Dissecting the molecular basis of high viscosity of monospecific and bispecific IgG antibodies

Figures & data

Figure 1. (a) Cartoon representation of monospecific and bispecific antibody formats investigated. The isotype for the anti-IL-17 antibody is IgG₁, whereas the anti-IL-13 and BsIgG antibodies are both IgG₄. All antibodies have κ light chains. Half-antibodies containing knobs-into-holes mutations³⁴ (‘knob’ mutation (T366W) in the anti-IL-13 arm or ‘hole’ mutations (T366S:L368A:Y407V) in the anti-IL-17arm) were expressed and purified separately before assembly in vitro as described previously.^35,36 A hinge stabilizing mutation (S228P) was introduced into the IgG₄ to attenuate Fab arm exchange.^37,38 (b) Viscosity of fragments (F(ab’)₂ and Fab) or full-length anti-IL-13/IL-17 bispecific or its parent antibodies are shown. (c) The effect of 150 mM NaCl or 150 mM Arg-HCl on solution viscosity is shown. The dotted line represents the viscosity of parent IgG antibody level as a reference. Data shown are the mean values (n = 3, ± SD) measured by rheometer at 23ºC and 150 mg/mL total protein concentration in 20 mM His-HCl pH 6.0 (buffer) in the absence or presence of excipients

Figure 2. X-ray crystallographic structures of anti-IL-13 (lebrikizumab)⁴⁰ and anti-IL-17 (MCAF5352A⁴¹) antibody Fabs complexed with their respective ligands displaying aromatic residues chosen for mutational studies. (a) View of anti-IL-13/IL-13 interface depicting important viscosity reducing residues (PDB 4I77).⁴⁰ The anti-IL13 V_H is cyan and V_L is light pink, with dark cyan and magenta side chains within 4 Å of IL-13. IL-13 is gray with yellow epitope within 4 Å of anti-IL-13. (b) View of anti-IL-17/IL-17F interface depicting important residues (PDB 6PPG). Anti-IL-17F V_H is cyan and V_L is light pink, with dark cyan and magenta side chains within 4 Å of IL-17. IL-17F is gray with yellow epitope within 4 Å of anti-IL-17F. See the Supplementary Materials for the sequences for the variable domains for the anti-IL-13 (Figure S6) and anti-IL17 (Figure S7) antibodies

Figure 3. MD simulations of (a) anti-IL-13 (lebrikizumab⁴⁰) and (b) anti-IL-17 (MCAF5352A⁴¹) variable heavy (V_H) and variable light (V_L) domains in the presence of arginine. Data shown are the mean number of arginine contacts from 20 independent simulations. Residues that show >2σ or >3σ deviations from the mean (μ) number of arginine contact number of all residues in chains are highlighted. Kabat numbering is used.^42.

Figure 4. Viscosity of antibody variants. Monospecific anti-IL-13 IgG₄ and anti-IL-17 IgG₁ antibody variants were mixed with the parent anti-IL-17 IgG₁ and anti-IL-13 IgG₄ antibody counterparts, respectively, at a 1:1 ratio and a final total concentration of 150 mg/mL. The viscosity of the solutions was then measured by rheometer at 23ºC. (a) Viscosity of mixtures of anti-IL-13 charged variants and the anti-IL-17 parent antibody. (b) Viscosity of mixtures of anti-IL-17 charged variants and the anti-IL-13 parent antibody. (c) Viscosity of mixtures of anti-IL-13 and anti-IL-17 aromatic variants with their parent IgG antibody counterpart. (d) Viscosity of anti-IL-13/IL-17 BsIgG₄ variants at 150 mg/mL final concentration. Data shown are the mean values (n = 2, ± SD) measured in 20 mM His-HCl buffer at pH 6.0. The dotted line shows the parent antibody viscosity level as a reference

Table 1. Antigen-binding kinetics of anti-IL-13 antibody variants. Binding kinetics of anti-IL-13 variants for IL-13 were measured by SPR at 37°C, pH 7.4 and data were fitted to a 1:1 binding model. *Variants that reduced viscosity and have comparable K_D as the parent antibody. Dissociation rate constants (k_off values) ≤ 1 × 10⁻⁴ s⁻¹ could not be reliably measured by SPR under the experimental conditions used

Variant	kon, s^−1 M^−1 x 10^6	koff, s^−1 x 10^−6	KD, pM
Parent	2.16 ± 0.04	≤ 1 × 10^−4	<10
VH Y97L*	2.08 ± 0.11	≤ 1 × 10^−4	<10
VL Y49Q*	2.34 ± 0.06	≤ 1 × 10^−4	<10
VH Y98L	1.16 ± 0.04	87.6 ± 2.7	75.3 ± 0.2
VL Y28Q	0.58 ± 0.05	159 ± 11	275 ± 3
VH Y100Q	1.00 ± 0.01	3,310 ± 99	3,320 ± 95

Table 2. Antigen-binding kinetics of anti-IL-17 antibody variants. Binding kinetics of the anti-IL-17 variants for IL-17FF were measured by SPR at 37°C, pH 7.4 and data fitted to a 1:1 binding model

Variant	kon, s^−1 M^−1 x 10^7	koff, s^−1 x 10^−4	KD, pM
Parent	5.68 ± 0.22	4.42 ± 0.30	7.78 ± 0.23
VL W90Q	3.92 ± 0.62	9.20 ± 2.12	23.4 ± 3.7
VH Y100cL	4.04 ± 0.14	10.8 ± 1.07	26.6 ± 1.8
VH W52aL	8.10 ± 0.39	26.5 ± 2.32	32.6 ± 1.9
VH F99Q	4.74 ± 1.37	73.8 ± 12.09	161 ± 38

Table 3. Antigen-binding kinetics and viscosity of anti-GCGR antibody variants. Binding kinetics of the anti-GCGR antibody variants for GCGR were measured by SPR at 37°C, pH 7.4 and data fitted to a 1:1 binding model. *Variants that reduced viscosity by >2-fold and maintained K_D within 2-fold of the parent antibody. The viscosity was measured at 180 mg/mL final concentration in 20 mM histidine-acetate buffer at pH 5.5

Variant	kon s^−1 M^−1 x 10^5	koff s^−1 x 10^−4	KD nM	Viscosity cP
Parent	5.66 ± 0.47	3.43 ± 0.08	0.61 ± 0.06	60.6 ± 9.9
VH Y59A	21.0 ± 2.1	6.60 ± 0.14	0.32 ± 0.03	84 ± 11
VL Y55A*	6.18 ± 0.50	4.53 ± 0.09	0.74 ± 0.05	27.0 ± 0.2
VL Y91A*	5.69 ± 0.56	5.63 ± 0.06	0.84 ± 0.08	20.0 ± 0.5
VL Y96A	6.74 ± 0.41	5.40 ± 0.08	0.93 ± 0.08	51.4 ± 0.6
VH F51A	4.47 ± 0.39	6.43 ± 0.08	1.45 ± 0.15	75.7 ± 2.8
VH Y79A	2.41 ± 0.54	10.40 ± 0.35	1.84 ± 0.14	42.2 ± 1.1
VH Y100dA	5.80 ± 0.67	29.0 ± 1.1	5.03 ± 0.41	23.1 ± 0.5
VH W100aA	2.41 ± 0.09	26.1 ± 0.3	10.8 ± 0.3	7.95 ± 0.1
VH F100eA	7.78 ± 0.86	109 ± 5	14.1 ± 0.9	65.2 ± 0.4
VH Y54A	0.57 ± 0.28	23.0 ± 3.5	46 ± 17	14.3 ± 0.3
VH Y33A	3.68 ± 0.47	193.0 ± 1.0	53.0 ± 7.0	35.3 ± 0.8
VH Y31A	0.24 ± 0.16	43.7 ± 16.1	206 ± 71	19.2 ± 0.8

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