The long-term objective of this proposal is to understand the mechanism of internalization of integrins and their utility for targeting drugs to specific cells. The short-term objective of this proposal is to study the mechanism of internalization of LFA-1 on T cells and to utilize this mechanism for targeting drugs to leukocytes. The central hypothesis is that an ICAM-1-derived peptide (cIBR) can bind to and be internalized by LFA-1. Therefore, a cytotoxic drug (methotrexate, MTX) will be linked with cIBR peptide to give MTX-cIBR conjugate; this conjugate will have dual functions: (a) to inhibit T-cell activation via inhibition of the cell adhesion signal and (b) to induce T-cell death by internalizing MTX. The MTX-cIBR conjugate has selective toxicity for LFA-1-expressing T cells but not LFA-1-deficient KB epithelial cells. MTXcIBR conjugate suppressed rheumatoid arthritis in the collagen-induced arthritis (CIA) mouse model better than MTX alone. Therefore, the first objective is to evaluate the mechanism of suppression of arthritis by MTX-cIBR conjugate (Specific Aim 1). The mechanism of MTX-cIBR conjugate in altering the activation of T cells from Th1 to Th2 response in the CIA mouse model will be elucidated. In addition, the long-term kinetic effects of MTX-cIBR on the T-cell commitment in the CIA mouse model will be determined. Secondly, the internalization of MTX-cIBR or FITC-cIBR conjugates will be released by LFA-1 in the cytoplasm; alternatively, it could (a) remain with the endosome, (b) be recycled to the cell surface, or (c) be targeted to other membrane compartments. Therefore, the internalization process and localization of these conjugates will be determined (Specific Aim 2). Thirdly, the internalization of MTX-cIBR may also be via reduced folate carrier (RFC) or membrane folate binding protein (mFBP).
In Specific Aim 3, MTX-cIBR will be compared using wild type and RFC-deficient mouse leukemia T cells. In addition, this conjugate will be compared with MTX in mFBP over-expressing leukemia mouse cells in the presence and absence of folic acid. Finally, the exact mechanism of the LFA-1 internalization and the identity of other proteins that are involved in this process are not well understood.
In Specific Aim 4, cIBR peptide will be modified and cross-linked to LFA-1 on activated T cells; the crosslink will be isolated along with proteins that interact with LFA-1. These isolated proteins will be identified.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063002-03
Application #
7209818
Study Section
Special Emphasis Panel (ZRG1-GDD (01))
Program Officer
Ferguson, Stacy E
Project Start
2005-07-01
Project End
2009-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
3
Fiscal Year
2007
Total Cost
$269,836
Indirect Cost
Name
University of Kansas Lawrence
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
076248616
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Badawi, Ahmed H; Kiptoo, Paul; Siahaan, Teruna J (2015) Immune Tolerance Induction against Experimental Autoimmune Encephalomyelitis (EAE) Using A New PLP-B7AP Conjugate that Simultaneously Targets B7/CD28 Costimulatory Signal and TCR/MHC-II Signal. J Mult Scler (Foster City) 2:
Büyüktimkin, Barlas; Kiptoo, Paul; Siahaan, Teruna J (2014) Bifunctional Peptide Inhibitors Suppress Interleukin-6 Proliferation and Ameliorates Murine Collagen-Induced Arthritis. J Clin Cell Immunol 5:
Buyuktimkin, Barlas; Manikwar, Prakash; Kiptoo, Paul K et al. (2013) Vaccinelike and prophylactic treatments of EAE with novel I-domain antigen conjugates (IDAC): targeting multiple antigenic peptides to APC. Mol Pharm 10:297-306
Kiptoo, P; Büyüktimkin, B; Badawi, A H et al. (2013) Controlling immune response and demyelination using highly potent bifunctional peptide inhibitors in the suppression of experimental autoimmune encephalomyelitis. Clin Exp Immunol 172:23-36
Badawi, Ahmed H; Siahaan, Teruna J (2013) Suppression of MOG- and PLP-induced experimental autoimmune encephalomyelitis using a novel multivalent bifunctional peptide inhibitor. J Neuroimmunol 263:20-7
Majumdar, Sumit; Siahaan, Teruna J (2012) Peptide-mediated targeted drug delivery. Med Res Rev 32:637-58
Büyüktimkin, Barlas; Wang, Qun; Kiptoo, Paul et al. (2012) Vaccine-like controlled-release delivery of an immunomodulating peptide to treat experimental autoimmune encephalomyelitis. Mol Pharm 9:979-85
Badawi, Ahmed H; Siahaan, Teruna J (2012) Immune modulating peptides for the treatment and suppression of multiple sclerosis. Clin Immunol 144:127-38
Manikwar, Prakash; Büyüktimkin, Barlas; Kiptoo, Paul et al. (2012) I-domain-antigen conjugate (IDAC) for delivering antigenic peptides to APC: synthesis, characterization, and in vivo EAE suppression. Bioconjug Chem 23:509-17
Majumdar, Sumit; Anderson, Meagan E; Xu, Christine R et al. (2012) Methotrexate (MTX)-cIBR conjugate for targeting MTX to leukocytes: conjugate stability and in vivo efficacy in suppressing rheumatoid arthritis. J Pharm Sci 101:3275-91

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