ABSTRACT
Immunoglobulin G (IgG) is transcytosed across intestinal epithelial cells of suckling mammals by the neonatal Fc receptor (FcRn); however, the contribution of FcRn vs. FcRn-independent uptake to serum IgG levels had not been determined in either rat pups or human (h)FcRn-expressing mice (Tg276 and Tg32). In isoflurane-anesthetized rodents, serum levels were determined after regional intestinal delivery of human monoclonal antibodies (hIgG) with either wild-type (WT) Fc sequences or variants engineered for different FcRn binding affinities. Detection of full-length hIgG was by immunoassay; intestinal hFcRn and hIgG localization was by immunocytochemistry. High (µg/ml) serum levels of hIgG were detected after proximal intestinal delivery (0.1-10 mg/kg) in 2-wk-old rats. Human FcRn was visualized in epithelial cells of Tg276 mice, but low serum hIgG levels (<10 ng/ml) were obtained. In rat pups, intraintestinal hIgG1 WT administration resulted in dose-related and saturable uptake, whereas uptake of a low FcRn-binding affinity variant was nonsaturable. There were no differences in hIgG levels from systemic and hepatic portal vein serum samples, and intense hIgG immunostaining was noted in villi enterocytes and within lymphatic lacteal-like vessels. This study demonstrated that FcRn-mediated uptake in rat pups accounted for ~80% of serum hIgG levels and that IgG enters the circulation via the lymph and not the hepatic portal vein. The remaining uptake though the immature intestine is nonreceptor mediated. Intestinal epithelial cell hFcRn expression occurred in Tg276 mice, but receptor-mediated transport of IgG was not observed. The suckling rat pup intestine is a mechanistic model of FcRn-IgG-mediated transcytosis.
Subject(s)
Animals, Suckling/metabolism , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Immunoglobulin G/metabolism , Intestinal Mucosa/metabolism , Receptors, Fc/genetics , Receptors, Fc/metabolism , Animals , Antibodies, Monoclonal/metabolism , Dose-Response Relationship, Drug , Enterocytes/metabolism , Epithelial Cells/metabolism , Female , Humans , Immunoassay , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Rats , Transcytosis/physiologyABSTRACT
A quantitative, one-step, competitive electrochemiluminescence (ECL)-based immunoassay for the determination of a fully human, anti-TNFalpha monoclonal antibody in human serum has been developed. A biotinylated, mouse anti-variable region-specific antibody and a ruthenium-labeled anti-TNFalpha antibody were the only specific reagents needed to develop the assay. A single incubation step of 2 h followed by ECL detection was used. The assay was capable of measuring the analyte in neat serum over approximately a 1600-fold range with higher concentrations measured following a single dilution. Assay accuracy, precision, and reproducibility were suitable to support pharmacokinetic studies of the analyte. This competitive assay format offers an alternative approach to the development of immunoassays for the measurement of macromolecules in complex matrices to support preclinical and clinical studies.
Subject(s)
Antibodies, Monoclonal/blood , Immunoassay/methods , Tumor Necrosis Factor-alpha/immunology , Animals , Antibody Specificity , Arthritis, Rheumatoid/blood , Biotin , Humans , Indicators and Reagents , Luminescent Measurements , Mice , Reference Standards , Reproducibility of Results , RutheniumABSTRACT
Despite major deficits in their immune system, SCID, Nude, and NIH III mice reject allo- and xenografts, particularly leukemic cell lines, albeit less readily than immunologically intact mice. Since variation among these immunodeficient mouse strains in rejection of a human lymphoid cell line (CCRF-CEM) parallels splenic non-MHC-mediated cytolytic activity, non-MHC-restricted cytolytic activity may be responsible for retained resistance to leukemic cell transplantation. SCID mice that had the least cytolytic activity accepted 100% of their grafts. The converse was true for NIH III mice that showed the greatest cytolytic activity and were relatively resistant to CEM cell engraftment. Different approaches to ablate NK activity and thus enhance engraftment led to variable results for each strain. A single dose (500 micrograms) of anti-asialoGM1 (AsGM1) markedly reduced NK activity in SCID and NIH III mice by 60 and 40%, respectively. A moderate 20% decrease was seen in Nude mice at this dose. In contrast, gamma irradiation suppressed NK activity by greater than 80% of baseline levels in all three strains. Of importance, total cytolytic activity in immunosuppressed Nude and NIH III mice, although significantly depressed compared to untreated mice of the same strain, still remained higher than that seen in nonimmunosuppressed SCID mice. Enhanced engraftment and systemic dissemination of CEM cells in immunosuppressed mice correlated directly with decreased total splenic cytolytic activity in all three strains. These results have implications for the use of immunodeficient models for transplantation, tumor immunobiology, and engraftment of a human immune system.