Novel monoclonal antibodies to putative selectin carbohydrate ligands thatinhibit selectin binding to myeloid cells

Four newly developed monoclonal antibodies (MAbs) are characterized using f lowcytometry, enzyme-linked immunoadsorbent assay (ELISA), immunoprecipitat ion and Western blots, carbohydrate epitope mapping, glycosidase cleavage, and competition binding assays. Their effects on selectin binding to myeloi d cells was tested, These MAbs react only with myeloid cells, MAbs CI-1, BU 60, and HIM95 recognize epitopes expressed by CD11/CD18 (beta(z)) integrins , while H1247 and CSLEX1 do not. The epitopes require Lewis x [Gal beta 1-4 (Fuc alpha 1-3)GlcNAc] based on reactivity with oligosaccharide-polyacryla mide-biotin or oligosaccharide-BSA conjugates, MAb HI247 recognizes a relat ed structure, sialyl-Lewis x, NeuAc alpha 2-3GaL beta 1-4(Fuc alpha 1-3)Glc NAc. The three MAbs against Lewis x show some minor differences in their re activity such as recognizing their antigens on CD11/CD18 integrins after en do-beta-galactosidase treatment and recognizing free Lewis x, The hydroxyl group on C-3 of the terminal galactose is important for recognition by MAb CI-1, BU60, and HIM95 as its substitution with sulfo group of sialic acid a bolishes the binding of these MAbs, The C-3 sialic acid is crucial for the binding of MAb HI247, Its replacement by sulphate or its cleavage by sialid ase eliminates recognition by this MAb, MAbs HI247 and CSLEX-1 did not reac t in ELISA with immobilized CD11/CD18, suggesting that the majority of sial yl Lewis x on CD11/CD18 molecules may have sialic acid 6-linked rather than 3-linked to galactose, Unexpectedly, MAb BU60 inhibited binding of P-selec tin mu chain chimera to HL-60 or U937 cells, while CI-1, HIM95 and three ot her defined anti-Lewis x MAbs (6C7, M6-1 and LeuM1) did not, MAb HI247 inhi bited binding of both E- and P-selectin chimeras to these cell lines more e ffectively than several characterized MAbs (CSLEX-1, FH6, HECA-452) to sial yl Lewis x and related oligosaccharides. Certain combinations of these anti carbohydrate MAbs had additive inhibitory effects on selectin binding, sugg esting a potential application of these new MAbs in cell adhesion/migration and tumor metastasis studies.