A420983, a Novel, Small Molecule Inhibitor of LCK Prevents Allograft Rejection W. Waegell, M. Babineau, M. Hart, K. Dixon, B. McRae, C. Wallace, M. Leach, S. Ratnofsky, A. Belanger, G. Hirst, A. Rossini, M. Appel, J. Mordes, D. Greiner, and S. Banerjee T HE SRC family kinase, Lck, that is expressed only in T cells and natural killer (NK) cells, plays a key role in T-cell activation due to its critical role in signal transduction through the T-cell receptor (TCR). 1,2 Upon TCR stimula- tion, Lck is rendered catalytically competent by tyrosine phosphorylation on the activation loop and this phosphor- ylates the TCR -chain leading to recruitment of ZAP-70 and its subsequent activation, also by Lck. Zap 70 activation is critical to downstream signaling events leading to T cell activation. 3,4 Mice lacking Lck have defective T-cell func- tion as evidenced by their impaired response to TCR stimulation 5 . Lck -/- mice are unable to reject fully major histocompatability complex (MHC)-mismatched allo- grafts 6,7 despite the presence of peripheral T cells. Since Lck expression is limited to cells of the T-cell and NK-cell lineage, it is an attractive target for selective immunosup- pression. We have developed a novel, small molecule inhibitor of Lck, A420983, which potently competes for the ATP binding pocket of the enzyme (IC 50 = 39 nM at 1 mmol/L ATP). This compound is active in vivo and has a biologic duration of action of greater than 12 hours after oral administration. In the present studies we demonstrate the ability of A420983 to inhibit several T-cell-driven responses and provide evidence that inhibition of Lck is an effective method of T-cell-specific immunosuppression. METHODS AND MATERIALS Animals Inbred C57BL/6 (H-2 b ) and BALB/c (H-2 d ) female mice, aged 6 to 12 weeks, were obtained from Jackson Laboratory (Bar Harbor, Maine), Taconic Farms (Germantown, NY), or the NCI (Freder- ick, Md). In cardiac transplantation studies, 1- to 2-day-old neo- nates were used as cardiac donors. Compounds A420983 was synthesized at Abbott Bioresearch Center, Worces- ter, Mass and was dissolved in sterile water. Cyclosporin A (Neoral, Novartis Pharmaceutical, East Hanover, NJ) was diluted with water prior to dosing. Anti-CD3-Induced Interleukin-2 Production Six- to 8-week-old BALB/c mice were dosed orally with A420983 30 minutes prior to intravenous injection of 75 ng hamster anti-mouse CD3 antibody, 145-2C11 (PharMingen, San Diego, Calif). Two hours after anti-CD3 injection mice were bled via cardiac puncture then serum was collected and assayed for interleukin (IL)-2 by enzyme-linked immunosorbent assay (ELISA) (Endogen, Woburn, Mass). Antigen-Induced Cytokine Production We utilized a modification of a method described by Magram et al. 8 Briefly, C57BL/6 mice were immunized intradermally on day 0 with 200 g MOG 35–55 (myelin oligodendrocyte glycoprotein peptide) (New England Peptide, Fitchburg, Mass) in a 1:1 emulsion with complete Freund’s adjuvant (Difco, Detroit, Mich). Mice were treated daily, orally with vehicle or A420983 from day -1 through day 6. On day 7 postimmunization, mice were euthanized using CO 2 inhalation. Draining lymph nodes were aseptically removed and placed in RPMI (Gibco BRL, Grand Island, NY) supple- mented with 10% fetal bovine serum (Hyclone, Logan, Utah), 5.510 -3 mmol/L -mercaptoethanol, 110 -3 mmol/L nonessen- tial amino acids, 110 -4 mmol/L sodium pyruvate, 510 -3 U/mL penicillin/ 510 -3 g/mL streptomycin, and 210 -4 mmol/L L- glutamine (Gibco). Cells were suspended at a concentration of 6  10 6 cells/ml and cultured in a 96 well plate (Corning, Corning, NY) with MOG 35–55 at a concentration of 50 or 100 g/mL. Plates were incubated at 37°C for 24 hours (for IL-2 measurement) or 48 hours (for interferon (IFN)- measurement). Cytokine levels were deter- mined by ELISA kit (IFN-. R&D Systems, Minneapolis, Minn; IL-2: Endogen, Woburn, Mass). Delayed Type Hypersensitivity We used a modification of a method described by Magram et al. 8,9 On day 0 C57BL/6 mice were immunized intradermal (ID) with 400 g methylated bovine serum albumin (mBSA) (Sigma, St. Louis, Mo) in a 1:1 emulsion with complete Freund’s adjuvant (Difco). Mice were treated orally once daily with vehicle or A420983 from day -1 through day 8. On day 7 postimmunization, mice were challenged in one hind footpad with 100 g of mBSA in 20 L of PBS and in the opposite footpad with PBS alone. Footpad swelling was measured 24 hours after challenge using a vernier caliper. From the Abbott Bioresearch Center, (W.W., M.B., M.H., K.D., B.M., C.W., M.L., S.R., A.B., G.H., S.B.), and the University of Massachusetts Medical Center (A.R., M.A., J.M., D.G.), Worces- ter, Massachusetts. Address reprint requests to Dr W. Waegell, Abbott Biore- search Center, 100 Research Dr, Worcester, MA 01605. © 2002 by Elsevier Science Inc. 0041-1345/02/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(02)02909-3 Transplantation Proceedings, 34, 1411–1417 (2002) 1411