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IN VITRO AND IN VIVO EVALUATION OF NOVEL I-DOMAIN CONJUGATES FOR DRUG TARGETING TO IMMUNE CELLS AND SUPPRESSION OF EAE IN MICE
Manikwar, Prakash
Manikwar, Prakash
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Abstract
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.
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Date
2010-06-23
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University of Kansas
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Keywords
Pharmaceutical chemistry, Conjugation, Experimental autoimmune encephalomyelitis, Fluorescein isothiocyanate, Leukocyte function associated antigen-1 (lfa-1), Lfa 1 i-domain, Proteolipid protein (plp)