Influence of Fc Modifications and IgG Subclass on Biodistribution of Humanized Antibodies Targeting L1CAM

Abstract

Immuno-positron emission tomography (immuno-PET) is a powerful tool to noninvasively characterize the in vivo biodistribution of engineered antibodies (Abs). Methods: L1 cell adhesion molecule (L1CAM)-targeting HuE71 immunoglobulin G1 (IgG1) and immunoglobulin G4 (IgG4) Abs bearing identical variable heavy and light chain sequences but different fragment crystallizable (Fc) portions were radiolabeled with Zirconium-89 ( 89 Zr) and the in vivo biodistribution was studied in SKOV3 ovarian cancer xenografted nude mice. Results: In addition to showing uptake in L1CAM-expressing SKOV3 tumors like its parental counterpart HuE71 IgG1, the afucosylated variant having enhanced Fc-receptor (FcR) affinity showed high nonspecific uptake in lymph nodes. On the other hand, aglycosylated HuE71 IgG1 with abrogated FcR binding did not show lymphoid uptake. The use of IgG4 subclass showed high nonspecific uptake in the kidneys, which was prevented by mutating serine at position 228 to proline (S228P) in the hinge region of the IgG4 Ab to mitigate in vivo fragment antigen-binding (Fab) arm exchange. Conclusion: Our findings highlight the influence of Fc-modifications and the choice of IgG subclass on the in vivo biodistribution of Abs and the potential outcomes thereof.

In Vivo Biorthogonal Antibody Click for Dual Targeting and Augmented Efficacy in Cancer Treatment

“…3 ). Interestingly, we observed lower liver uptake of click antibodies in immunocompetent mice (8.9±1.5 %ID/g) when compared with immunocompromised mice (4.5±2.4 %ID/g), as immunocompetent mice have circulating IgG ( 37, 38 ) ( Supplementary Fig. 4) .…”

“…3 ). Antibody click in blood circulation likely enhances the interaction of the Fc domain of mAbs with Fc receptors in the liver of immunocompromised mice ( 37 ). Additional studies demonstrated that pre-injection of a 15-fold excess of cold IgG significantly lowered liver accumulation ( Extended Fig.…”

“…Additionally, liver uptake of clicking mAbs was ∼3-fold higher when compared with [ 89 Zr]Zr-DFO-Trast-Tz no click ( Supplementary Fig. 21 ) likely due to bioorthogonal click reaction occurring in blood circulation that results in an enhancement in the interaction of the Fc domain of antibodies with Fc receptors in the liver of immunocompromised mice 31 . Additional studies demonstrated that pre-injection of a 100-fold excess of cold IgG significantly lowered liver accumulation by blocking the Fc-mediated interaction of the antibodies with Fc receptors in liver cells ( Extended Fig.…”

Antibody Engineering for Nuclear Imaging and Radioimmunotherapy

“…More recently, Sharma et al demonstrated the power of this approach using two glycoengineered variants of the L1CAM-targeting radioimmunoconjugate [ 89 Zr]Zr-DFO-HuE71 (Fig. 4B) (28). An afucosylated variant with enhanced FcgRIIIA binding exhibited reduced tumoral uptake and enhanced accretion in the liver and lymphoid tissues compared with the parent radioimmunoconjugate.…”

“…In the study from Sharma et al mentioned above, the authors also explored 89 Zr-immunoPET with two L1CAM-targeting IgG 4 -based radioimmunoconjugates, as this subclass has attracted attention in the immunotherapeutics community because it elicits reduced effector functions relative to other subclasses (Fig. 4D) (28). Although a radioimmunoconjugate based on the wild-type IgG 4 framework produced high levels of nonspecific uptake in the kidneys, this behavior was eliminated in a variant with S228P mutations in the hinge region that facilitated the formation of IgG 1 -like interchain disulfides and thus mitigated in vivo Fab arm exchange.…”