Molecular and Functional Characterization of Cynomolgus Monkey IgG Subclasses

Abstract

Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc–FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.

Different Adaptations of IgG Effector Function in Human and Nonhuman Primates and Implications for Therapeutic Antibody Treatment

“…Some of our findings are in contradiction with an earlier study from Jacobsen et al (20) showing that cynomolgus IgG1 displayed the highest binding on cynomolgus monocytes compared with other human and cynomolgus IgG isotypes. In addition, cynomolgus IgG1 was far more potent in mediating ADCC in cynomolgus whole blood.…”

“…There was no sign of reaggregation during the course of the study, as assessed by analytical size exclusion chromatography. Analysis of the protein size under reducing and nonreducing conditions revealed human IgG4 half-molecule formation and cynomolgus IgG4 H-L chain dimer formation similar to a recent report (20), and all Abs showed comparable binding to CD20-expressing cells (Supplemental Fig. 1).…”

“…4A, 4B). The cynomolgus IgG4 H chain is missing a cysteine at position 131 in comparison with human IgG4, and thus, the H and L chains are only held together via noncovalent interactions (20,28). However, introduction of the cysteine (S131C) did not result in enhancement of Fab arm exchange (Fig.…”

Cell Contact–Dependent Priming and Fc Interaction with CD32+ Immune Cells Contribute to the TGN1412-Triggered Cytokine Response

“…As an immediate reaction to the incident, regulatory bodies implemented a new guideline for risk mitigation of early clinical trials (12), which turned out to be helpful. Nevertheless, the predictive value of experiments with nonhuman primates is still being debated (11,(13)(14)(15) and the need to establish new experimental settings based on human cells is evident.…”

Novel Drug and Soluble Target Tolerant Antidrug Antibody Assay for Therapeutic Antibodies Bearing The P329G Mutation

“…The high-sequence homology of the Fc region between human and cynomolgus monkey IgG [28] allows the usage of the same assay for the analysis of preclinical and clinical samples [12]. In addition to the PG, several other Fc modifications affect the affinity of mAbs to both the Fc receptors and complement [16,[29][30][31].…”