The isolation of novel phage display-derived human recombinant antibodies against CCR5, the major co-receptor of HIV.
Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest.
Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
Moria
M
Shimoni
Alon
A
Herschhorn
Yelena
Y
Britan-Rosich
Moshe
M
Kotler
Itai
I
Benhar
Amnon
A
Hizi
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology; D015704:CD4 Antigens / Q000096:biosynthesis; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D015658:HIV Infections / Q000276:immunology* / Q000517:prevention & control; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D008565:Membrane Proteins / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D019713:Receptors, CCR5 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D015728:Receptors, HIV / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology
D000911:Antibodies, Monoclonal; C584933:CCR5 protein, human; D015704:CD4 Antigens; D007074:Immunoglobulin G; D008565:Membrane Proteins; D019151:Peptide Library; D019713:Receptors, CCR5; D015728:Receptors, HIV; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins