Properties and pharmacokinetics of two humanized antibodies specific for L-selectin.
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.


OBJECTIVE
For this purpose, two mouse monoclonal antibodies, DREG-55 and DREG-200, that block human L-selectin were humanized and characterized.


METHODS
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.


CONCLUSIONS
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.
Co
M S
MS
Protein Design Labs, Inc., Fremont, CA 94555, USA.
Landolfi
N F
NF
Nagy
J O
JO
Tan
J H
JH
Vexler
V
V
Vasquez
M
M
Roark
L
L
Yuan
S
S
Hinton
P R
PR
Melrose
J
J
Klingbeil
C
C
Queen
C
C
Berg
E L
EL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000493:pharmacokinetics*; D000915:Antibody Affinity; D000918:Antibody Specificity; D002448:Cell Adhesion; D003001:Cloning, Molecular; D003429:Cross Reactions; D004729:Endothelium, Lymphatic / Q000378:metabolism; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019041:L-Selectin / Q000276:immunology*; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008253:Macaca mulatta; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D013045:Species Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D008081:Liposomes; D011994:Recombinant Proteins; D019041:L-Selectin