Subsequently, primary antibodies against Clec4F or cytokeratin 8 were added for 2 hours before detection with AlexaFluor 549 or AlexaFluor 568 conjugated secondary antibodies ( online supplementary table 1). Following blocking, primary antibodies against FcRn or FcγRII were added overnight before detection with Alexafluor 488-labeled secondary antibody ( online supplementary table 1) for 45 min. 8 µm sections were cut and transferred to Superfrost plus slides (Thermo Scientific), air dried overnight and fixed in 100% acetone. Liver tissue from BALB/c or NSG mice was embedded in OCT (CellPath) and frozen in isopentane on dry ice. Additionally, we identified a reduced level of FcRn expression in NOD SCID mice, leading us to propose a novel hypothesis for how mAb half-life is regulated in these strains and means through which it can be overcome. Employing genetically altered mice, we showed the rapid mAb clearance was dependent on the expression of the inhibitory FcγR, FcγRII. Using a Eμ-Tcl1 hCD20 +tumor model we found this rapid clearance resulted in reduced efficacy of clinically relevant mAb, such as rituximab. 17 This suggests further work is required to understand the limitations of these models and develop strategies to overcome their shortcomings to make more translationally relevant preclinical tumor models.ĭuring a recent project examining the efficacy of a tumor targeting antibody in NOD SCID mice, we noted rapid mAb clearance of human (h) IgG1 and mouse (m) IgG2a isotypes. 14–16 More recently, it was reported that NOD SCID mice display an anomalous biodistribution of therapeutic antibodies, including reduced tumor targeting. In addition to the potential issue of altered efficacy arising from the lack of endogenous IgG (and reduced competition for FcγR with therapeutic mAb) in NOD SCID mice, previous reports indicate that immune-compromised mice, such as NOD SCID and NSG, have reduced mAb half-life compared with related strains. The pH-dependent nature of FcRn-IgG interactions allows the receptor to scavenge IgG from lysosomes at an acidic pH, releasing it back into the circulation at neutral pH, providing the long in vivo half-life of antibodies. 10 Another receptor capable of interacting with IgG in both humans and mice is FcRn, which is widely expressed throughout the body. It is composed of six receptors in humans and four in mice, which vary in expression pattern and affinity for IgG subclass. The primary receptors responsible for mediating IgG mAb activity are the Fc gamma receptor (FcγR) family. While the effector function defects of NOD SCID mice and their related strains are often considered, one aspect regularly overlooked is mAb clearance, despite the fact that genetic alterations, as well as the lack of endogenous IgG in immune deficient strains, could readily impact on mAb pharmacokinetics, resulting in altered efficacy. 6 While these immune-deficient phenotypes make NOD SCID mice attractive recipients for cell transfers (such as human peripheral blood mononuclear cells (PBMC) and tumor xenografts), they may be further enhanced by additional genetic deletions such as the interleukin-2 γ-chain (NSG). 4 5 The NOD phenotype results in reduced NK cell frequency and function and the absence of hemolytic complement activity. The SCID mutation occurs in the Prkdc gene and impairs V(D)J recombination, leading to an absence of functional B and T cells and resulting in mice lacking endogenous IgG. 2 3Ĭommonly used models include non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice. 1 These models have increasingly made use of immune-compromised mice for growing patient-derived tumor xenografts and engrafting human immune or stem cells. The growth in the numbers of monoclonal antibodies (mAb) being developed for the clinic, particularly for use in cancer, has led to the concurrent development of in vivo models enabling their preclinical evaluation.
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