Functional humanization of immunoglobulin heavy constant gamma 1 Fc domain human FCGRT transgenic mice

Abstract

A major asset of many monoclonal antibody (mAb)-based biologics is their persistence in circulation. The MHC class I family Fc receptor, FCGRT, is primarily responsible for this extended pharmacokinetic behavior. Engagement of FCGRT with the crystallizable fragment (Fc) domain protects IgG from catabolic elimination, thereby extending the persistence and bioavailability of IgG and related Fc-based biologics. There is a need for reliable in vivo models to facilitate the preclinical development of novel IgG-based biologics. FcRn-humanized mice have been widely accepted as translationally relevant surrogates for IgG-based biologics evaluations. Although such FCGRT-humanized mice, especially the mouse strain, B6.Cg-Fcgrt tm1Dcr Tg(FCGRT)32Dcr (abbreviated Tg32), have been substantially validated for modeling humanized IgG-based biologics, there is a recognized caveat – they lack an endogenous source of human IgG that typifies the human competitive condition. Here, we used CRISPR/Cas9-mediated homology-directed repair to equip the hFCGRT Tg32 strain with a human IGHG1 Fc domain. This replacement now results in mice that produce human IgG1 Fc-mouse IgG Fab 2 chimeric antibodies at physiologically relevant levels, which can be further heightened by immunization. This endogenous chimeric IgG1 significantly dampens the serum half-life of administered humanized mAbs in an hFCGRT-dependent manner. Thus, such IgG1-Fc humanized mice may provide a more physiologically relevant competitive hFCGRT-humanized mouse model for the preclinical development of human IgG-based biologics.

A Single Multipurpose FSH–Blocking Therapeutic for Osteoporosis and Obesity

“…Of note is that the β phase t ½ for i.v. trastuzumab, currently in human use, is ∼8.5 days in Tg32 mice (31)––this latter β decay translates into 21–day dosing intervals.…”

Prevention of diabetes-associated fibrosis: Strategies in FcRn-targeted nanosystems for oral drug delivery

“…Interestingly, a modified version of the Tg32 strain has been recently reported to produce hIgG1 Fc combined with mIgG Fab arms, which is rescued by hFcRn (Fig. 5B) [219]. The levels of endogenous chimeric mouse-human IgG1 were shown to be greatly increased upon immunization, which modulated the half-life of injected hIgG.…”

“…To solve this preclinical challenge, several genetically engineered mouse strains have been developed where the mFcRn heavy chain has been replaced with the human counterpart, either under the control of mouse or human promoter elements [32,74,181,216,[219][220][221][222][223]. These strains are the current gold standards for the evaluation of hFcRn-targeting strategies addressing pharmacokinetics, pharmacodynamics and delivery across selective mucosal barriers prior to studies in non-human primates.…”

“…These conditions must be taken into consideration in the preclinical evaluation of FcRn-targeted strategies, since the outcome can be greatly affected by a lack of competition for receptor binding, as in the case of the IgG binding site, which is in contrast to strong competition for the albumin-binding site. To address how competition for the IgG binding site of FcRn affects targeting approaches, mice may be preloaded with excess amounts of hIgG prior to administration, which has been shown to modulate the half-life of injected IgG variants [181,219,226]. Interestingly, a modified version of the Tg32 strain has been recently reported to produce hIgG1 Fc combined with mIgG Fab arms, which is rescued by hFcRn (Fig.…”

Perspective: Quality Versus Quantity; Is It Important to Assess the Role of Enhancers in Complex Disease from an In Vivo Perspective?

“…CRISPR-driven humanization of large regions of the mouse genome represents a challenging process that, in addition to CAS9 and gRNA, requires the co-injection of repair template DNA, designed to trigger the cells' homology-directed repair pathway, into the nucleus of a 1-cell mouse zygote. Whilst this approach has been successfully used to humanize gene coding regions in mice [62][63][64], it has yet to be used to humanize enhancer regions. The alternative, and more conventional, strategy to humanization involves embryonic stem (ES)-cell targeting and blastocyst microinjection [65].…”