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 g/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.

Evaluation of Fab and F(ab′) 2 Fragments and Isotype Variants of a Recombinant Human Monoclonal Antibody against Shiga Toxin 2

“…One of these antibodies, 5C12, was shown to protect mice and gnotobiotic piglets from the fatal complications of Stx2 (13,24,25). A recombinant 5C12 antibody also demonstrated similar protective activity in these two animal models (1). We have recently shown that 5C12 neutralizes the toxicity of Stx2 for HeLa cells by blocking the retrograde transport of the toxin to the cytosol where the A subunit inhibits protein synthesis (11).…”

“…The sequences of genes encoding the heavy-and light-chain variable regions of the 5H8 IgG1 were determined from the 5H8-producing hybridoma cell line as previously described (1). The 5C12 heavy-and light-chain variable region cassettes in p5C12IgG1 and p5C12Fab were replaced with the corresponding 5H8 heavy-and light-chain variable cassettes as AflII/NheI and DraIII/BsiWI fragments.…”

“…Selection in the presence of G418 and methotrexate and the screening and production of these antibodies have been previously described (1). Each antibody was scaled up to a CELLineAD1000 Bioreactor (Integra Biosciences, Inc., East Dundee, IL) as previously described (1) for in vitro and in vivo testing of these antibodies.…”

“…The 5C12 isotype variants and the Fab and F(abЈ) 2 expression vectors were transfected into CHO DG44 cells, a double DHFR mutant cell line (kindly provided by L. Chasin, Columbia University, New York, NY) (32,33), as previously described (1). Selection in the presence of G418 and methotrexate and the screening and production of these antibodies have been previously described (1).…”

“…The A subunit possesses RNA N-glycosidase activity, which cleaves a specific adenine residue in the 28S rRNA, resulting in the inhibition of protein synthesis. The B subunits are responsible for binding to the host cell receptor, globotriaosyl ceramide (Gb3; Gal␣ [1][2][3][4]-Gal␤[1-4]-Glc␤1-ceramide). The A subunit is cleaved as it is internalized through clathrin-dependent or independent endocytosis and translocated via the retrograde pathway to the cytosol where it inhibits protein synthesis (3,4,19).…”