Novel neutralizing human monoclonal antibodies against tetanus neurotoxin

Abstract

Tetanus is a fatal disease caused by tetanus neurotoxin (TeNT). TeNT is composed of a light chain (Lc) and a heavy chain, the latter of which is classified into two domains, N-terminus Hn and C-terminus Hc. Several TeNT-neutralizing antibodies have been reported, but it remains unclear which TeNT domains are involved in neutralization. To further understand the mechanism of these antibodies, we isolated TeNT-reactive human antibody clones from peripheral blood mononuclear cells. We then analyzed the reactivity of the isolated antibody clones to each protein domain and their inhibition of Hc-ganglioside GT1b binding, which is critical for TeNT toxicity. We also investigated the TeNT-neutralizing ability of isolated antibody clones and showed that an Hn-reactive clone protected strongly against TeNT toxicity in mice. Furthermore, combination treatment of Hn-reactive antibody clones with both Hc-reactive and TeNT mix (the mixture of Hc, Hn, and Lc proteins)–reactive antibody clones enhanced the neutralizing effect. These results indicated that antibody clones targeting Hn effectively neutralized TeNT. In addition, the use of a cocktail composed of Hc-, Hn-, and TeNT mix–reactive antibodies provided enhanced protection compared to the use of each antibody alone.

Optimization of anti-ADAMTS13 antibodies for the treatment of ADAMTS13-related bleeding disorder in patients receiving circulatory assist device support

“…To design humanized A10 antibodies, the amino acid sequences of the heavy and light chain variable domains (V H and V L , respectively) of mouse anti-ADAMTS13 antibody mA10 were compared with those of previously constructed human antibody frameworks, 8A7 and 16E8 16 . After determining the CDRs and framework regions (FRs) in the V H and V L of mA10, 8A7, and 16E8 in accordance with IMGT (the international ImMunoGeneTics information system) numbering scheme 17 , a global alignment was manually conducted by adjusting the positions and sequence motifs around CDRs between the amino acid sequences (Figs.…”

“…Previously constructed human antibody clones, 8A7 and 16E8 16 , were used as human antibody frameworks and negative controls in this study.…”

“…In the RT-PCR procedure, Not I and Eco RI sites were added to the 5′ and 3′ end of the mA10 genes, respectively. The PCR products were then digested with Not I and Eco RI, and the heavy and light chain genes of mA10 were inserted into pQEFIP or pQEFIN vectors 16 , respectively.…”

“…The constructed mA10 plasmids were used to amply the V H and V L genes of mA10. Previously constructed 8A7 plasmids 16 were used to amplify the C H and C L genes because 8A7 has typical sequences of C H and C L for immunoglobulin gamma 1 (IgG1) and immunoglobulin kappa (Igκ), respectively. Using a PCR-based method with target specific primers (Supplemental Table S2 ), the expression vectors were also amplified along with the C H and C L genes for cloning, and approximately 20-nt overlaps were added to the both 5′and 3′ end of the target genes, respectively.…”

“…Currently, complementary determining region (CDR) grafting 14 or its-related techniques 15 have been widely used for mAb humanization. In this study, we constructed humanized A10 antibodies by CDR grafting with two human antibody frameworks, 8A7 and 16E8 16 , to develop an applicable therapeutic agent for the treatment of patients with ADAMTS13-related bleeding disorder. The characteristics of the two humanized A10 antibodies, namely A10/8A7 and A10/16E8, were assessed in vitro as well as in silico.…”