A human immune system (HIS) mouse model that dissociates roles for mouse and human FcR+ cells during antibody-mediated immune responses.

Human immune system (HIS) mice provide a model to study human immune responses in vivo. Currently available HIS mouse models may harbor mouse Fc Receptor (FcR)-expressing cells that exert potent effector functions following administration of human Ig. Previous studies showed that the ablation of the murine FcR gamma chain (FcR-γ) results in loss of antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vivo. We created a new FcR-γ-deficient HIS mouse model to compare host (mouse) versus graft (human) effects underlying antibody-mediated immune responses in vivo. FcR-γ-deficient HIS recipients lack expression and function of mouse activating FcRs and can be stably and robustly reconstituted with human immune cells. By screening blood B-cell depletion by rituximab Ig variants, we found that human FcγRs-mediated IgG1 effects, whereas mouse activating FcγRs were dominant in IgG4 effects. Complement played a role as an IgG1 variant (IgG1 K322A) lacking complement binding activity was largely ineffective. Finally, we provide evidence that FcγRIIIA on human NK cells could mediate complement-independent B-cell depletion by IgG1 K322A. We anticipate that our FcR-γ-deficient HIS model will help clarify mechanisms of action of exogenous administered human antibodies in vivo.

Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions.

The quality control testing of chemical degradations in the bio-pharmaceutical industry is currently under controversial debate. Here we have systematically applied in vitro and in vivo stress conditions to investigate the influence of protein degradation on structure-function. Extensive purification and characterization enabled identification and functional assessment of the physiological degradation of chemical modification sites in the variable complementarity-determining regions (CDRs) and conserved region of trastuzumab. We demonstrate that the degradation of the solvent-accessible residues located in the CDR and the conserved fragment crystallizable region (Fc) occurs faster in vivo (within days) compared to the levels observed for bio-process and real-time storage conditions. These results hence question the rationality of extreme monitoring of low level alterations in such chemical modifications as critical patient safety parameters in product quality control testing, given that these modifications merely mirror the natural/physiological aging process of endogenous antibodies.