Isolation of high affinity human antibodies directly from large synthetic repertoires.

Abstract

Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 × 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH‐26 (DP‐47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.

Antigen specificity and tumour targeting efficiency of a human carcinoembryonic antigen-specific scFv and affinity-matured derivatives

“…The dissociation constant of CEA6 for CEA is in the nanomolar range (7.7 x 10-9 M), which is typical of antibodies isolated from very large repertoires (Perelson and Oster, 1979;Griffiths et al, 1994;Vaughan et al, 1996). Our early work showed that the antibody binds CEA-expressing cells by flow cytometry and immunocytochemistry, and localizes to CEA-expressing human tumour xenografts in nude mice.…”

Phage antibodies against an unstable hapten: Oxygen sensitive reduced flavin

“…2A, curves b,c). This was unexpected as the rotational correlation time of a scFv molecule can be calculated according to Visser et al [17] as [10][11][12][13][14][15][16][17][18][19][20] ns. It appears that this discrepancy may be due to association of scFv molecules by hydrophobic interactions in the presence of salt, as in experiments performed in the absence of NaC1 (%crv shifts to about 20 ns (data not shown).…”

Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library

“…The theoretical advantages of recombinant human antibodies over mouse monoclonals as therapeutic agents are well recognized [1][2][3][4][5]. However, the optimal source of immunoglobulin genes for the construction of these libraries remains uncertain.…”

“…Moreover, it has recently been demonstrated that the retrieval of specific antibodies can be enhanced by increasing the library size and by using synthetic immunoglobulin genes [3,4,8]. The diversity and size of synthetic libraries, and thus the affinity of particular antibodies, can be further increased by combinatorial infection and in vitro recombination strategies [3,9,10]. However, this artificial construction of large non-immune libraries is technically challenging and introduces a number of potential problems.…”