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

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

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

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

Both reversible self-association and structural changes underpin molecular viscoelasticity of mAb solutions

The role of antibody structure (conformation) in solution rheology is probed. It is demonstrated here that pH-dependent changes in the tertiary structure of 2 mAb solutions lead to viscoelasticity and not merely a shear viscosity (η) increase. Steady shear flow curves on mAb solutions are reported over broad pH (3.0 ≤ pH ≤ 8.7) and concentration (2 mg/mL ≤ c ≤ 120 mg/mL) ranges to comprehensively characterize their rheology. Results are interpreted using size exclusion chromatography, differential scanning calorimetry, analytical ultracentrifugation, near-UV circular dichroism, and dynamic light scattering. Changes in tertiary structure with concentration lead to elastic yield stress and increased solution viscosity in solution of “mAb1.” These findings are supported by dynamic light scattering and differential scanning calorimetry, which show increased hydrodynamic radius of mAb1 at low pH and a reduced melting temperature Tm, respectively. Conversely, another molecule at 120 mg/mL solution concentration is a strong viscoelastic gel due to perturbed tertiary structure (seen in circular dichroism) at pH 3.0, but the same molecule responds as a viscous liquid due to reversible self-association at pH 7.4 (verified by analytical ultracentrifugation). Both protein–protein interactions and structural perturbations govern pH-dependent viscoelasticity of mAb solutions

Effects of Oxidative Stress on The Cytoskeletal Crosslinker Protein, Plectin

Oxidative stress has been implicated in the onset of various diseases, including renal failure and neurodegenerative diseases. Increasing number of studies suggest the involvement of multiple processes in the pathogenesis of cell injury during oxidative stress. An important event caused by oxidants is the loss of cellular morphology and function, which may be due to disruption of the cytoskeleton. However, we still do not have a detailed understanding of the mechanisms by which cytoskeleton disruption takes place. Hence, an understanding of the mechanisms would be very important to explore agents that can prevent damage caused by free radicals in renal and neurodegenerative diseases. These studies were performed in monkey kidney cells (CV-1) to investigate mechanisms for kidney disorders and in human cortical neurons (HCN2) to investigate mechanisms for neurodegenerative diseases. This study has two objectives. First, is to determine effects of oxidative stress on the crosslinker protein ‘plectin’ and to visualize and compare the distribution patterns of plectin and actin distributions under conditions of oxidative stress in monkey kidney cells. Second, to determine the role of caspases in plectin breakdown in neuronal cells. Previous studies from our laboratory showed that CV-1 (monkey kidney) cells and human cortical neurons (HCN2) subjected to oxidative stress, showed damage to all the three major cytoskeletal structures. It is well understood that crosslinker proteins, like plectin, interconnect the three major cytoskeleton filaments. Hence, it is possible that early disruption of plectin results in the subsequent breakdown of all cytoskeleton components. This study examines the effect of free radical generatin toxin, tertiary-butyl hydroperoxide (t-BuOOH) on plectin in CV-1 cells, CV-1 cells were treated with t-BuOOH at times before significant cell death and apoptosis was observed. Immunofluorescence studies showed that actin co-localized with plectin in control cells. However, in cells treated with t-BuOOH for as early as 30 mins, loss of plectin was observed in areas with intact actin filaments and indicated that plectin breakdown preceded actin actin disruption. Changes in gene expression were measured by RT-PCR; proteins levels were measured by SDS-PAGE and Western blot analysis. Results showed reduced levels of plectin protein even in 30 mins-treated cells as compared to controls though there was no significant reduction of plectin gene expression. TUNEL staining studies on HCN2 cells treated with free radical toxin for various time points (30 mins, 1 or 3 hrs) showed DNA fragmentation only after 3 hrs. Previous studies in our laboratory showed breakdown of plectin proteins as early as 30 mins in neurons treated with free radical toxin (t-BuOOH). These findings suggest that plectin proteins as early as 30 mins in neurons treated with free radical toxin (t-BuOOH). These findings suggest that plectin damage takes place well before the DNA fragmentation. Further, we investigated the role of caspases in the breakdown of plectin in human neuronal cells. HCN2 cells treated with 100 uM t-BuOOH for 15 or 30 mins showed increased levels of pro-caspase-8 (active form) is seen significantly increased at 30 mins. Both caspase-9 and caspase-3 levels not altered in HCN2 cells treated with t-BuOOH for 15 and 30 mins compared to control. So cleaved products of caspase-9 or 3 were observed. These results suggested that caspase-8 is activated at 30 mins treatment even before caspase-9 and caspase-3 during t-BuOOH-induced oxidative stress in human cortical neurons. SDS-PAGE and Western blot analysis performed using pancaspase inhibitor indicated involvement of capases in plectin breakdown, studies performed using specific caspase-8 inhibitor suggested plectin cleavage by caspase-8 at 30 mins in t-BuOOH oxidative stress in human cortical neurons. In addition, the results obtained from immunofluorescence studies of plectin using specific caspase-8 inhibitor supported the biochemical analysis. This indicated that the early breakdown of plectin by caspase-8 even before activation of caspase-9 and caspase-3 could result in subsequent collapse of the cytoskeleton, ultimately cell death