Reduced elimination of IgG antibodies by engineering the variable region

Abstract

Fc engineering to increase the binding affinity of IgG antibodies to FcRn has been reported to reduce the elimination of IgG antibodies. Herein, we present a novel non-FcRn-dependent approach to reduce the elimination of IgG antibodies. Pharmacokinetic studies conducted in normal mice of various humanized IgG4 antibodies, which had identical constant regions but different variable region sequences, revealed that an antibody with a lower isoelectric point (pI) has a longer half-life. These antibodies exhibited comparable binding affinity to FcRn, and with the antibodies with lower pIs, a longer half-life was also observed in beta2-microglobulin knockout mice, suggesting that differences in the pharmacokinetics were due to a non-FcRn-dependent mechanism. On the basis of our findings, we attempted to engineer the pharmacokinetic properties of a humanized anti-IL6 receptor IgG1 antibody. Selected substitutions in the variable region, without substitution in the Fc region, lowered the pI but did not reduce the biological activity and showed a significant reduction in the clearance of the antibody in cynomolgus monkey. These results suggest that lowering the pI by engineering the variable region could reduce the elimination of IgG antibodies and could provide an alternative to Fc engineering of IgG antibodies.

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

“…This fast clearance in mice could be due to the high isoelectric point of the mAb (pI = 9.2), a parameter that has been shown to negatively affect half-life and clearance. 45 In this set-up, all IgG variants displayed substantial serum half-life increase with ratios (half-life variant/WT) between 1.8 and 2.8. 25 Similarly, IgG-YTE had a ratio between 2.3 and 3.3, slightly lower than the published values of ~3.8.…”

“…In addition, other factors than FcRn binding have been shown to influence PK of natural mAbs: overall protein structure, glycosylation status, isoelectric point, 45 methionine oxidation, 59 interaction with the target but also specificity. Indeed, mAbs with the same wild-type human Fc sequences but different Fv domains were shown to bind FcRn with considerable differences.…”

pH-dependent Binding Engineering Reveals an FcRn Affinity Threshold That Governs IgG Recycling

“…antibody stability and variability in antigen distribution) also play a role in determining the ultimate PK outcome of an engineered antibody. In this regard, we do envision that extremely long half-lives of Fc-containing molecules could be achieved by combining FcRn binding engineering with FcRn-independent half-life-extending technologies, such as pI optimization (39) and pH-dependent antigen binding engineering (40,41).…”

Target-independent variable region mediated effects on antibody clearance can be FcRn independent

“…12,18,[38][39][40] These results are also consistent with a similar study using the IgG4 isotype, in which a higher pI led to a shortened half-life and higher clearance rates regardless of presence of FcRn. 15 Studies on larger sets of antibodies suggest that the correlation between pI and clearance rates is not generalizable; 24,25 however, a localized patch of positive charge may be sufficient to increase interactions with the negatively charged surface of cell membranes. Nonspecificity driven clearance is not solely charge dependent, and other factors, including hydrophobicity, have been implicated in driving accelerated clearance rates.…”

“…[9][10][11][12][13][14] In addition to Fc engineering efforts, multiple studies have reported disparate PK parameters in antibodies with identical Fc regions but different variable regions. [15][16][17][18][19][20] In particular, clearance rates are increased in antibodies with polyreactive [21][22][23] or cross-reactive 24,25 profiles.…”