(T1430-09-51) Comparability of Glycosylation, Antigen Binding and Fc Effector Function for Three Anti-TNF-a Therapeutics

Purpose: Of the 80+ known autoimmune diseases, numerous conditions can be treated with an anti-TNF-⍺ biopharmaceutical therapy. Perhaps one of the most well-known anti-TNF-⍺ biopharmaceuticals in the US is Humira^®. There are two other full-length mAbs approved for similar indications – Remicade^® and Simponi Aria^®. While these drug products are indicated for similar diseases including rheumatoid arthritis, psoriatic arthritis, and ulcerative colitis, and claim to work via the same mechanisms of action, (TNF-⍺ neutralization and Fc-mediated effector functions) they have notable differences across multiple properties. Some known differences include their binding affinity – for target TNF-⍺ cells and for Fc𝛾RIIIa on NK cells involved in ADCC, their pharmacokinetics, their PTMs, and their effector function. However, these differences were derived from a number of different methods, sites, and researchers, which translates to inherent variability in the published results. Therefore, to continue elucidating the physicochemical and function differences of these anti-TNF-⍺ mAbs in a more controlled setting, we have conducted a head-to-head analytical comparison studying the N-glycosylation profile, TNF-⍺ and Fc𝛾RIIIa binding affinity, and ADCC potency in Humira^®, Remicade^® and Simponi Aria^®. The results of this study will be helpful in guiding physicians as they prescribe treatments, especially for IBD patients where Fc𝛾RIIIa binding affinity, and, subsequently, ADCC potency are critical for maintaining the drug’s efficacy.

Methods: For N-glycosylation, we did bottom-up proteomics with LC-MS/MS quantification. We digested 20 ug of mAbs with Lys-C and trypsin overnight at a final concentration of 1:10 protease: mAb. LC was completed on a C18 column interfaced to a Waters Acquity UPLC. MS was conducted on a ThermoFisher Orbitrap Fusion. Data was processed using Protein Metrics’ Byos PTM suite. For TNF-a binding affinity, we utilized ELISA. Plates were coated with TNF-⍺ and mAb samples (starting from a concentration of 0.01 µg/mL and diluted two-fold for a total of 12 times) were added to each well. After the addition of HRP conjugate then TMB substrate, plates were developed and read on a SpectraMax M3 plate reader (Molecular Devices). To study the Fc𝛾RIIIa binding affinity, we performed AlphaLISA. Samples were prepared and analyzed using the protocol provided by AlphaLISA FCGR3A kit (Perkin Elmer). The sample plate was incubated for 90 minutes at room temperature in the dark and luminescence was read on a Synergy Neo2 plate reader (BioTek). For ADCC, we performed the ADCC Reporter Bioassay developed by Promega according to the manufacturer’s protocol. A final antibody concentration of 5nM was selected and serially diluted 2.25-fold using the assay buffer. Luminescence was measured using a GloMax Explorer plate reader (Promega).

Results: TNF-⍺ ELISA data shows that Humira^® has the lowest average EC50 value (1.98 pM), thus the highest soluble TNF-⍺ binding affinity and, subsequently, TNF-⍺ neutralization, of the three drugs. With regards to glycosylation, there were similar trends for high abundance glycans but differences in the total abundance and total number of glycans for each mAb. Remicade^® has the highest percentage of afucosylated glycans (15.5%) and the largest total number of unique glycans identified, 28. A larger % of afucoyslated glycans is desirable since afucoyslated glycans have been linked to higher binding affinity. While there were fewer glycans identified (15), many of Humira^®’s glycans are mannosylated and mannosylated glycans have been linked to increased clearance rates of drugs in vivo. Simponi Aria^® had 21 unique glycans identified and very few of those were mannosylated and/or afucosylated - only 8.9% were afucosylated. The AlphaLISA data for Fc𝛾RIIIa binding (V variant) showed that Humira^® had the lowest average IC50 value (10.59 nM), thus statistically significant higher binding affinity to Fc𝛾RIIIa. A higher Fc𝛾RIIIa binding affinity is beneficial when it comes to eliciting an effector function because the mAb needs to bind to Fc𝛾RIIIa on effector cells in order initiate ADCC. Therefore, we expected that Humira^® would have the lowest EC50 in the ADCC assay, indicating higher potency. This prediction was supported by the results from the ADCC Reporter Bioassay. Humira^®’s average EC50 was measured as 0.55 nM, which is significantly lower than Remicade^® (0.64 nM) and Simponi Aria^® (0.67 nM).

Conclusion: Humira^® yielded lower EC50/IC50 values than Remicade^® and Simponi Aria^® for the TNF-⍺ binding ELISA, Fc𝛾RIIIa binding AlphaLISA and ADCC bioassay. This data, along with the fewer glycans identified, suggest that Humira^® may have higher efficacy in vivo compared to its competitors. This hypothesis would be expected to hold true in IBD patients where effector function (ADCC) is believed to be a secondary mechanism of action. It is important to note though that these are only expected trends and, due to extraneous factors such as patient HLA type, prior therapeutic exposure, etc., they cannot be taken at face-value for actual clinical outcomes. Nevertheless, the methods used in this research are useful in that they show how testing anti-TNF-⍺ biotherapeutics under the same conditions, allows us elucidate differences in their physicochemical and functional properties.

Acknowledgements: Jill Coghlan was supported by the Cellular Biotechnology Training Program under the National Institutes of Health, USA award number (NIH T32GM008353).