Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays

Abstract

The gene VII protein (pVII) and gene IX protein (pIX) are associated closely on the surface of filamentous bacteriophage that is opposite of the end harboring the widely exploited pIII protein. We developed a phagemid format wherein antibody heavy-and light-chain variable regions were fused to the amino termini of pVII and pIX, respectively. Significantly, the fusion proteins interacted to form a functional Fv-binding domain on the phage surface. Our approach will be applicable to the display of generic peptide and protein libraries that can form combinatorial heterodimeric arrays. Consequently, it represents a first step toward artificial antibodies and the selection of novel biological activities.Now that combinatorial antibody libraries have been secured (1-9), a powerful next step is the evolution toward artificial antibody constructs. Artificial antibodies are defined here as protein motifs of large diversity that use the functional strategy of the antibody molecule, but can be free of loop and framework structural constraints. When reduced to its essence, the antibody molecule is a biological device for the presentation of a combinatorial array of peptide elements in threedimensional space. The essential feature is that while CDRs (complementarity-determining regions) cooperate to form a binding site, their interaction is dynamic and functional with little structural association between the CDRs themselves. In this way, the full complement of amino acid residues is available for antigen recognition at a minimum energetic cost for binding. We propose that the ability to control the combinatorial design not only of sequence space but also of three-dimensional space would recapitulate and ultimately transcend the natural design of the immune repertoire.Although phage display has been investigated intensively, many details of the phage particle itself have not been fully elucidated, and the possibility of alternative display formats also remain to be explored. The filamentous bacteriophage fd, and, similarly, M13, consists of a circular, single-stranded DNA molecule surrounded by a cylinder of coat proteins (Fig. 1). The molecular mass of a particle is about 1.6 ϫ 10 7 Da, of which 88% is protein and 12% is DNA (10). There are about 2,700 molecules of the major coat protein pVIII that envelope the phage. At one end of the particle, there are five copies each of gene VII and VI proteins (pIII and pVI) that are involved in host-cell binding and in the termination of the assembly process. The other end contains five copies each of pVII and pIX that are actually hydrophobic peptides of 33 and 32 aa, respectively, required for the initiation of assembly and for maintenance of virion stability. Whereas pIII, pVI, and pVIII have been used to display biological molecules, pVII and pIX have not been utilized (1, 11).Substantial information has been accumulated about the structural and the phage display characteristics of pIII and pVIII. Yet, the data concerning the minor proteins encoded by genes VII and IX are...

A method for the generation of combinatorial antibody libraries using pIX phage display

“…However, a similar body of data concerning the proteins pVII and pIX remains to be acquired. Recently, we elucidated the orientation of pVII and pIX on the phage surface and, for the first time, showed that pVII and pIX could be used to individually display fusion proteins, notably the V H and V L of the antibody Fv fragment (33). Encouraged by these results, we explored the further application of pIX to display a library of human scFvs.…”

“…Significantly, large, nonimmune or ''naïve'' antibody-phage libraries displayed on pIII have proven to be a general method to readily isolate high-affinity and specific human antibodies against a variety of target antigens (28)(29)(30)(31)(32). We previously showed that pVII and pIX could be used to display the antibody variable heavy-chain region (V H ) and variable light-chain region (V L ), respectively, and that this heterodimeric presentation afforded a viable Fv with fully functional binding and catalytic activities (33). In the present study, we used a pIX display alone for the efficient construction of a large, naïve human antibody library based on the fusion of the scFv format to the N terminus of pIX.…”

Gelatinase-mediated migration and invasion of cancer cells

“…All the other coat proteins of the filamentous bacteriophage can and have been utilized for phage display. The pVII and pIX minor coat proteins have been used together for two-chain antibody display (Gao et al, 1999) and pVI display for peptide and cDNA display. In contrast to the pIII and pVIII display systems, the C-terminus of the pVI protein projects outwards and allows display of peptides and proteins with a free C-terminus (Hufton et al, 1999).…”

Efficient phage display of polypeptides fused to the carboxy‐terminus of the M13 gene‐3 minor coat protein

“…A single report has detailed the display of proteins fused to the carboxy-terminus of the gene-6 minor coat protein, but display levels were about 100-fold lower than those observed with aminoterminal P3 display [5]. Another report has indicated that the two remaining coat proteins (the gene-7 and gene-9 minor coat proteins) both support amino-terminal fusions but not carboxy-terminal fusions [6]. Crameri et al [7] have also developed a heterodimeric display system in which one member of a heterodimer pair is displayed as a fusion to the aminoterminus of P3, and the second member is co-expressed with a carboxy-terminal fusion.…”

“…could be useful in studies of heterodimeric protein^protein interactions [6] and enzyme^substrate interactions [21].…”