(M0930-10-58) Protein Engineering Strategies to Overcome High Target-Independent Clearance of Monoclonal Antibodies Due to Charge-Driven Liabilities

Purpose: The disposition of immunoglobulins (IgGs) is influenced by both target-dependent and target-independent mechanisms. Target-independent clearance (CL_ind) of monoclonal antibodies (mAbs) is primarily determined by propensity for non-specific endocytosis (NSE) and the efficiency of subsequent pH-dependent cellular recycling mediated by the neonatal Fc receptor (FcRn).¹ These processes were evident in a human IgG2 mAb targeted against the interleukin-4 receptor alpha chain (anti-IL-4Rα WT mAb) that exhibited high  CL_ind (11 mL/d/kg) in humans.¹ Structurally, we identified four arginine (R) residues in the mAb’s variable domain (Fv) affecting substantial NSE and compromised FcRn engagement.^1,2 Here, we aimed to expand our understanding of the cellular mechanisms driving the CL_ind of Fc-bearing biologics by determining the extent to which Fc-engineered half-life extension (HLE) strategies could successfully overcome variable degrees of non-specific binding character imparted by Fv  and decrease CL_ind. We elaborated upon our previous Fv-engineered anti-IL-4Rα mutant panel to broaden the range of NSE character.¹ Additionally, we introduced clinically validated Fc mutations to increase mAb affinity for FcRn at acidic pH while maintaining minimal binding at near neutral pH.^3,4

Methods: We introduced FcRn-binding enhanced M252Y/S254T/T256E triple point mutations (YTE) to each Fv-engineered mutant mAbs.^3,4 Cellular NSE measurements were performed in parental CHO-K1 cells at pH 5.8, as previously reported.¹ FcRn affinity chromatography experiments were performed according to a method reported previously.⁵ Cell-based hFcRn recycling studies were conducted with six pairs of anti-IL-4Rα WT and YTE mAbs. The FcRn recycling efficiency metric (FREM) score was calculated by analyzing mAb uptake, recycled mAb, and residual mAb samples, and was based on either or both FcRn recycling efficiency (RE) and non-specific uptake coefficient (NUC) scores as we have described previously.^2,6 The assay was optimized to allow comparison of  both Fc-engineered YTE and WT mAbs in the same experiment by normalizing all results to the anti-IL-4Rα EEES-YTE mAb. Single dose PK studies were performed in a male SCID variant of the humanized FcRn Tg32 transgenic mouse model.¹ The proteins were administered to mice by intravenous bolus dose (1 mg/kg) via the lateral tail vein. The serum samples were analyzed as reported earlier.¹

Results: Reducing the positive charge patch cluster by double point mutations at both complementarity determining regions, CDR1 and CDR3, reduced NSE in the anti-IL-4Rα mAb-33S,110L to a greater degree when compared to single point mutations in anti-IL-4Rα mAb-20S or -33S alone (Figure 1B). By mutating the four arginines with three glutamic acids (E) and one serine (S) in anti-IL-4Rα mAb-EEES, the highest reduction of non-specificity was observed at pH 5.8 among all WT mutants as judged by CHO-K1 NSE assay. Overall, the addition of the YTE mutations maintained the general non-specificity trends between mAbs and led to decreased non-specificity at pH 5.8 when compared to their WT parent mAbs. Affinity chromatography demonstrated that exposed positive charge directly affected the pH-sensitive interaction between the mAbs and FcRn whereby mAbs with higher NSE exhibited prolonged retention times. The trends of relative retention time between WT and YTE pairs were mostly conserved but couldn’t be compared simultaneously due to a shift of relative retention times in YTE mAbs from the enhanced FcRn affinity. Higher non-specificity as measured by elevated NSE corresponded to lower FcRn recycled efficiency as indicated by decreased FREM scores (Figure 2). Double point mutations in anti-IL-4Rα mAb-33S,110L drastically improved the FREM score (0.504 ± 0.085) compared to mAbs with single point mutations. Anti-IL-4Rα mAb-EEES exhibited the highest FREM score (0.584 ± 0.127) among all WT mAbs. The YTE mAbs exhibited better FREM scores relative to their WT counterparts, except minimal improvement for the SSLS-YTE mutant. The anti-IL-4Rα mAb-EEES-YTE demonstrated the highest FREM score (1.000 ± 0.196). It was evident from the PK results that mAbs with higher NSE and lower FcRn recycling efficiency had overall poorer PK (Figure 2). The FREM scores were utilized to rank order both the WT and YTE mAbs simultaneously. We observed a strong correlation between the murine CL_ind and cellular FREM scores for all mAbs, with lower FREM scores being associated with higher CL_ind.

Conclusion: Our study shows that reducing the Fv exposed positive charge can significantly reduce elevated NSE of a high-risk mAb. Additionally, surface positive charge also directly impacts the pH-sensitivity of the FcRn-mAb interaction. Furthermore, accounting for both NSE and FcRn engagement via calculation of cellular FREM scores strengthened our ability to rank order both the WT and YTE mutant mAbs simultaneously according to their PK outcomes. Our results show that targeted Fv mutations to reduce general non-specificity can have a greater impact on mAb CL_ind when compared to YTE mutations alone. But incorporating both Fv and Fc mutations result in better FREM scores and reduced CL_ind as observed for the YTE mutants compared to their WT counterparts.

References: 1. Bryniarski, M. A., Tuhin, M. T. H., Shomin, C. D., Nasrollahi, F., Ko, C. E., Soto, M., ... & Conner, K. P. (2024). Utility of Cellular Measurements of Non-Specific Endocytosis to Assess the Target-Independent Clearance of Monoclonal Antibodies. bioRxiv, 2024-04.
2. Bryniarski, M. A., Tuhin, M. T. H., Acker, T. M., Wakefield, D. L., Sethaputra, G., ... & Conner, K. P. (2024). Cellular neonatal Fc receptor recycling efficiencies can differentiate target-independent clearance mechanisms of monoclonal antibodies. Journal of Pharmaceutical Sciences, 2024.
3. Dall'Acqua, W. F., Kiener, P. A., & Wu, H. (2006). Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn). Journal of Biological Chemistry, 281(33), 23514-23524.4. Borrok, M. J., Wu, Y., Beyaz, N., Yu, X. Q., Oganesyan, V., Dall'Acqua, W. F., & Tsui, P. (2015). pH-dependent binding engineering reveals an FcRn affinity threshold that governs IgG recycling. Journal of Biological Chemistry, 290(7), 4282-4290.
4. Chung, S., Nguyen, V., Lin, Y. L., Lafrance-Vanasse, J., Scales, S. J., Lin, K., ... & Song, A. (2019, July). An in vitro FcRn-dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans. In MAbs (Vol. 11, No. 5, pp. 942-955). Taylor & Francis.
5. Schlothauer, T., Rueger, P., Stracke, J. O., Hertenberger, H., Fingas, F., Kling, L., ... & Papadimitriou, A. (2013, July). Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies. In MAbs (Vol. 5, No. 4, pp. 576-586). Taylor & Francis.
6. Grevys, A., Frick, R., Mester, S., Flem-Karlsen, K., Nilsen, J., Foss, S., ... & Andersen, J. T. (2022). Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life. Iscience, 25(2).

Acknowledgements:

Funding: All studies were supported by Amgen Inc.
Conflict of Interest: All authors are paid employees of Amgen Inc.

Figure 1: (A) Anti-IL-4Rα WT and –YTE mAb cartoon figures with mutation sites. Antibody homology modeling of anti-IL-4Rα mAb in MOE software identified several charge patches on the anti-IL-4Rα mAb variable region (Fv), including two positive patches in heavy chain (HC) complementarity determining regions, CDR1 (R33) and CDR3 (R110), and one positive charge patch (R95) at the light chain (LC) framework. Additional isolated positive surface charge area in HC Fv (R20) was also mutated which doesn’t form a patch. (B) Non-specific endocytosis measurements of anti-IL-4Rα mAb WT and YTE mAb mutant panel in CHO-K1 cells at pH 5.8. The non-specific endocytosis of all WT and YTE mutants was significantly different when compared to the control anti-IL-4Rα wild-type (WT) mAb at pH 5.8 (ordinary one-way ANOVA with Dunnett’s multiple comparisons test; *: P ≤ 0.0001). Each bar graph represents mean ± SD, N = 3-4 per group.

Figure 2: Poor hFcRn recycling scores correspond to high CLind in hFcRn SCID Tg32 mice PK. Brown symbols represent FREM scores of the mutant mAbs which are connected by teal lines. The scores were normalized to anti-IL-4Rα mAb-EEES-YTE. The assay was optimized to simultaneously compare both WT (i.e. hIgG2) and Fc-engineered charge mutant mAbs. The half-life extending YTE mAbs exhibited better FREM scores relative to their WT counterparts. Each brown symbol represents mean ± SD, and N= 6-8 replicates per group, except for anti-IL-4Rα mAb-EEES-YTE, where N = 16. YTE mutant mAbs were tested in two batches in two different days, and FREM scores from each day were normalized to the EEES-YTE mutant mAb from that day as the relative control. The blank and grey bars represent the target independent clearance (CLind) of anti-IL-4Rα mAb WT and YTE mutant mAbs, respectively (N=3 mice per group). The figure shows the comparison between CLind and hFcRn recycling efficiency metric (FREM) scores. It was observed that mAbs with relatively lower FREM scores demonstrated higher CLind and vice versa.