Directed selection of MIP-1 alpha neutralizing CCR5 antibodies from a phage display human antibody library.
The seven trans-membrane chemokine receptor CCR-5 is a coreceptor for macrophage tropic HIV-1 strains. CCR-5 responds to a number of chemokines, including macrophage inflammatory protein (MIP)-1 alpha. We describe the use of MIP-1 alpha in a biotin tyramine-mediated proximity selection to guide the selection of CCR-5-specific phage antibodies from a large phage display human library. Proximity based selections resulted in a population of antibodies recognizing CCR-5 on primary CD4+ lymphocytes, none of which blocked MIP-1 alpha binding to cells. The selected population of phage antibodies were subsequently used as guide molecules for a second phase of selection that was carried out in the absence of MIP-1 alpha. This generated a panel of CCR-5-binding antibodies, of which around 20% inhibited MIP-1 alpha binding to CD4+. The single chain Fvs (scFv) generated by this step-back selection procedure also inhibited MIP-1 alpha-mediated calcium signaling.
Cambridge Antibody Technology Limited, Melbourn, Cambridgeshire, U.K. jane.osbourn@camb-antibody.co.uk
J K
JK
Osbourn
J C
JC
Earnshaw
K S
KS
Johnson
M
M
Parmentier
V
V
Timmermans
J
J
McCafferty
D000818:Animals; D019138:Antibodies, Blocking / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D001435:Bacteriophages / Q000235:genetics; D001667:Binding, Competitive; D019921:Biotinylation; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D054407:Chemokine CCL4; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000302:isolation & purification / Q000378:metabolism; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism* / Q000494:pharmacology; D019151:Peptide Library; D019713:Receptors, CCR5 / Q000276:immunology* / Q000378:metabolism; D014162:Transfection
D019138:Antibodies, Blocking; D054407:Chemokine CCL4; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019402:Macrophage Inflammatory Proteins; D019151:Peptide Library; D019713:Receptors, CCR5; D002118:Calcium