Making antibody fragments using phage display libraries

Abstract

To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.

A peptide derived from an autoantibody can stimulate t cells in the (nzb × nzw)f1 mouse model of systemic lupus erythematosus

“…Of 697 murine Ig H and L chains stored in GenBank, only 2 (0.3%) contained an amino acid sequence identical to A6H 58-69. One of those is an autoantibody from an MRLllpr mouse (26), the other is an anti-2-phenyloxazole-5-thione from a BALBlc mouse (27). The 5-mer KFKGK is more frequent, with identity found in 7.5% of H or L chains in the database.…”

Targeting TARP, a novel breast and prostate tumor‐associated antigen, with T cell receptor‐like human recombinant antibodies

“…However, in the last 2-3 years we and others utilized the selection power of the phage display approach [51][52][53][54] together with the availability of very large human naive antibody libraries. These enabled us to screen these large libraries of such vast diversity and size in order to isolate these rare and unique TCR-like recombinant antibodies targeting human tumor and viral epitopes [21,22,[24][25][26].…”

Intrabody-mediated phenotypic knockout of major histocompatibility complex class I expression in human and monkey cell lines and in primary human keratinocytes

“…81 Positive clones were further screened by BstNI fingerprinting after reamplification of the inserts using primers 7452 and 11816. 82 …”