Higher Binding Affinity and In Vitro Potency of Reslizumab for Interleukin-5 Compared With Mepolizumab

Abstract

Reslizumab and mepolizumab are recently approved monoclonal antibodies for the treatment of severe (uncontrolled) eosinophilic asthma. Both are effective in neutralizing the function of interleukin-5 (IL-5). This study is the first to compare the binding affinity and in vitro potency of both antibodies in head-to-head assays. Two assays assessed binding affinity (using the equilibrium dissociation constant [K D ]) of each drug for human IL-5. In the Biacore surface plasmon resonance assay, the association constant (k on ) values for human IL-5 for reslizumab and mepolizumab were 3.93 × 10 6 and 1.83 × 10 5 , respectively. The dissociation constant (k off ) values were 4.29 × 10 −4 and 2.14 × 10 −4 , respectively. Calculated K D values for human IL-5 for reslizumab and mepolizumab were 109 and 1,170 pM, respectively, representing an approximately 11-fold stronger binding affinity with reslizumab. In the Kinetic Exclusion Assay, the k on values for human IL-5 for reslizumab and mepolizumab were 3.17 × 10 6 and 1.32 × 10 5 , respectively. The k off values were 1.36 × 10 −5 and 1.48 × 10 −5 , respectively. Measured K D values for human IL-5 for reslizumab and mepolizumab were 4.3 and 112 pM, respectively, representing an approximately 26-fold stronger binding affinity for reslizumab. A human-IL-5-dependent cell proliferation assay was developed to assess in vitro potency, based on a human cell line selected for enhanced surface expression of IL-5 receptor-alpha and consistent proliferation response to IL-5. The concentration at which 50% inhibition occurred (IC 50 ) was determined for both antibodies. Reslizumab and mepolizumab inhibited IL-5-dependent cell proliferation, with IC 50 values of approximately 91.1 and 286.5 pM, respectively, representing on average 3.1-fold higher potency with reslizumab. In conclusion, comparative assays show that reslizumab has higher affinity binding for and in vitro potency against human IL-5 compared with mepolizumab. However, these results do not take into consideration the different methods of administration of reslizumab and mepolizumab.

Kinetic Exclusion Assay of Biomolecules by Aptamer Capture

“…The curves corresponding to the three different FA concentrations were simultaneously analyzed with the n-curve analysis module [ 54 , 67 , 70 , 76 ] included with the KinExA Pro software package to determine the equilibrium dissociation constant K d . The calculated percent of free FA vs. thrombin concentration and the theoretical curves for the three FA concentrations for a 1:1 binding model (Equation (A1)) are shown in Figure 4 a.…”

“…Details of the signal equation used for fitting the 1:1 binding model [ 55 , 59 , 75 , 76 ], n-curve analysis module [ 54 , 67 , 70 , 76 ], mixed model [ 75 , 77 ], and logistic equation for concentration determination [ 61 , 78 ] are provided in Appendix A . The data were analyzed and plotted with KinExA Pro software (version 4.4.36, Sapidyne Instruments), and Origin 8.5.1 software (Origin Labs, Northampton, MA, USA).…”

“…This equation is fitted by the method of least squares to the experimental data obtained by using FA as CPB and thrombin as titrant. The n-curve analysis simultaneously fits Equation (A1) to multiple signal curves generated by using different CBP concentrations [ 54 , 67 , 70 , 76 ]. For the two-population aptamer mixture, the total concentration of FA , [ FA ] 0 , is assumed to be composed of two fractions [ FA ] 1 = f × [ FA ] 0 , and [ FA ] 2 = (1 − f ) × [ FA ] 0 , where f is the fraction of the tightest binder in the population.…”

“…This equation is fitted by the method of least squares to the experimental data obtained by using FA as CPB and thrombin as titrant. The n-curve analysis simultaneously fits Equation (A1) to multiple signal curves generated by using different CBP concentrations [ 54 , 67 , 70 , 76 ].…”

“…Although this technology has been chiefly used with antibodies as biorecognition elements [ 46 , 54 , 55 , 56 , 70 ], the only restriction on recognition molecules is that at least one of them needs to be fluorescently detectable, either directly or indirectly [ 55 ]. We used this technology to interrogate an aptamer-thrombin binding system, and our protocols make full use of the advantages offered by the KinExA technology in terms of automation, accuracy, reliability, and adaptability.…”