Immunotechnology 4 (1999) 253 – 266 Properties and pharmacokinetics of two humanized antibodies specific for L-selectin Man Sung Co a , Nicholas F. Landolfi a , Jon O. Nagy b , Jennifer H. Tan b , Vladimir Vexler a , Max Vasquez a , Linda Roark a , Shauna Yuan a , Paul R. Hinton a , Jennifer Melrose a , Corine Klingbeil a , Cary Queen a , Ellen L. Berg a, * a Protein Design Labs, Inc., 34801 Campus Dr., Fremont, CA 94555, USA b Life Sciences Diision, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd., Berkeley, CA 94720, USA Received 5 October 1998; received in revised form 14 December 1998; accepted 16 December 1998 Abstract Background : The participation of L-selectin in leukocyte recruitment during inflammation has suggested the use of L-selectin inhibitors as potential anti-inflammatory therapeutics. Blocking monoclonal antibodies could serve as such therapeutic agents, particularly if humanized to reduce their immunogenicity and improve their serum half-life. Objecties : For this purpose, two mouse monoclonal antibodies, DREG-55 and DREG-200, that block human L-selectin were humanized and characterized. Study design : The resulting humanized antibodies, HuDREG-55 and HuDREG-200, constructed with human IgG4 constant regions, were evaluated for their specificity, affinity and ability to block L-selectin-dependent adhesion in in vitro assays. Their pharmacokinetic behavior in rhesus monkeys was also studied. Results : HuDREG-55 and HuDREG-200 were found to retain the specificity and affinity, within 2-fold, of the parent murine antibodies. HuDREG-55 and HuDREG-200 block L-selectin-dependent adhesion of human lymphocytes to high endothelial venules in frozen sections of lymph nodes. In addition, HuDREG-55 and HuDREG-200 are inhibitory in a novel L-selectin-dependent adhesion assay. This assay utilizes flow cytometry to measure binding of polymerized liposomes containing an analog of sialyl Lewis X, sialyl Lewis X glycoliposomes, to peripheral blood neutrophils and lymphocytes. Studying the pharmacokinetics of HuDREG-55 and HuDREG-200 in rhesus monkeys showed terminal elimination half-lives at 12.0 and 20.3 days, respectively. Conclusion : The shorter terminal elimination half-life of HuDREG-55 in rhesus monkeys may be due to the ability of HuDREG-55 but not HuDREG-200 to bind rhesus monkey L-selectin. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Adhesion; Antibodies; Humanization; Pharmacokinetics; L-Selectin Abbreiations: DMEM, Dulbecco’s modified Eagle’s medium; CDR, complementarity determining regions; FBS, fetal bovine serum; HAMA, human anti-mouse antibody; HBSS, Hank’s balanced salt solution; HEV, high endothelial venules; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PLN, peripheral lymph node. * Corresponding author. Tel.: +1-510-574-1400; fax: +1-510-574-1500. 1380-2933/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII:S1380-2933(98)00024-4