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SUMMARYC8/119S is a mutant of recombinant Der f 2 (rDer f 2), and lacks a disulphide bond possessed by wildtype rDer f 2. In humans and mice, C8/119S has a very weak IgE-binding capacity compared with the wild-type, but possesses a T cell reactivity comparable to that of the wild-type. C8/119S may thus be a safe immunotherapeutic agent for house dust mite allergy. The aim of the present study was to evaluate whether the intranasal administration of C8/119S could suppress an immediate allergic reaction in mice sensitized with wild-type rDer f 2, possessing an allergic activity comparable to native counterparts purified from mite extract. Seven-week-old male A/J mice were immunized with wild-type rDer f 2 four times, and then intranasally administered 0·2-2 mg of wild-type, 0·2-20 mg of C8/119S, or PBS alone, three times a week for 4 weeks. Seven days after the last administration, the mice were examined for an immediate allergic reaction. The animals administered 2 mg of C8/119S (C2.0 group) showed significantly reduced immediate bronchoconstriction provoked by the i.v. injection of 1 and 10 mg of wild-type rDer f 2, compared with the PBS-treated mice. Similar results were obtained when we examined mice 10 weeks after the last administration. The reactions in the other groups given wild-type or C8/119S also tended to decrease in severity in comparison with the animals of the PBS group. The allergic phenotypes of the T cells, B cells, and basophils in the C2.0 group were shifted to that of naive mice without immunization. We conclude that C8/119S has hyposensitizing activities in mice sensitized with wild-type rDer f 2. C8/119S may be useful for immunotherapy of house dust mite allergy.

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We describe the preparation of Fab fragments of a humanized anti-human high-affinity IgE receptor (Fc epsilonRIalpha) antibody potentially useful for treatment of IgE-mediated allergic diseases. IgE-binding capacities of sixteen combinations of light and heavy chains of four recombinant anti-Fc epsilonRIalpha antibodies, chimeric CRA2, humanized CRA2, chimeric CRA4, and humanized CRA4, were compared. A combination in which both chains were of humanized CRA2 had the highest activity. Stable transfectant clones of four kinds of host cells expressing recombinant antibodies were established. CHO-K1 cells were the most productive. Serum-free media suitable for culture of the stable CHO-transfectant clones were screened. The concentration of the humanized CRA2, which the most productive clone secreted into the chosen serum-free medium, was approximately 100 microg/ml. A procedure for the purification of the antibody, papain-digestion, and purification of Fab fragments was established. The highly purified humanized Fab fragments are suitable for use to examine their in vivo activity and immunogenicity in primates.