Production of IgA using nasal associated lymphoid tissues toward development of therapeutic antibodies for oral administration.

可以口服并作用于粘膜表面的IgA类抗体可能是有用的治疗性抗体。用鼻腔免疫小鼠获得足够的IgA应答，然后用鼻腔相关淋巴组织(NALT)制备IgA单抗。作为抗原，我们表达并纯化了与糖类配体结合的重组志贺毒素B亚单位(STX1B)。由于STX1B对小鼠的免疫原性很低，很难产生针对STX1B的特异性IgA。我们发现，化学交联、适当大小的聚苯乙烯微球的吸附以及脂质体的掺入大大提高了STX1B对黏膜免疫的免疫原性。一种抗STX1B的IgA单抗有效地抑制了固定化STX1B与多赖氨酸骨架上存在球状三糖的聚合物碳水化合物配体之间的相互作用。相反，相同的单抗可部分抑制可溶性STX1B与细胞表面天然配体的结合。为了研究这一差异，我们通过表面等离子体共振研究了可溶性STX1B与固定化单抗IgA的结合。与能有效中和STX生物活性的Ig G mAb相比，Ig A mAb结合速度较慢，解离动力学较快。我们从抗STX1B的IgA和Ig G单抗中克隆了编码多肽的全长c DNA，以制备重组抗体(包括“植物抗体”)。我们能够获得抗霍乱毒素的IgA单抗和抗卵白蛋白的单抗，这表明我们的方法适用于生产一般的IgA单抗。

Antibodies of IgA class that can be orally administered and act on the mucosal surface may be useful as therapeutic antibodies. Mice were intranasally immunized to obtain sufficient IgA response, and then IgA monoclonal antibodies were produced by the use of nasal-associated lymphoid tissue (NALT). As an antigen, we expressed and purified recombinant Shiga toxin B subunits (Stx1B) that are responsible for the binding to carbohydrate ligands. Because of very low immunogenicity of Stx1B to mice, it was difficult to produce specific IgA against Stx1B. We found that chemical cross-linking, adsorption on polystyrene microspheres of appropriate size, and incorporation into liposome greatly enhanced immunogenicity of Stx1B upon mucosal immunization. An IgA mAb against Stx1B efficiently inhibited interaction between immobilized Stx1B and polymer-based carbohydrate ligands, in which globotriose is present on the poly-lysine backbone. In contrast, the same mAb partially inhibited the binding of soluble Stx1B to the cell surface natural ligands. To investigate this difference, we examined binding of soluble Stx1B to the immobilized mAb IgA by means of surface plasmon resonance studies. The IgA mAb showed slower association and faster dissociation kinetics as compared with an IgG mAb that can efficiently neutralize biological activity of Stx. We cloned full-length cDNA encoding polypeptides from IgA and IgG mAbs against Stx1B to produce recombinant antibodies (including "plantibodies") in future. We were able to obtain IgA mAbs against cholera toxin and those against ovalbumin, suggesting the applicability of our procedure to produce IgA mAbs in general.