Understanding the structure–property relationship of bispecific monoclonal antibodies with Fc site-specific substitutions

Figures & data

Figure 1. Schematic of the REGN bsAb format with site-specific substitutions at the Fc domain. The star represents the position of HY to RF substitutions in the three-dimensional structure of REGN bsAbs. EU numbering was used to label antibody sequences.

Cartoon of a “Y”-shape REGN bispecific antibody. The bispecificity is visualized by using distinct colors and shapes to represent two complementary determining regions (CDRs). The specific HY to RF mutation at the binding site is denoted by a star positioned between the CH2 and CH3 regions of the HC*, indicating the precise location of the mutation within the higher order structure of the bispecific antibody. Below the illustration, the aligned sequences of the generic hIgG4 HC and hIgG3 HC* at the C-terminus are provided to demonstrate the presence of the HY to RF mutations.

Figure 2. (a) Structural homology of the Fc domain of REGN bsAb-1. Regions showing statistically significant differences in deuterium uptake (HC* > HC) are in red. Regions with unchanged deuterium uptake after substitutions are in gray. Representative HDX-MS kinetic plots are shown for (b) C_H peptide ²⁴¹FLFPPKPKDTLM²⁵², (c) C_H peptide ⁴²⁷VMHEALHNHYTQK⁴³⁹ (for HC) and ⁴²⁷VMHEALHNRFTQK⁴³⁹ (for HC*) and (d) C_H peptide ²⁹⁸STYRVVSVL³⁰⁶. The means and error bars (standard deviations) are based on triplicated experiments.

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(a) A homology model of the Fc domain REGN bsAb-1 displays the structural characteristics. Various regions at the CH2-CH3 interface of HC* are highlighted in red, indicating statistically significant differences in deuterium uptake. (b) The kinetic plot of the peptide FLFPPKPKDTLM reveals a dynamic pattern with the following trend: HC/HC homodimer > HC/HC* heterodimer > HC*/HC* homodimer. (c) The kinetic plot of the peptide VMHEALHNHY(RF)TQK demonstrates that the deuterium uptake in the HC/HC homodimer is consistently higher than in the HC*/HC* homodimer across all time points. The trace of ”HC from heterodimer” coincides with the HC/HC homodimer, while the trace of ”HC* from heterodimer” aligns with the HC*/HC* homodimer. (d) When examining the peptide STYRVVSVL, the deuterium uptake is similar between the HC/HC homodimer and the HC*/HC* homodimer, except for the 14400s time point.

Figure 3. DSC thermograms of Fc subunit of (a) HC/HC homodimer, (b) HC/HC* heterodimer, (c) HC*/HC* homodimer of REGN bsAb-1 in 150 mM ammonium acetate, pH 6.0. Black trace represents the experimental data. The blue dotted traces represent the thermal transition fits and the red trace represents the cumulative fit data.

Displayed in a top-to-bottom arrangement are the stacked DSC thermograms of the Fc subunits for the following configurations: (a) HC/HC homodimer, (b) HC/HC* heterodimer, and (c) HC*/HC* homodimer. The thermograms reveal two distinct thermal transitions, labeled as Tm1 and Tm2. These transitions exhibit a consistent trend where the HC*/HC* Fc has the lowest Tm values, followed by the HC/HC* Fc, and finally, the HC/HC Fc with the highest Tm values.

Table 1. Thermal stability of Fc subunit samples indicated by the melting onset temperature (T_onset) and the melting transitions (T_m and T_m) as measured by DSC. The mean and standard deviations (SD) are based on triplicate measurements.

Sample description	Tonset (°C)		Tm1 (°C)		Tm2 (°C)
	Mean	SD	Mean	SD	Mean	SD
HC/HC Fc	58.8	0.3	65.5	0.1	68.0	<0.1
HC/HC* Fc	57.0	1.4	65.0	0.1	67.7	<0.1
HC*/HC* Fc	54.3	<0.1	64.1	<0.1	66.8	<0.1

Figure 4. Quantification of (a) HC Met²⁵²/HC* Met²⁵² and (b) HC Met⁴²⁸/HC* Met⁴²⁸ oxidation under H₂O₂ stress; (c) HC Met²⁵²/HC* Met²⁵² and (d) HC Met⁴²⁸/HC* Met⁴²⁸ oxidation under thermal stress in bsAb-1 heterodimer and homodimers. The levels of Met oxidation generated in stressed samples at different time points were normalized by subtracting the initial Met oxidation level for the same Met residue in each sample prior to incubation. The means and error bars (standard deviations) are based on triplicated experiments.

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(a) The line graph shows the increase in methionine oxidation in the peptide DTLMISR under oxidative stress conditions over 120 minutes. Similar rates of oxidation were observed for the HC*/HC* homodimer, HC/HC* heterodimer, and HC/HC homodimer samples. (b) The line graph shows the increase in methionine oxidation in the peptide WQEGNVFSCSVMHEALHNHYTQK (from HC) or WQEGNVFSCSVMHEALHNR (from HC*) under oxidative stress conditions over 120 minutes. The trend indicates that the HC*/HC* homodimer = HC* from heterodimer > HC from heterodimer = HC/HC homodimer. (c) The line graph shows the increase in methionine oxidation in the peptide DTLMISR under thermal stress conditions over a duration of 14 days. The trend shows that the HC*/HC* homodimer configuration exhibits higher levels of oxidation compared to the HC/HC* heterodimer and HC/HC homodimer samples. (d) The line graph shows the increase in methionine oxidation in the peptide WQEGNVFSCSVMHEALHNHYTQK (from HC) or WQEGNVFSCSVMHEALHNR (from HC*) under thermal stress conditions over a 14-day period. The trend indicates that the HC*/HC* homodimer configuration exhibits the highest rate of oxidation, followed by HC* from heterodimer, HC from heterodimer, and HC/HC homodimer configurations, in descending order. Top of Form.

Figure 5. (a) Significant differences in deuterium uptake for the C_H peptide ²⁴¹FLFPPKPKDTLM²⁵² between mAb 1–3 and bsAb 2–4. (b) Normalized oxidation level of HC Met²⁵²/HC* Met²⁵² in thermal stressed samples. (c) Deuterium uptake of the C_H peptide ⁴²⁷VMHEALHNHYTQK⁴³⁹ with or without HY to RF substitutions. (d) Normalized oxidation level of HC Met²⁵²/HC* Met²⁵² in thermal stressed samples. The means and error bars (standard deviations) are based on triplicated experiments. Statistical testing was demonstrated in Figure S7.

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(a) The bar graph shows the deuterium uptake of the peptide FLFPPKPKDTLM in three monoclonal antibodies (mAbs) and three bispecific antibodies (bsAbs). The three bsAbs exhibit similar and higher levels of deuterium uptake compared to the three mAbs. (b) The line graph shows the increase in methionine oxidation in the peptide DTLMISR under thermal stress conditions over a 14-day period in three mAbs and three bsAbs. Methionine oxidation occurs at a faster rate in bsAbs compared to mAbs. (c) The bar graph presents the deuterium uptake of the peptide VMHEALHNHY(RF)TQK in three mAbs and three bsAbs. Peptides with RF substitutions exhibit higher levels of deuterium uptake compared to those without substitutions. (d) The line graphs showcase the increase in methionine oxidation in the peptide WQEGNVFSCSVMHEALHNHYTQK (from HC) or WQEGNVFSCSVMHEALHNR (from HC*) under thermal stress conditions over a 14-day duration for three mAbs and three bsAbs. The rates of oxidation follow the trend: HC* from bsAbs > HC from bsAbs > mAbs.

Figure 6. BLI sensorgrams of FcRn binding to Fc subunit of (a) HC/HC homodimer, (b) HC/HC* heterodimer, (c) HC*/HC* homodimer of bsAb-1.

Bio-Layer Interferometry (BLI) sensorgrams of FcRn binding to Fc subunits (a) HC/HC homodimer, (b) HC/HC* heterodimer, (c) HC*/HC* homodimer of bsAb-1. Based on the steady-state signals observed in the sensorgrams, the binding strength follows the trend where the HC/HC Fc subunit shows the strongest binding to FcRn, followed by the HC/HC* Fc subunit, and finally the HC*/HC* Fc subunit, which exhibits the weakest binding to FcRn.

Table 2. Steady state analysis of the bindings between human FcRn and REGN bsAb-1 Fc subunit samples.

Sample description	Steady state analysis
	KD (nM)	Rmax (nm)	R^2
HC/HC Fc	250	0.174	0.927
HC/HC* Fc	350	0.165	0.928
HC*/HC* Fc	560	0.139	0.972