Humanized porcine VCAM-specific monoclonal antibodies with chimeric IgG2/G4 constant regions block human leukocyte binding to porcine endothelial cells.
Discordant xenografts surviving the initial hyperacute rejection phase may be subject to cellular rejection processes mediated by infiltrating leukocytes including T cells, NK cells and monocytes. The stable adhesion of these cell types to endothelial cells is due to the molecular interaction of the integrins VLA-4 and LFA-1 with their ligands vascular cell adhesion molecule (VCAM) and ICAM-1 present on the endothelial cells. Human VLA-4 binds to porcine VCAM, and blocking mAbs specific for porcine VCAM have been developed. We have localized the epitope of the anti-porcine VCAM blocking mAbs 2A2 and 3F4 to domains 1 and 2, respectively. Humanized antibodies (IgG4 isotype) were constructed from these anti-porcine VCAM antibodies and demonstrated to inhibit adhesion of Ramos, Jurkat and YT cells, as well as purified resting and activated human T cells, to porcine aortic endothelial cells (PAEC). These cell types express both LFA-1 as well as VLA-4, suggesting blockade of human VLA-4 interaction with porcine VCAM may alone be sufficient to significantly impair adhesion of human leukocytes to porcine endothelial cells. The chimeric anti-porcine VCAM (pVCAM) HuG4 antibodies promoted increased adhesion of Fc receptor (FcR) positive cells such as U937 monocytic cells to PAEC. In contrast, chimeric anti-porcine VCAM antibodies created using the CH1 and hinge region from human IgG2 and the CH2 and CH3 regions from human IgG4 (HuG2/G4 antibodies) inhibited binding of FcR positive cells to PAEC. These chimeric anti-pVCAM antibodies should allow delineation of the in vivo role of VLA-4/VCAM interaction in porcine-to-primate xenotransplants. Further, the design of the HuG2/G4 antibodies should render them efficacious in multiple settings requiring elimination of FcR binding.
Mueller
J P
JP
Alexion Pharmaceuticals Inc., Department of Immunobiology, New Haven, CT 06511, U.S.A. muellerj@alxn.com
Giannoni
M A
MA
Hartman
S L
SL
Elliott
E A
EA
Squinto
S P
SP
Matis
L A
LA
Evans
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D002448:Cell Adhesion; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology*; D018604:Epitope Mapping; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007962:Leukocytes / Q000166:cytology*; D008969:Molecular Sequence Data; D011961:Receptors, Fc / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013552:Swine; D019010:Vascular Cell Adhesion Molecule-1 / Q000276:immunology
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D019010:Vascular Cell Adhesion Molecule-1