Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Abstract

BackgroundPassive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.Methodology/Principal FindingsAfter transplantation with CD34+CD38− human hematopoietic progenitor cells, BALB/c Rag2−/−IL-2Rγc−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively.Conclusion/SignificanceThis work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.

Dendritic Cell–Mediated Immune Humanization of Mice: Implications for Allogeneic and Xenogeneic Stem Cell Transplantation

“…Plasma was harvested from HSC-NRG mice 20 wk after reconstitution and screened by ELISA for the presence of total human IgM an total human IgG as described elsewhere (21). Total IgM and IgG determination was performed by coating 96-well plates either with AffiniPure F(ab9) 2 fragment goat anti-human IgM (Fc5m specific; Jackson ImmunoResearch Laboratories) or AffiniPure goat anti-human IgG (Fcg fragment specific; Jackson ImmunoResearch Laboratories) at a concentration of 1.2 mg/ml.…”

A novel humanized mouse model with significant improvement of class-switched, antigen-specific antibody production

“…Although antigenspecific human IgM antibody responses are generated, the achievement of affinity maturation and class-switching from the IgM to the IgG isotype has been particularly difficult. 5,6 Several reports demonstrated that antigen-specific human IgG can be detected in humanized mice by enzyme-linked immunosorbent assay (ELISA). 7,8 The B-cell response was nevertheless not robust and the scarcity of IgG 1 memory B cells makes them extremely difficult to be identified and isolated.…”

High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Igκ/Igλ Loci Bearing the Rat CH Region

“…There have been major recent improvements in the way in which genetic information to encode Ag-specific mAbs can be obtained directly from human B cells (3)(4)(5). However, such approaches are largely restricted to Ags such as infectious agents where individuals mounting a specific immune response can be identified and provide a source of Ag-specific B cells.…”