56 research outputs found
IN VITRO AND IN VIVO EVALUATION OF NOVEL I-DOMAIN CONJUGATES FOR DRUG TARGETING TO IMMUNE CELLS AND SUPPRESSION OF EAE IN MICE
The long-term objective of this project is to utilize the I-domain of leukocyte function associated antigen-1 (LFA-1) to target antigenic peptides and drugs to intercellular adhesion molecule-1 (ICAM-1) expressed on the surface of immune cells. The objectives of the dissertation are: 1) to characterize the binding properties of the I-domain to ICAM- 1 receptors on the surface of lymphocytes (Raji cells), and 2) to evaluate the efficacy of PLP-I-domain conjugates in suppressing experimental autoimmune encephalomyelitis (EAE) in the mouse model, an animal model for multiple sclerosis. To accomplish these objectives, the I-domain protein was labeled with fluorescein isothiocyanate (FITC) at several lysine residues to produce the FITC-I-domain. Along with trypsin digestion and peptide mapping, we utilized a specific fragmentation of the fluorochrome moiety from the modified residues in the electrospray ionization-mass spectrometry (ESI-MS) to identify the conjugation sites more quickly (Chapter 2). The FITC-I-domain binds to ICAM-1 in a calcium-, time- and energy-dependent manner. It enters the Raji cells via receptor-mediated endocytosis. FITC-I-domain binding to ICAM-1 was blocked by anti- I-domain mAb; in contrast, anti-ICAM-1 mAb to D1 and D2-domain enhance FITC-I- domain binding to ICAM-1 on the cell surface (Chapter 3). The I-domain protein was conjugated to an antigenic peptide, PLP139-151, to produce PLP-I-domain-1 and -2 conjugates. We evaluated the biological activities of the conjugates in female SJL/J mice induced with EAE (Chapter 4). The in vivo studies showed that PLP-I-domain-1 has excellent efficacy in suppressing EAE, similar to that of the best positive control (i.e., Ac-PLP-cIBR1-NH2). Although PLP-I-domain-2 could delay the onset of EAE compared to PBS, it was not as potent as PLP-I-domain-1. The chemical structure differences between PLP-I-domain-1 and -2 were determined using tryptic digestion followed by mass spectroscopic analysis. The number of conjugation sites in PLP-I-domain-1 is higher than in PLP-I-domain-2; this suggests that these additional sites in PLP-I-domain- 1 contribute to its biological activity. In conclusion, the I-domain protein binds to and is internalized by ICAM-1 receptors on the surface of immune cells. The proper conjugation of PLP peptides to I-domain (i.e., PLP-I-domain-1) is necessary for suppressing EAE in the animal model
Vaccine-like and Prophylactic Treatments of EAE with Novel IDomain 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-NH2 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 i.v or s.c. injections of IDAC in a prophylactic or a vaccine-like dosing schedule.
IDAC-3 was better than IDAC-1 in suppressing and delaying the onset of EAE when delivered in
prophylactic and vaccine-like manners. IDAC-3 also suppressed subsequent relapse of the disease.
The production of IL-17 was lowered in the IDAC-33 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-33treated mice. In conclusion, the Idomain
can effectively deliver antigenic peptides in a vaccine-like or prophylactic manner for
inducing immunotolerance in the EAE mouse model
cIBR effectively targets nanoparticles to LFA-1 on acute lymphoblastic T cells
Leukocyte function associated antigen-1 (LFA-1) is a primary cell adhesion molecule of leukocytes required for mediating cellular transmigration into sites of inflammation via the vascular endothelium. A cyclic peptide, cIBR, possesses high affinity for LFA-1 and conjugation to the surface of poly(dl-lactic-co-glycolic acid) nanoparticles can specifically target and deliver the encapsulated agents to T cells expressing LFA-1. The kinetics of targeted nanoparticle uptake by acute lymphoblastic leukemia T cells was investigated by flow cytometry and microscopy and compared to untargeted nanoparticles. The specificity of targeted nanoparticles binding to the LFA-1 integrin was demonstrated by competitive inhibition using free cIBR peptide or using the I domain of LFA-1 to inhibit the binding of targeted nanoparticles. The uptake of targeted nanoparticles was concentration and energy dependent. The cIBR-conjugated nanoparticles did not appear to localize with lysosomes whereas untargeted nanoparticles were detected in lysosomes in 6 hrs and steadily accumulated in lysosomes for 24 hrs. Finally, T-cell adhesion to epithelial cells was inhibited by cIBR-nanoparticles. Thus, nanoparticles displaying the cIBR ligand may offer a useful targeted drug delivery system as an alternative treatment of inflammatory diseases involving transmigration of leukocytes
Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes
The long-term objective of this project is to utilize the I-domain protein for the α-subunit of LFA-1 to target drugs to lymphocytes by binding to ICAM receptors on the cell surface. The short-term goal is to provide proof-of-concept that I-domain conjugated to small molecules can still bind to and uptake by ICAM-1 on the surface of lymphocytes (i.e., Raji cells). To accomplish this goal, the I-domain protein was labeled with FITC at several lysine residues to produce the FITC-I-domain and CD spectroscopy showed that the FITC-I-domain has a secondary structure similar to that of the parent I-domain. The FITC-I-domain was taken up by Raji cells via receptor-mediated endocytosis and its uptake can be blocked by anti-I-domain mAb but not by its isotype control. Antibodies to ICAM-1 enhance the binding of I-domain to ICAM-1, suggesting it binds to ICAM-1 at different sites than the antibodies. The results indicate that fluorophore modification does not alter the binding and uptake properties of the I-domain protein. Thus, I-domain could be useful as a carrier of drug to target ICAM-1-expressing lymphocytes
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 PLP139-151 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-NH2-2 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
Single-Step Grafting of Aminooxy-Peptides to Hyaluronan: A Simple Approach to Multifunctional Therapeutics for Experimental Autoimmune Encephalomyelitis
The immune response to antigens is directed in part by the presence or absence of costimulatory signals. The ability to coincidently present both antigen and, for example, a peptide that inhibits or activates the costimulatory pathway, would be a valuable tool for tolerization or immunization, respectively. A simple reaction scheme utilizing oxime chemistry was identified as a means to efficiently conjugate different peptide species to hyaluronan. Peptides synthesized with an aminooxy N-terminus reacted directly to hyaluronan under slightly acidic aqueous conditions without the need for a catalyst. The resulting oxime bond was found to rapidly hydrolyze at pH 2 releasing peptide, but was stable at higher pH values (5.5 and 7). Two different peptide species, a multiple sclerosis antigen (PLP) and an ICAM-1 ligand (LABL) known to block immune cell stimulation, were functionalized with the aminooxy end group. These peptides showed similar reactivity to hyaluronan and were conjugated in an equimolar ratio. The resulting hyaluronan with grafted PLP and LABL significantly inhibited disease in mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Aminooxy-peptides facilitate simple synthesis of multifunctional hyaluronan graft polymers, thus enabling novel approaches to antigen-specific immune modulation
Suppression of EAE and Prevention of Blood-Brain Barrier Breakdown after Vaccination with Novel Bifunctional Peptide Inhibitor
The efficacy of bifunctional peptide inhibitor (BPI) in preventing blood-brain barrier (BBB) breakdown during onset of experimental autoimmune encephalomyelitis (EAE) and suppression of the disease was evaluated in mice. The mechanism that defines how BPI prevents the disease was investigated by measuring the in vitro cytokine production of splenocytes. Peptides were injected 5 to 11 days prior to induction of EAE, and the severity of the disease was monitored by a standard clinical scoring protocol and change in body weight. The BBB breakdown in diseased and treated mice was compared to that in normal control mice by determining deposition of gadolinium diethylenetriaminepentaacetate (Gd-DTPA) in the brain using magnetic resonance imaging (MRI). Mice treated with PLP-BPI showed no or low indication of EAE as well as normal increase in body weight. In contrast, mice treated with the control peptide or PBS showed a decrease in body weight and a high disease score. The diseased mice had high deposition of Gd-DTPA in the brain, indicating breakdown in the BBB. However, the deposition of Gd-DTPA in PLP-BPI-treated mice was similar to that in normal control mice. Thus, PLP-BPI can suppress EAE when administered as a peptide vaccine and maintain the integrity of the BBB
Rapid Identification of Fluorochrome Modification Sites in Proteins by LC ESI-Q-TOF Mass Spectrometry
Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5′-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain
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
Synthetic glycopolymers as modulators of protein aggregation: influence of chemical composition, topology and concentration
Novel drug excipients are required to achieve stable formulations of protein drug candidates. Synthetic glycopolymers have been shown in some cases to improve protein formulation stability, although their structure function relationship remains unknown. Here we report the synthesis of linear or 4-arm star glycopolymers with different molecular topology and chemical composition, with mannose, galactose, arabinose, N-acetyl glucosamine, lactose and trehalose pendant units - and investigate their modulation of conformational stability and aggregation propensity of a model monoclonal antibody (mAb1). Mono-and di-saccharides with free reducing ends are not frequently utilised as protein stabilisers, due to potential reactivity with a protein’s amine groups. In this study this was circumvented through the use of a stable acetal linker connecting the polymer backbone to the sugar pendant residues, which made the latter virtually non-reactive with amines. The general destabilisation the antibody was determined as anunfolding transition temperature (Tm) of CH2 and Fab structural domains, and aggregation temperature (Tagg). The most prominent effect of the glycopolymers on a temperature induced stress in a low concentration solutions was a decrease in Tm and Tagg, regardless of sugar composition or glycopolymer topology - in contrast to the stabilising effect of the corresponding mono- and di-saccharide constituents. The exceptions of linear-lactose and star-trehalose glycopolymers, which increased Tm of the mAb Fab region and Tagg, however, highlights a more complex structure-function relationship. Accelerated stability studies of the high concentrated mAb solutions (50 mg mL-1) revealed that the increased glycopolymer concentrations generally decreased the mAb stability, as judged by the amount of mAb1 ‘monomer’ molecules in solution, with star- and linear-trehalose glycopolymers further generating visible aggregates. Interestingly the latter effect could not have been predicted from the Tm or Tagg experiments conducted at a low concentration regime. Taken together, the data demonstrate a complex interplay of sugar chemistry and molecular topology of the synthetic glycopolymers on their modulation of protein conformational stability and aggregation propensity. Solution concentration was also an important parameter contributing to the stability modulation, and suggests that the stabilising properties of a sugar as the mono- or di-saccharide cannot be extrapolated to the corresponding glycopolymers
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