Two-Phase Approach for the Expression of High-Affinity Human Anti-Human Immunodeficiency Virus Immunoglobulin Fab Domains in Escherichia coli

Abstract

We describe here a two-phase approach for the development of high -affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system. The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgpl60. One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4x10^-10 M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gpl20 with a dissociation constant on the order of 8 x 10^-10 M, but was unable to recognize cell surface antigen. In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector. mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA. The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector. The recombinant Fabs (rFabM7B5 and rFabT15Gl) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the γ chain isotypes and the V_H and V_L chain gene usage. The binding specificities of rFabM7B5 and rFabT15Gl were indistinguishable from their respective intact MAbs. The dissociation constants for the rFabs were approximately 1.4 x 10^-9 M for rFabM7B5 and 2 x 10^-9 M for rFabT15Gl. Our studies show that human Fab domains, which maintain their binding specificities and have high binding affinities for HIV, can be rescued from hybridoma cell lines and expressed in bacteria.