Delivering therapeutic antibodies into the brain across the blood–brain barrier at a therapeutic level is a promising while challenging approach in the treatment of neurological disorders. Here, we present a polymeric nanomicelle (PM) system capable of delivering therapeutically effective levels of 3D6 antibody fragments (3D6-Fab) into the brain parenchyma for inhibiting Aβ aggregation. PM assembly was achieved by charge-converting 3D6-Fab through pH-sensitive citraconylation to allow complexation with reductive-sensitive cationic polymers. Brain targeting was achieved by functionalizing the PM surface with glucose molecules to allow interaction with recycling glucose transporter (Glut)-1 proteins. Consequently, 41-fold enhanced 3D6-Fab accumulation in the brain was achieved by using the PM system compared to free 3D6-Fab. Furthermore, therapeutic benefits were obtained by successfully inhibiting Aβ1–42 aggregation in Alzheimer’s disease mice systemically treated with 3D6-Fab-loaded glucosylated PM. Hence, this nanocarrier system represents a promising method for effectively delivering functional antibody agents into the brain and treating neurological diseases.
Current xenogeneic mouse models cannot evaluate on-target off-tumor adverse effect, hindering the development of chimeric antigen receptor (CAR) T cell therapies for solid tumors, due to limited human/mouse cross-reactivity of antibodies used in CAR and sever graft-versus-host disease induced by administered human T cells. We have evaluated safety and antitumor efficacy of CAR-T cells targeting glypican-1 (GPC1) overexpressed in various solid tumors. GPC1-specific human and murine CAR-T cells generated from our original anti-human/mouse GPC1 antibody showed strong antitumor effects in xenogeneic and syngeneic mouse models, respectively. Importantly, the murine CAR-T cells enhanced endogenous T cell responses against a non-GPC1 tumor antigen through the mechanism of antigen-spreading and showed synergistic antitumor effects with anti-PD-1 antibody without any adverse effects in syngeneic models. Our study shows the potential of GPC1 as a CAR-T cell target for solid tumors and the importance of syngeneic and xenogeneic models for evaluating their safety and efficacy.
Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents. Despite their small size, the high affinity and specificity displayed by VHHs for antigen molecules rival those of IgGs. How such small antibodies achieve that level of performance? Structural studies have revealed that VHHs tend to recognize concave surfaces of their antigens with high shape-complementarity. However, the energetic contribution of individual residues located at the binding interface has not been addressed in detail, obscuring the actual mechanism by which VHHs target the concave surfaces of proteins. Herein, we show that a VHH specific for hen egg lysozyme, D3-L11, not only displayed the characteristic binding of VHHs to a concave region of the surface of the antigen, but also exhibited a distribution of energetic hot-spots like those of IgGs and conventional protein-protein complexes. The highly preorganized and energetically compact interface of D3-L11 recognizes the concave epitope with high shape complementarity by the classical lock-and-key mechanism. Our results shed light on the fundamental basis by which a particular VHH accommodate to the concave surface of an antigens with high affinity in a specific manner, enriching the mechanistic landscape of VHHs.
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.
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