020, 17 (1), 357, DOI: 10.1186/s12974-020-02021-x
Google Scholar
There is no corresponding record for this reference.
31
Xu, H.; Agalioti, T.; Zhao, J.; Steglich, B.; Wahib, R.; Vesely, M. C. A.; Bielecki, P.; Bailis, W.; Jackson, R.; Perez, D. The induction and function of the anti-inflammatory fate of T(H)17 cells. Nat. Commun. 2020, 11 (1), 3334, DOI: 10.1038/s41467-020-17097-5
Google Scholar
There is no corresponding record for this reference.
32
Worzner, K.; Zimmermann, J.; Buhl, R.; Desoi, A.; Christensen, D.; Dietrich, J.; Nguyen, N.; Lindenstrom, T.; Woodworth, J. S.; Alhakeem, R. S. Repeated immunization with ATRA-containing liposomal adjuvant transdifferentiates Th17 cells to a Tr1-like phenotype. J. Autoimmun 2024, 144, 103174 DOI: 10.1016/j.jaut.2024.103174
Google Scholar
There is no corresponding record for this reference.
33
Vieira, P. L.; Christensen, J. R.; Minaee, S.; O’Neill, E. J.; Barrat, F. J.; Boonstra, A.; Barthlott, T.; Stockinger, B.; Wraith, D. C.; O’Garra, A. IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells. J. Immunol 2004, 172 (10), 5986– 5993, DOI: 10.4049/jimmunol.172.10.5986
Google Scholar
There is no corresponding record for this reference.
34
Maimone, D.; Guazzi, G. C.; Annunziata, P. IL-6 detection in multiple sclerosis brain. J. Neurol Sci. 1997, 146 (1), 59– 65, DOI: 10.1016/S0022-510X(96)00283-3
Google Scholar
There is no corresponding record for this reference.
Kebir, H.; Ifergan, I.; Alvarez, J. I.; Bernard, M.; Poirier, J.; Arbour, N.; Duquette, P.; Prat, A. Preferential recruitment of interferon-gamma-expressing TH17 cells in multiple sclerosis. Ann. Neurol. 2009, 66 (3), 390– 402, DOI: 10.1002/ana.21748
Google Scholar
There is no corresponding record for this reference.
35
Prendergast, C. T.; Anderton, S. M. Immune cell entry to central nervous system--current understanding and prospective therapeutic targets. Endocr Metab Immune Disord Drug Targets 2009, 9 (4), 315– 327, DOI: 10.2174/187153009789839219
Google Scholar
There is no corresponding record for this reference.
36
Lin, W.; Kemper, A.; Dupree, J. L.; Harding, H. P.; Ron, D.; Popko, B. Interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress. Brain 2006, 129 (Pt 5), 1306– 1318, DOI: 10.1093/brain/awl044
Google Scholar
There is no corresponding record for this reference.
37
Buyuktimkin, B.; Manikwar, P.; Kiptoo, P. K.; Badawi, A. H.; Stewart, J. M., Jr; Siahaan, T. J. Vaccinelike and prophylactic treatments of EAE with novel I-domain antigen conjugates (IDAC): targeting multiple antigenic peptides to APC. Mol. Pharm. 2013, 10 (1), 297– 306, DOI: 10.1021/mp300440x
Google Scholar
There is no corresponding record for this reference.
38
Martin, T. R.; Galli, S. J.; Katona, I. M.; Drazen, J. M. Role of mast cells in anaphylaxis. Evidence for the importance of mast cells in the cardiopulmonary alterations and death induced by anti-IgE in mice. J. Clin Invest 1989, 83 (4), 1375– 1383, DOI: 10.1172/JCI114025
Google Scholar
There is no corresponding record for this reference.
39
Smith, C. E.; Eagar, T. N.; Strominger, J. L.; Miller, S. D. Differential induction of IgE-mediated anaphylaxis after soluble vs. cell-bound tolerogenic peptide therapy of autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (27), 9595– 9600, DOI: 10.1073/pnas.0504131102
Google Scholar
There is no corresponding record for this reference.
40
Miller, S. D.; Turley, D. M.; Podojil, J. R. Antigen-specific tolerance strategies for the prevention and treatment of autoimmune disease. Nat. Rev. Immunol 2007, 7 (9), 665– 677, DOI: 10.1038/nri2153
Google Scholar
There is no corresponding record for this reference.
41
Sabatos-Peyton, C. A.; Verhagen, J.; Wraith, D. C. Antigen-specific immunotherapy of autoimmune and allergic diseases. Curr. Opin Immunol 2010, 22 (5), 609– 615, DOI: 10.1016/j.coi.2010.08.006
Google Scholar
There is no corresponding record for this reference.
42
Pirzer, T.; Becher, K. S.; Rieker, M.; Meckel, T.; Mootz, H. D.; Kolmar, H. Generation of Potent Anti-HER1/2 Immunotoxins by Protein Ligation Using Split Inteins. ACS Chem. Biol. 2018, 13 (8), 2058– 2066, DOI: 10.1021/acschembio.8b00222
Google Scholar
There is no corresponding record for this reference.
43
Ludwig, C.; Pfeiff, M.; Linne, U.; Mootz, H. D. Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing. Angew. Chem., Int. Ed. Engl. 2006, 45 (31), 5218– 5221, DOI: 10.1002/anie.200600570
Google Scholar
There is no corresponding record for this reference.
44
Manikwar, P.; Tejo, B. A.; Shinogle, H.; Moore, D. S.; Zimmerman, T.; Blanco, F.; Siahaan, T. J. Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes. Theranostics 2011, 1, 277– 289, DOI: 10.7150/thno/v01p0277
Google Scholar
There is no corresponding record for this reference.
45
Manikwar, P.; Buyuktimkin, B.; Kiptoo, P.; Badawi, A. H.; Galeva, N. A.; Williams, T. D.; Siahaan, T. J. I-domain-antigen conjugate (IDAC) for delivering antigenic peptides to APC: synthesis, characterization, and in vivo EAE suppression. Bioconjug Chem. 2012, 23 (3), 509– 517, DOI: 10.1021/bc200580j
Google Scholar
There is no corresponding record for this reference.

Cited By

Click to copy section linkSection link copied!

This article has not yet been cited by other publications.

Get e-Alerts

Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2025, 36, 12, 2700–2709

Click to copy citationCitation copied!

https://doi.org/10.1021/acs.bioconjchem.5c00518

Published November 21, 2025

Request reuse permissions

Article Views

Altmetric

Citations

Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

Abstract
High Resolution Image
Download MS PowerPoint Slide
Figure 1
Figure 1. Structure of MOG-Fc-BPI consists of an Fc-domain with the LABL peptide at the N-terminus and the MOG-R₅ peptide at the C-terminus.
High Resolution Image
Download MS PowerPoint Slide
Figure 2
Figure 2. MS spectra of the purified LABL-Fc-ST protein. (A) Intact-MS spectrum of the reduced and deglycosylated LABL-Fc-ST protein with [M + H]: 27,024 amu. (B) The MS spectrum of fully glycosylated LABL-Fc-ST with the Man8 glycan (expected [M + Na]: 28,749 amu; observed mass: 28,749 amu) as well as the Man9 glycan (expected [M + Na]: 28,910 amu; observed mass: 28,911 amu).
High Resolution Image
Download MS PowerPoint Slide
Figure 3
Figure 3. MS spectrum of G₃MOGR₅ peptide showed peaks for [M + 5H] (512.18 m/z), [M + 4H] (639.98 m/z), and [M + 3H] (853.09 m/z) with an overall observed mass of 2555.9 amu, which was similar to the expected mass of 2555.94 amu.
High Resolution Image
Download MS PowerPoint Slide
Figure 4
Figure 4. Synthetic scheme for the production of MOG-Fc-BPI. LABL-Fc-ST contains the C-terminal sortase recognition tag, LPETGGG. The G₃-MOG-R₅ peptide was conjugated to the C-terminus of LABL-Fc-ST using sortase A, which recognizes the LPETGGG sequence and cleaves between the threonine and glycine residues to form a thioester with threonine while releasing the C-terminal triglycine peptide. The N-terminal triglycine residues of G₃-MOG-R₅ act as a nucleophile in the sortase A reaction to attack the thioester and produce the desired product, MOG-Fc-BPI.
High Resolution Image
Download MS PowerPoint Slide
Figure 5
Figure 5. MS spectra of MOG-Fc-BPI. (A) The intact-MS spectrum of the reduced and deglycosylated MOG-Fc-BPI protein with [M + H] = 29,391 amu. (B) MS spectrum of purified glycosylated MOG-Fc-BPI with major species such as (a) Man8 glycoform of MOG-Fc-BPI (expected = 31,093.8 amu, observed = 31,092.8 amu); (b) MOG-Fc-BPI with no glycan (expected = 29,372.3 amu, observed = 29,392.1 amu); and (c) LABL-Fc-ST (expected = 28,726.9 amu, observed = 28,726.7 amu).
High Resolution Image
Download MS PowerPoint Slide
Figure 6
Figure 6. Activity of MOG-Fc-BPI to suppress EAE was determined by comparing (A) daily clinical scores, (B) AUC of clinical scores, (C) daily body weights, and (D) AUC of body weights to those of PBS control. (A) The MOG-Fc-BPI-treated mice received 2 doses of 35 nmol/dose injections on days 4 and 7 and showed lower clinical scores than those of PBS-treated mice. (B) AUC of clinical scores for PBS-treated mice was significantly higher than that of MOG-Fc-BPI-treated mice over 25 days (p < 0.001). (C) The differences in the increase body weights over 25 day period for MOG-Fc-BPI- and PBS-treated mice. There is inhibition in the increase of body weights on days 14–25 in PBS-treated mice but not MOG-Fc-BPI-treated mice. (D) A significantly higher ACU of body weights of MOG-Fc-BPI-treated mice compared to PBS-treated mice (p = 0.0274).
High Resolution Image
Download MS PowerPoint Slide
Figure 7
Figure 7. Proposed mechanisms of action for MOG-Fc-BPI suppression of EAE disease involving iDC and mDC. (A) MOG-Fc-BPI binds simultaneously to ICAM-1 and MHC-II using LABL and MOG peptides, respectively, on iDC to block Signal-2 in the presence of Signal-1. and this also prevents immunological synapse (IS) formation. The presence of Signal-1 in the absence of Signal-2 causes naïve T cells to become Treg cells. (B) MOG-Fc-BPI binds to ICAM-1 and MHC-II on the surface of mDC. The TCRs on naïve T cells recognize the MOG/MHC-II complexes (Signal-1) with blocked Signal-2; as a result, this prevents the differentiation of naïve T cells to inflammatory Th17 or Th1 cells.
High Resolution Image
Download MS PowerPoint Slide
References
This article references 45 other publications.
1. 1
  Brandstadter, R.; Katz Sand, I. The use of natalizumab for multiple sclerosis. Neuropsychiatr Dis Treat 2017, 13, 1691– 1702, DOI: 10.2147/NDT.S114636
  There is no corresponding record for this reference.
2. 2
  Mahadik, R.; Kiptoo, P.; Tolbert, T.; Siahaan, T. J. Immune Modulation by Antigenic Peptides and Antigenic Peptide Conjugates for Treatment of Multiple Sclerosis. Med. Res. Arch. 2022, 10 (5), 2804, DOI: 10.18103/mra.v10i5.2804
  There is no corresponding record for this reference.
3. 3
  Ghasemi, N.; Razavi, S.; Nikzad, E. Multiple Sclerosis: Pathogenesis, Symptoms, Diagnoses and Cell-Based Therapy. Cell J. 2017, 19 (1), 1– 10, DOI: 10.22074/cellj.2016.4867
  There is no corresponding record for this reference.
4. 4
  Kammona, O.; Kiparissides, C. Recent Advances in Antigen-Specific Immunotherapies for the Treatment of Multiple Sclerosis. Brain Sci. 2020, 10 (6), 333, DOI: 10.3390/brainsci10060333
  There is no corresponding record for this reference.
5. 5
  Manikwar, P.; Kiptoo, P.; Badawi, A. H.; Buyuktimkin, B.; Siahaan, T. J. Antigen-specific blocking of CD4-specific immunological synapse formation using BPI and current therapies for autoimmune diseases. Med. Res. Rev. 2012, 32 (4), 727– 764, DOI: 10.1002/med.20243
  There is no corresponding record for this reference.
6. 6
  Badawi, A. H.; Siahaan, T. J. Immune modulating peptides for the treatment and suppression of multiple sclerosis. Clin Immunol 2012, 144 (2), 127– 138, DOI: 10.1016/j.clim.2012.05.010
  There is no corresponding record for this reference.
7. 7
  Moral, M. E. G.; Siahaan, T. J. Conjugates of Cell Adhesion Peptides for Therapeutics and Diagnostics Against Cancer and Autoimmune Diseases. Curr. Top Med. Chem. 2018, 17 (32), 3425– 3443, DOI: 10.2174/1568026618666180118154514
  There is no corresponding record for this reference.
8. 8
  Kobayashi, N.; Kiptoo, P.; Kobayashi, H.; Ridwan, R.; Brocke, S.; Siahaan, T. J. Prophylactic and therapeutic suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor. Clin Immunol 2008, 129 (1), 69– 79, DOI: 10.1016/j.clim.2008.06.002
  There is no corresponding record for this reference.
9. 9
  Kobayashi, N.; Kobayashi, H.; Gu, L.; Malefyt, T.; Siahaan, T. J. Antigen-specific suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor. J. Pharmacol Exp Ther 2007, 322 (2), 879– 886, DOI: 10.1124/jpet.107.123257
  There is no corresponding record for this reference.
10. 10
  Badawi, A. H.; Siahaan, T. J. Suppression of MOG- and PLP-induced experimental autoimmune encephalomyelitis using a novel multivalent bifunctional peptide inhibitor. J. Neuroimmunol 2013, 263 (1–2), 20– 27, DOI: 10.1016/j.jneuroim.2013.07.009
  There is no corresponding record for this reference.
11. 11
  Murray, J. S.; Oney, S.; Page, J. E.; Kratochvil-Stava, A.; Hu, Y.; Makagiansar, I. T.; Brown, J. C.; Kobayashi, N.; Siahaan, T. J. Suppression of type 1 diabetes in NOD mice by bifunctional peptide inhibitor: modulation of the immunological synapse formation. Chem. Biol. Drug Des 2007, 70 (3), 227– 236, DOI: 10.1111/j.1747-0285.2007.00552.x
  There is no corresponding record for this reference.
12. 12
  Buyuktimkin, B.; Kiptoo, P.; Siahaan, T. J. Bifunctional Peptide Inhibitors Suppress Interleukin-6 Proliferation and Ameliorates Murine Collagen-Induced Arthritis. J. Clin. Cell. Immunol. 2014, 5 (6), 1000273 DOI: 10.4172/2155-9899.1000273
  There is no corresponding record for this reference.
13. 13
  Ridwan, R.; Kiptoo, P.; Kobayashi, N.; Weir, S.; Hughes, M.; Williams, T.; Soegianto, R.; Siahaan, T. J. Antigen-specific suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor: structure optimization and pharmacokinetics. J. Pharmacol Exp Ther 2010, 332 (3), 1136– 1145, DOI: 10.1124/jpet.109.161109
  There is no corresponding record for this reference.
14. 14
  White, D. R.; Khedri, Z.; Kiptoo, P.; Siahaan, T. J.; Tolbert, T. J. Synthesis of a Bifunctional Peptide Inhibitor-IgG1 Fc Fusion That Suppresses Experimental Autoimmune Encephalomyelitis. Bioconjug Chem. 2017, 28 (7), 1867– 1877, DOI: 10.1021/acs.bioconjchem.7b00175
  There is no corresponding record for this reference.
15. 15
  Xiao, J.; Chen, R.; Pawlicki, M. A.; Tolbert, T. J. Targeting a homogeneously glycosylated antibody Fc to bind cancer cells using a synthetic receptor ligand. J. Am. Chem. Soc. 2009, 131 (38), 13616– 13618, DOI: 10.1021/ja9045179
  There is no corresponding record for this reference.
16. 16
  Zou, Z.; Ji, Y.; Schwaneberg, U. Empowering Site-Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase-Mediated Ligation. Angew. Chem., Int. Ed. Engl. 2024, 63 (12), e202310910 DOI: 10.1002/anie.202310910
  There is no corresponding record for this reference.
17. 17
  Antos, J. M.; Truttmann, M. C.; Ploegh, H. L. Recent advances in sortase-catalyzed ligation methodology. Curr. Opin Struct Biol. 2016, 38, 111– 118, DOI: 10.1016/j.sbi.2016.05.021
  There is no corresponding record for this reference.
18. 18
  Heistinger, L.; Gasser, B.; Mattanovich, D. Microbe Profile: Komagataella phaffii: a methanol devouring biotech yeast formerly known as Pichia pastoris. Microbiology (Reading) 2020, 166 (7), 614– 616, DOI: 10.1099/mic.0.000958
  There is no corresponding record for this reference.
19. 19
  Miller, D. H.; Leary, S. M. Primary-progressive multiple sclerosis. Lancet Neurol 2007, 6 (10), 903– 912, DOI: 10.1016/S1474-4422(07)70243-0
  There is no corresponding record for this reference.
20. 20
  Lamb, Y. N. Ocrelizumab: A Review in Multiple Sclerosis. Drugs 2022, 82 (3), 323– 334, DOI: 10.1007/s40265-022-01672-9
  There is no corresponding record for this reference.
21. 21
  Hauser, S. L.; Kappos, L.; Montalban, X.; Craveiro, L.; Chognot, C.; Hughes, R.; Koendgen, H.; Pasquarelli, N.; Pradhan, A.; Prajapati, K. Safety of Ocrelizumab in Patients With Relapsing and Primary Progressive Multiple Sclerosis. Neurology 2021, 97 (16), e1546– e1559, DOI: 10.1212/WNL.0000000000012700
  There is no corresponding record for this reference.
22. 22
  Kiptoo, P.; Buyuktimkin, B.; Badawi, A. H.; Stewart, J.; Ridwan, R.; Siahaan, T. J. Controlling immune response and demyelination using highly potent bifunctional peptide inhibitors in the suppression of experimental autoimmune encephalomyelitis. Clin. Exp. Immunol. 2013, 172 (1), 23– 36, DOI: 10.1111/cei.12029
  There is no corresponding record for this reference.
23. 23
  Badawi, A. H.; Kiptoo, P.; Wang, W. T.; Choi, I. Y.; Lee, P.; Vines, C. M.; Siahaan, T. J. Suppression of EAE and prevention of blood-brain barrier breakdown after vaccination with novel bifunctional peptide inhibitor. Neuropharmacology 2012, 62 (4), 1874– 1881, DOI: 10.1016/j.neuropharm.2011.12.013
  There is no corresponding record for this reference.
24. 24
  Littman, D. R.; Rudensky, A. Y. Th17 and regulatory T cells in mediating and restraining inflammation. Cell 2010, 140 (6), 845– 858, DOI: 10.1016/j.cell.2010.02.021
  There is no corresponding record for this reference.
25. 25
  Moorman, C. D.; Bastian, A. G.; DeOca, K. B.; Mannie, M. D. A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4(+) CD25(+) FOXP3(+) Tregs that inhibit experimental autoimmune encephalomyelitis (EAE). J. Neuroinflammation 2020, 17 (1), 180, DOI: 10.1186/s12974-020-01856-8
  There is no corresponding record for this reference.
26. 26
  Gagliani, N.; Amezcua Vesely, M. C.; Iseppon, A.; Brockmann, L.; Xu, H.; Palm, N. W.; de Zoete, M. R.; Licona-Limon, P.; Paiva, R. S.; Ching, T. Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation. Nature 2015, 523 (7559), 221– 225, DOI: 10.1038/nature14452
  There is no corresponding record for this reference.
27. 27
  Mari, E. R.; Rasouli, J.; Ciric, B.; Moore, J. N.; Conejo-Garcia, J. R.; Rajasagi, N.; Zhang, G. X.; Rabinovich, G. A.; Rostami, A. Galectin-1 is essential for the induction of MOG35–55 -based intravenous tolerance in experimental autoimmune encephalomyelitis. Eur. J. Immunol. 2016, 46 (7), 1783– 1796, DOI: 10.1002/eji.201546212
  There is no corresponding record for this reference.
28. 28
  Leno-Duran, E.; Ng, S. L.; Strominger, J. L. Regulation of EAE by spontaneously generated IL-10-secreting regulatory T cells in HLA-DR15/TCR.Ob1A12 double transgenic mice. Immunology 2021, 163 (3), 338– 343, DOI: 10.1111/imm.13321
  There is no corresponding record for this reference.
29. 29
  Raverdeau, M.; Christofi, M.; Malara, A.; Wilk, M. M.; Misiak, A.; Kuffova, L.; Yu, T.; McGinley, A. M.; Quinn, S. M.; Massilamany, C. Retinoic acid-induced autoantigen-specific type 1 regulatory T cells suppress autoimmunity. EMBO Rep. 2019, 20 (5), e47121 DOI: 10.15252/embr.201847121
  There is no corresponding record for this reference.
30. 30
  Loos, J.; Schmaul, S.; Noll, T. M.; Paterka, M.; Schillner, M.; Loffel, J. T.; Zipp, F.; Bittner, S. Functional characteristics of Th1, Th17, and ex-Th17 cells in EAE revealed by intravital two-photon microscopy. J. Neuroinflammation 2020, 17 (1), 357, DOI: 10.1186/s12974-020-02021-x
  There is no corresponding record for this reference.
31. 31
  Xu, H.; Agalioti, T.; Zhao, J.; Steglich, B.; Wahib, R.; Vesely, M. C. A.; Bielecki, P.; Bailis, W.; Jackson, R.; Perez, D. The induction and function of the anti-inflammatory fate of T(H)17 cells. Nat. Commun. 2020, 11 (1), 3334, DOI: 10.1038/s41467-020-17097-5
  There is no corresponding record for this reference.
32. 32
  Worzner, K.; Zimmermann, J.; Buhl, R.; Desoi, A.; Christensen, D.; Dietrich, J.; Nguyen, N.; Lindenstrom, T.; Woodworth, J. S.; Alhakeem, R. S. Repeated immunization with ATRA-containing liposomal adjuvant transdifferentiates Th17 cells to a Tr1-like phenotype. J. Autoimmun 2024, 144, 103174 DOI: 10.1016/j.jaut.2024.103174
  There is no corresponding record for this reference.
33. 33
  Vieira, P. L.; Christensen, J. R.; Minaee, S.; O’Neill, E. J.; Barrat, F. J.; Boonstra, A.; Barthlott, T.; Stockinger, B.; Wraith, D. C.; O’Garra, A. IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells. J. Immunol 2004, 172 (10), 5986– 5993, DOI: 10.4049/jimmunol.172.10.5986
  There is no corresponding record for this reference.
34. 34
  Maimone, D.; Guazzi, G. C.; Annunziata, P. IL-6 detection in multiple sclerosis brain. J. Neurol Sci. 1997, 146 (1), 59– 65, DOI: 10.1016/S0022-510X(96)00283-3
  There is no corresponding record for this reference.
  Kebir, H.; Ifergan, I.; Alvarez, J. I.; Bernard, M.; Poirier, J.; Arbour, N.; Duquette, P.; Prat, A. Preferential recruitment of interferon-gamma-expressing TH17 cells in multiple sclerosis. Ann. Neurol. 2009, 66 (3), 390– 402, DOI: 10.1002/ana.21748
  There is no corresponding record for this reference.
35. 35
  Prendergast, C. T.; Anderton, S. M. Immune cell entry to central nervous system--current understanding and prospective therapeutic targets. Endocr Metab Immune Disord Drug Targets 2009, 9 (4), 315– 327, DOI: 10.2174/187153009789839219
  There is no corresponding record for this reference.
36. 36
  Lin, W.; Kemper, A.; Dupree, J. L.; Harding, H. P.; Ron, D.; Popko, B. Interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress. Brain 2006, 129 (Pt 5), 1306– 1318, DOI: 10.1093/brain/awl044
  There is no corresponding record for this reference.
37. 37
  Buyuktimkin, B.; Manikwar, P.; Kiptoo, P. K.; Badawi, A. H.; Stewart, J. M., Jr; Siahaan, T. J. Vaccinelike and prophylactic treatments of EAE with novel I-domain antigen conjugates (IDAC): targeting multiple antigenic peptides to APC. Mol. Pharm. 2013, 10 (1), 297– 306, DOI: 10.1021/mp300440x
  There is no corresponding record for this reference.
38. 38
  Martin, T. R.; Galli, S. J.; Katona, I. M.; Drazen, J. M. Role of mast cells in anaphylaxis. Evidence for the importance of mast cells in the cardiopulmonary alterations and death induced by anti-IgE in mice. J. Clin Invest 1989, 83 (4), 1375– 1383, DOI: 10.1172/JCI114025
  There is no corresponding record for this reference.
39. 39
  Smith, C. E.; Eagar, T. N.; Strominger, J. L.; Miller, S. D. Differential induction of IgE-mediated anaphylaxis after soluble vs. cell-bound tolerogenic peptide therapy of autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (27), 9595– 9600, DOI: 10.1073/pnas.0504131102
  There is no corresponding record for this reference.
40. 40
  Miller, S. D.; Turley, D. M.; Podojil, J. R. Antigen-specific tolerance strategies for the prevention and treatment of autoimmune disease. Nat. Rev. Immunol 2007, 7 (9), 665– 677, DOI: 10.1038/nri2153
  There is no corresponding record for this reference.
41. 41
  Sabatos-Peyton, C. A.; Verhagen, J.; Wraith, D. C. Antigen-specific immunotherapy of autoimmune and allergic diseases. Curr. Opin Immunol 2010, 22 (5), 609– 615, DOI: 10.1016/j.coi.2010.08.006
  There is no corresponding record for this reference.
42. 42
  Pirzer, T.; Becher, K. S.; Rieker, M.; Meckel, T.; Mootz, H. D.; Kolmar, H. Generation of Potent Anti-HER1/2 Immunotoxins by Protein Ligation Using Split Inteins. ACS Chem. Biol. 2018, 13 (8), 2058– 2066, DOI: 10.1021/acschembio.8b00222
  There is no corresponding record for this reference.
43. 43
  Ludwig, C.; Pfeiff, M.; Linne, U.; Mootz, H. D. Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing. Angew. Chem., Int. Ed. Engl. 2006, 45 (31), 5218– 5221, DOI: 10.1002/anie.200600570
  There is no corresponding record for this reference.
44. 44
  Manikwar, P.; Tejo, B. A.; Shinogle, H.; Moore, D. S.; Zimmerman, T.; Blanco, F.; Siahaan, T. J. Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes. Theranostics 2011, 1, 277– 289, DOI: 10.7150/thno/v01p0277
  There is no corresponding record for this reference.
45. 45
  Manikwar, P.; Buyuktimkin, B.; Kiptoo, P.; Badawi, A. H.; Galeva, N. A.; Williams, T. D.; Siahaan, T. J. I-domain-antigen conjugate (IDAC) for delivering antigenic peptides to APC: synthesis, characterization, and in vivo EAE suppression. Bioconjug Chem. 2012, 23 (3), 509– 517, DOI: 10.1021/bc200580j
  There is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.5c00518.
- Procedure to express and purify the LABL-Fc-ST protein as a starting material; experimental method to produce sortase A enzyme; WES capillary cartridge analysis of the MOG-Fc-BPI molecule in reduced and intact forms (Figure S1) (PDF)
- bc5c00518_si_001.pdf (90.62 kb)
Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Overcoming Synthetic Challenges and Evaluating in Vivo Efficacy of the MOG-Fc-Bifunctional Peptide Inhibitor for EAE in MiceClick to copy article linkArticle link copied!

Bioconjugate Chemistry

Abstract

Introduction

Figure 1

Materials and Methods

Expression and Purification of LABL-Fc-ST

Figure 2

Mass Spectrometry Analysis

Synthesis and Purification of the MOG Peptide

Figure 3

Sortase-Mediated Ligation with LABL-Fc-ST and MOG Peptide

Efficacy of MOG-Fc-BPI in EAE Mice

Results

Synthesis of MOG-Fc-BPI

Sortase-Mediated Ligation

Figure 4

Figure 5

MOG-Fc-BPI Formulation

Animal Studies

Figure 6

Discussion

Figure 7

Conclusions

Supporting Information

Terms & Conditions

Author Information

Acknowledgments

Abbreviations

References

Cited By

Bioconjugate Chemistry

Article Views

Altmetric

Citations

Recommended Articles

Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

References

Supporting Information

Supporting Information

Terms & Conditions

Overcoming Synthetic Challenges and Evaluating in Vivo Efficacy of the MOG-Fc-Bifunctional Peptide Inhibitor for EAE in Mice
Click to copy article linkArticle link copied!