Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens

Abstract

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 x 10(9) members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody-antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

Figure 1

Schematic outline of the approach used for library construction. A library of V_H and genes was generated from rearranged human V-genes and cloned into the plasmid pCITE3A. The V_L genes used for scFv assembly were derived from a previously constructed scFv library contained in the plasmid pHEN1 (12). The vector containing the V_L repertoire also contained the scFv linker DNA 5′ to the V_L genes. Primers for reamplification of the V-gene repertoires were derived from sequences several hundred bp 5′ (the V_H genes) or 3′ (the V_L genes) of the scFv gene cloning sites. This approach facilitated the efficiency of PCR assembling a new scFv repertoire and increasing the efficiency of cutting assembled scFv genes with restriction enzymes. (A) V_H and linker-V_L gene repertoires were generated by PCR from the plasmid DNA of the separate libraries. The V_H genes wereamplified by using a plasmid specific primer    and an equimolar mixture of HuJ_H primers    . The linker DNA and V_L genes were amplified by using a plasmid specific primer   and an equimolar mixture of RHuJ_H primers     . The RHuJH primers are complementary to the HuJ_H primers. (B) The V_H and linker DNA-V_L gene repertoires were PCR assembled into a scFv gene repertoire. (C) The assembled scFv gene repertoire was cut with the restriction enzymes NcoI and NotI and cloned into the plasmid pHEN1 (17) for phage display.

Figure 2

V-gene usage and V_H CDR3 length of unselected and antigen-specific scFv. The V_H and V_L genes were sequenced and the germ-line gene was assigned based on homology to a database (vbase) of germ-line V-genes compiled by Tomlinson et al. (25). Specific V_H, V_κ, and V_λ genes are listed on the ordinate, with the V_H, V_κ, or V_λ germ-line gene family indicated below. Only V-genes in unselected or selected clones are listed.

Figure 3

Specificity of anti-Botulinum neurotoxin scFv. Representative scFv (2H6, 3D1, 3B12, and 3C8) isolated respectively from selections on BoNT serotypes A, B, C, and E were studied. Specificity was determined by ELISA.

Figure 4

Staining of HeLa cells infected with C. trachomatis with the scFv 2A10. The scFv specifically stains C. trachomatis elementary bodies (c) within infected HeLa cells but does not stain uninfected cells. n = nucleus.