Recombinant Antibodies Expressed In Mammalian Cells For Analysis Of Antigenic Determinants On Mite Allergens

Human IgE antibodies are polyclonal and in low abundance in blood, making it impossible to purify homogeneous and high amounts of monoclonal antibodies (mAb) for crystallization. The goal was to engineer recombinant antibodies to determine the structural basis of antigenic determinants on mite allergens. Monoclonal antibodies were sequenced by Rapid Amplification of cDNA Ends (RACE) from hybridoma cell lines. Human IgE mAbs were obtained from fusion of human B cells from allergic patients with myeloma (HMMA2.5) cells using electrical cytofusion. IgG and IgE mAbs were expressed in ExpiCHO mammalian cells and purified using Protein G affinity chromatography. Purity was assessed by SDS-PAGE. Stability of recombinant and hybridoma-derived IgG mAb was compared by nanoDSF. Relative binding of the mAbs was assessed by two-site immunoassays. Twenty-two murine IgG and human IgE mAbs were sequenced and three antibodies targeting mite allergens (anti-Der p 1 IgG mAb r4C1, and two anti-Der p 2 IgE mAbs r1B8 and r2F10) were expressed in mammalian cells. The mAb r4C1 (from the supernatant of CHO cell cultures) and hybridoma-derived mAb 4C1 showed similar stability and interacted with Der p 1 in an equivalent manner by immunoassays. Human IgE mAbs 1B8 and 2F10 had unique amino acid sequences and bound different epitopes on Der p 2, making them prime candidates for structural studies. Expression of murine IgG and human IgE monoclonal antibodies targeting dust mite allergens will allow identification of antigenic determinants for analysis of the human IgE repertoire and for design of hypoallergens for immunotherapy.