Characterization of a human monoclonal antibody against Shiga toxin 2 expressed in Chinese hamster ovary cells

Abstract

Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 microg/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.

FIG. 1.

Schematic of the p5C12IgG1 and pDHFRExpress vectors for the expression of recombinant HuMAbs in CHO cell lines using a DHFR-CHO gene amplification system. (A) The pIgG1 expression vector for the expression of Stx2-specific HuMAbs. VL/CK, variable and constant region of the κ light chain; VH/Cgamma, variable and IgG1 constant region of the heavy chain; pCMV, CMV promoter; 5H8 pA, polyadenylation signal from the 5H8 HuMAb light chain; BGH pA, polyadenylation signal from bovine growth hormone gene; f1 ori, origin of replication from f1; SV40 ori, SV40 origin of replication; NPT gene, neomycin phosphotransferase gene; ColE1 ori, origin of replication from ColE1; Bla gene, β-lactamase gene. (B) The pDHFRExpress vector, which has a functional mouse DHFR gene in place of the antibody expression cassettes and the xanthine-guanine phosphoribosyltransferase (XGPRT) instead of the NPT gene, but otherwise is identical to the p5C12IgG1 vector. The DHFR gene is expressed from the β-globin major promoter.

FIG. 2.

5C12 production in the CHO (▪) and hybridoma (▴) cell lines. Both cell lines were grown as suspension cultures in 75-cm² flasks in duplicate and were monitored over a 5-day period. (A) Each flask was initially seeded at a density of 3 × 10⁵ cells/ml. Viable cell number was determined by trypan blue exclusion and cells were counted using a hemacytometer. (B) IgG1 production was measured by the IgG quantitation ELISA. Each error bar represents the standard error of the mean.

FIG. 3.

Comparison of the binding affinity and specificity of r5C12 (▪) to the parent 5C12 (•) using an Stx2-specific ELISA. Both antibodies were diluted twofold from 1 μg/ml to 0.4 ng/ml. Plates were coated with Stx2 and blocked, and 5C12 HuMAb (r5C12 or the parent 5C12) was added. Anti-human IgG-Fc coupled to horseradish peroxidase was added, the plates were developed with ο-phenylenediamine solution, the colorimetric reaction was stopped by the addition of sulfuric acid, and the optical density was measured at 490 nm (OD₄₉₀). Each data point represents the average of four replicates (two independent assays). The standard deviation of duplicate data points for each assay was <0.075.

FIG. 4.

Neutralization of Stx2 in a Vero cytotoxicity assay. The percent neutralization of 5 ng of Stx2 by various amounts (1.6 to 50 ng) of r5C12 (filled bars) or the parent 5C12 (open bars) was measured using a Vero cytotoxicity assay. Neutralization curves from two independent assays (triplicate wells) were determined for each antibody and used as a basis for the mean percent neutralization values shown here. Each error bar represents the standard error of the mean.