Overview

    Antibodies (Abs) play important roles in protective immunity in vertebrate bodies by specifically binding to target antigens including viruses, microbes, parasites, cancers, and toxins. Abs generally comprise two polypeptide chains, heavy (H) and light (L) chains, and mainly six complementarity-determining regions, CDR1, CDR2, and CDR3, in the variable (V) domains of H and L chains (VH and VL) involve the binding to the individual antigens and determine the antigen-specificity. V domains are constructed through DNA rearrangement, which generates huge repertoire of antibodies. Since V domains are highly diversified, animals can make numerous kinds of Ab against various antigens. Ab-producing B cells which bind to the specific antigen are selected from naive repertoires of B cells, proliferate, and produce the antibodies for the antigens in vivo. A mixture of various kinds of antibodies is secreted into the animal's serum, so called polyclonal antibodies. Cell fusion technology was developed in1975, and enabled to produce monoclonal antibodies (mAbs) by generating Ab-producing hybridomas (Köhler and Milstein, 1975). By using this technology, many kinds of murine mAb have been widely produced, while not so many human mAbs have been made because it was more difficult to generate human hybridoma than murine hybridoma.
    Replacing the method of generating hybridomas for production of mAbs, a novel method of using expression system for antibodies in E. coli was developed around 1990. In this method, filamentous phages harboring the mAb genes against the specific antigens are isolated from artificially constructed Ab phage-display libraries. In the libraries, Ab repertoires are expressed on the surface of filamentous phages as a consequence of fusion with the coat protein, namely, pIII (McCafferty et al., 1990; Clackson et al., 1991; Barbas III et al., 1991). The repertoires of V region genes for Ab have been prepared from B lymphocytes of immunized and of non-immunized animals (Winter and Milstein, 1991). By using the phage-display technology, various Ab phage-display libraries have been constructed, and many kinds of mAbs have been isolated from the libraries widespread. Since the phage-display method made easier to make human mAbs compared to the hybridoma method, many kinds of human mAbs have been isolated from Ab phage-display libraries.
    We have also constructed various Ab phage libraries, and isolated many kinds of mAbs from the libraries. First, we established a system for isolation of mAbs from Ab phage libraries. We constructed phagemid vectors that permit the simultaneous introduction of highly diverged sequences into six CDRs of an Ab by the polymerase chain reaction (PCR) with degenerate oligodeoxynucleotide primers to give a small Ab library. We successfully isolated mAbs against hen egg lysozyme (HEL) from the Ab library by panning method (Iba et al., 1997). Furthermore, we made two kinds of library in the same method, and isolated mAbs for 17-a-hydroxyprogensterone and cortisol (Iba et al., 1998) or mAbs for 11-deoxycortisol and cortisol (Miyazaki et al., 1999) from each library. After establishing the method for isolating mAbs for specific antigens by using the phage display-technology, we have made several kinds of human Ab phage-display library. From these libraries, we have isolated many kinds of human mAbs against diphtheria toxoid (Kakita et al., 2006); extracellular proteins on the cancer cell surface (Kurosawa et al., 2008; Akahori et al., 2009; Kitamura et al., 2009; Kurosawa et al., 2009); and viruses including rotaviruses (Higo-Moriguchi et al., 2004), cytomegaloviruses (Ohta et al., 2009), and human influenza viruses (Okada et al., 2010; Okada et al., 2011; Ohshima et al., 2011; Ohshima et al., 2013; Iba et al., 2013).
    Anti-influenza virus antibodies can protect animals from influenza virus infection in vivo, and influenza hemagglutinin (HA) is the main target for virus-neutralizing antibodies (Gerhard et al., 1997). HA is the major surface glycoprotein of influenza virus responsible for virus entry into target cells, which mediates virus binding to cell surface receptor, sialic acid, and the ensuing pH-dependent membrane fusion. There are two mechanisms for neutralization of influenza virus by antibodies; blocking receptor-binding and preventing the membrane fusion. Majority of neutralizing antibodies against HA are thought to be antibodies blocking receptor-binding, and the epitopes recognized by these antibodies are located at defined sites near the sialic acid-binding pocket (Wiley et al., 1981; Underwood, 1982). Antibodies against these sites are very potent and mutations can be introduced into these sites without losing the receptor-binding activity. Under the pressure of neutralizing antibodies, variant viruses that have acquired resistance to these antibodies become dominant, and the escape mutants cause annual epidemics, resulting in generation of many kinds of strain. This phenomenon is called as antigenic drift. Caused by the antigenic drift, anti-HA mAbs generally have strain specificities. We analyzed the strain specificities of mAbs which were isolated from human Ab phage libraries. Of the mAbs, a few mAbs had broad strain specificities. Furthermore, we determined the three dimensional structures of one of the broadly neutralizing Ab in complex with HA by X-ray analysis, and elucidated mechanism for the mAb neutralizing various strains of influenza virus.

Phage Abs

Anti-HA Abs

References