Two-Phase Approach for the Expression of High-Affinity Human Anti-Human Immunodeficiency Virus Immunoglobulin Fab Domains inEscherichia 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 rgp160. 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 4 x 10(-10) M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gp120 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 rFabT15G1) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the gamma chain isotypes and the VH and VL chain gene usage. The binding specificities of rFabM7B5 and rFabT15G1 were indistinguishable from their respective intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

A Nonneutralizing Anti-HIV-1 Antibody Turns into a Neutralizing Antibody When Expressed on the Surface of HIV-1-Susceptible Cells: A New Way to Fight HIV

“…The mAb derived from one of these hybridomas displays high affinity binding to the cluster II determinant of the ectodomain of gp41 with a K d value of 4 ϫ 10 Ϫ10 M (20). This human anti-gp41 Ab or its recombinant Fab or single-chain fragment variable region (scFv) 3 have been repeatedly shown to bind to HIV-1 gp160 or gp41, but fail to block viral entry (20,21). Thus, it is truly a nonneutralizing Ab.…”

The Glycosylphosphatidylinositol-Anchored Variable Region of Llama Heavy Chain-Only Antibody JM4 Efficiently Blocks both Cell-Free and T Cell-T Cell Transmission of Human Immunodeficiency Virus Type 1

“…Moreover, anti-V1/V2 loop GPI-HCDR3 PG9 and PG16 have potent and broad neutralization activities similar to those of the soluble whole antibodies PG9 and PG16 (26,28,46). Furthermore, anti-C-helix GPI-scFv TG15 exhibited moderate neutralization against 8 out of 12 HIV-1 strains tested, whereas soluble whole antibody TG15, which was originally defined as a nonneutralizing antibody, does not have any neutralization activity (27,48). Finally, anti-MPER GPI-scFv 4E10 significantly increases neutralization potency compared to that of soluble whole antibody 4E10 (27,46,50).…”

“…This may block further conformational change of the HIV-1 envelope protein, thereby preventing HIV-1 envelopemediated membrane fusion and virus entry. Table 2 summarizes neutralization breadth and potency of GPI-anchored antibody derivatives (scFv, HCDR3, and VHH) that we have developed in this study or previous studies (26)(27)(28) compared to those of soluble whole antibodies, scFvs and VHHs, that serve as sources for GPI-anchored antibody derivatives (2,(45)(46)(47)(48)(49)(50). GPI-anchored antibody derivatives that we have tested include CD4BS, coreceptor-binding site (CRBS), V1/V2 loop, Chelix, and MPER antibodies.…”

A Nonneutralizing Anti-HIV Type 1 Antibody Turns into a Broad Neutralizing Antibody When Expressed on the Surface of HIV Type 1-Susceptible Cells. II. Inhibition of HIV Type 1 Captured and Transferred by DC-SIGN

“…The monoclonal antibody (TG15) derived from one of these hybridomas exhibits high affinity binding to the ectodomain of gp41 with a K d value of 4 ϫ 10 Ϫ10 M. 9 Using a competition assay by a panel of monoclonal antibodies with known specificity, it was found that only antibodies against cluster determinant II of gp41 can compete the binding of antibody (TG15) to HIV-1 envelope protein. This human monoclonal antibody and its recombinant Fab or scFv have been repeatedly shown to bind to HIV-1 gp160 or gp41, but fail to block viral entry.…”

“…This human monoclonal antibody and its recombinant Fab or scFv have been repeatedly shown to bind to HIV-1 gp160 or gp41, but fail to block viral entry. 9,10 Thus, it is a true nonneutralizing antibody.…”