Pharmacokinetics of novel Fc-engineered monoclonal and multispecific antibodies in cynomolgus monkeys and humanized FcRn transgenic mouse models

Figures & data

Table 1. Panel of antibodies used in the study. All tested antibodies were of the IgG1 backbone type. The listed mAbs are not parental to the bi- or trispecific molecules

Antibody	Antibody format	Fc-backbone mutation
mAb1	mAb	(WT)
(bevacizumab)	mAb	LS
	mAb	NNAS
	mAb	DQ
	mAb	DW
	mAb	YD
mAb2	mAb	(WT)
	mAb	LS
mAb3	mAb	(WT)
(adalimumab)	mAb	LS
	mAb	NNAS
bi-Ab4	bi-Ab	mut
bi-Ab5	bi-Ab	(WT)
tri-Ab6	tri-Ab	(WT)
	tri-Ab	LS
mAb7	mAb	ADE

bi-Ab = bispecific antibody, mAb = monoclonal antibody, mut = mutated, tri-Ab = trispecific antibody, WT = wild type.

Table 2. In vitro characterization of antibodies carrying Fc-backbone modifications. Binding affinities to the FcRn receptor of humans and cynomolgus monkeys; binding to FcγRIIIa, and pI

		Human FcRn	Cyno FcRn	Human FcRn	Cyno FcRn	Human FcRn	Cyno FcRn	FcγRIIIa
Antibody	Fc-backbone mutation	KD,app (nM), at pH 6.0		Fold enhancement in KD (relative to WT)		Steady State RU, at pH 7.4		KD,app (nM)	pI
mAb1 (Bevacizumab)	WT	500 ± 37	1440 ± 360	1	1	1.3 ± 0.2	1.3 ± 0.3	377 ± 1	8.18
	NNAS	787 ± 33	2400 ± 800	0.6	0.6	0.6 ± 0.1	1.0 ± 0.1	*	7.73^†
	LS	198 ± 33	210 ± 43	2.5	6.8	44 ± 4	38.3 ± 6.4	307 ± 4	8.17
	DQ	89 ± 2	110 ± 42	5.6	13	14 ± 1	10.9 ± 0.4	361 ± 4	7.89
	DW	126 ± 32	99 ± 9	4.0	14.5	18 ± 2	14.8 ± 0.5	329 ± 1	7.89
	YD	86 ± 4	120 ± 6	5.8	12	27 ± 2	22.2 ± 3.0	645 ± 15	7.91
mAb2 (Adalimumab)	WT	645 ± 99	1100 ± 270			2.7 ± 0.2	2.5 ± 0.3	194 ± 6	8.74
	NNAS	1410 ± 350	2880 ± 1210	0.5	0.4	1.3 ± 0.1	1.5 ± 0.2	*	8.30^†
	LS	500 ± 33	48 ± 9	1.3	23	68.4 ± 5.5	55.7 ± 5.4	196 ± 3	8.75
mAb3	WT	2070 ± 1200	1040 ± 290			1.6 ± 0.1	1.5 ± 0.1	363 ± 3	8.64
	LS	415 ± 72	329 ± 49	5.0	3.2	53.8 ± 5.5	42.6 ± 2.0	248 ± 2	8.63
tri-Ab6	WT	1540 ± 270	2040 ± 290			3.2 ± 0.3	3.3 ± 0.1	391 ± 1	8.54
	LS	83 ± 2	61 ± 4	18.6	33	64.0 ± 5.6	62.9 ± 2.9	168 ± 13	8.54

*For NNAS molecules, no binding to FcγRIIIa receptor was detectable (silent backbone).

^†pI value was heterogeneous due to variations in glycosylation pattern.

Cyno = cynomolgus monkey, FcRn = neonatal Fc receptor, K_D = dissociation constant, mAb = monoclonal antibody,

pI = isoelectric point, tri-Ab = trispecific antibody, WT = wild type.

Table 3. Summary of PK parameters of the different antibodies tested in cynomolgus monkeys and Tg32 and Tg276 homozygous mice

				Cynomolgus monkeys				Tg32 mice				Tg276 mice
Antibody	Modality	Fc variant	Dose (mg/kg)	AUC (µg∙d/mL)	CL (mL/d/kg)	Vss (mL/kg)	t1/2 (d)	AUC (µg∙d/mL)	CL (mL/d/kg)	Vss (mL/kg)	t1/2 (d)	AUC (µg∙d/mL)	CL (mL/d/kg)	Vss (mL/kg)	t1/2 (d)
mAb1 (bevacizumab)	mAb	WT	2.5	457	5.5	70.6	9.8	339	7.4	107	11.7	292	8.6	62.3	6.4
		LS	2.5	1220	2.1	64.8	22.5	547	4.6	117	19.5	143**	NC	NC	NC
		NNAS	2.5	677	3.7	41.8	8.6	342	7.3	78.2	8.2	157**	NC	NC	NC
		DQ	2.5	726	3.4	97.3	20.8	790	3.2	108	24.5	381	6.6	60.6	6.2
		DW	2.5	995	2.5	70.0	20.4	725	3.5	98.6	20.1	398	6.3	65.8	7
		YD	2.5	1070	2.4	81.4	23.5	558	4.5	111	17.5	364	6.9	97.3	10.5
mAb2	mAb	WT	2.5	685	3.7	114	22.7	794	3.2	78.2	17.3	406	6.2	78.8	8.1
		LS	2.5	1280	2.0	73.8	27.1	695	3.6	93.7	18.7	311**	NC	NC	NC
mAb3 (adalimumab)	mAb	WT	10	1620	6.6	51.3	7.6	1220	8.2	95.1	8.7	–	–	–	–
		LS	10	2780	3.7	78.1	14.7	1900	5.3	73	10.6	–	–	–	–
		NNAS	10	1530	6.6	50.9	8.3	2260	4.4	64.2	10.0	–	–	–	–
bi-Ab4	bispecific	mut	0.003*/2.5^†	0.00471	548	400	3.7	231	10.8	66.6	4.4	–	–	–	–
bi-Ab5	bispecific	WT	3	210	14.5	62.7	4.5	157	16.2	100	5.4	–	–	–	–
tri-Ab6	trispecific	WT	2.5	155	16.2	92.5	6.3	168	14.9	107	5.5	53.6	46.6	69.3	1.4
		LS	2.5	466	5.4	83.9	11.9	198	12.6	152	9.2	105	23.9	89.2	3.6
mAb7	mAb	ADE	5	1190	4.2	68.9	11.7	570	9.3	137	10.5	–	–	–	–

*For cynomolgus monkeys.

^†For Tg32 mice.

**AUC_all instead of AUC due to anti-drug antibody formation.

–: not performed.

n = 3 monkeys and 6 mice per group.

ADA = anti-drug antibody, AUC = area under the curve, bi-Ab = bispecific antibody, CL = clearance, mAb = monoclonal antibody, mut = mutated, NC = not calculable due to the presence of ADA, PK = pharmacokinetics, t_1/2 = half-life, Vss = apparent volume of distribution at steady state, tri-Ab = trispecific antibody, WT = wild type.

Figure 1. Comparison of pharmacokinetic profiles in cynomolgus monkeys and homozygous Tg32 and Tg276 mice following administration of (a) mAb1-WT or (b) with the LS mutation; (c) tri-Ab6-WT or (d) with the LS mutation. For mouse studies, pooled plasma samples were plotted per sampling time. Abbreviations: ADA = anti-drug antibody, mAb = monoclonal antibody, tri-Ab = trispecific antibody, WT = wild type

Figure 2. Comparison of pharmacokinetic profiles in (A/C) Tg32 mice and (B/D) cynomolgus monkeys. Administration of (A/B) FcRn-modulating LS and YD mutations; (C/D) mAb1 or mAb3, WT and mutated Fc backbones, Fc-silencing FcγRIIIa (NNAS). Abbreviations: mAb = monoclonal antibody, WT = wild type

Figure 3. (a) Interspecies clearance comparison of the panel of mAbs and bi-Abs/tri-Abs tested. (b) Relationship between antibody clearance in cynomolgus monkeys/Tg32 mice and binding affinity (K_D pH6) for human FcRn. Abbreviations: bi-Ab = bispecific antibody, FcRn = neonatal Fc receptor, K_D = dissociation constant, mAb = monoclonal antibody, tri-Ab = trispecific antibody, WT = wild type

Figure 4. Correlation of antibodies’ (a) elimination half-lives and (b) clearance between homozygous Tg32 mice and cynomolgus monkeys

Figure 5. Estimation of α and β allometric exponents for CL and Vss, respectively, to scale from Tg32 mice to cynomolgus monkeys (NHP) using the equation Y_NHP = Y_Tg32 * (body weight_NHP/body weight_Tg32)^{α, β, γ}. Abbreviations: bi-Ab = bispecific antibody, CL = clearance, NHP = non-human primate, tri-Ab = trispecific antibody, Vss = volume of distribution at steady state, WT = wild type, Y = variable of interest

Figure 6. Ratios of predicted versus observed cynomolgus monkey Vss and CL values for the antibodies shown in Table 1. Only antibodies administered with a saturating dose to assess linear pharmacokinetics are presented; therefore, the bi-Ab4 antibody was excluded from the analysis. The predicted values were determined using the allometric equation with the α (for CL) or β (for Vss) allometric exponent estimated in Figure 5. Abbreviations: bi-Ab = bispecific antibody, CL = clearance, mAb = monoclonal antibody, tri-Ab = trispecific antibody, Vss = volume of distribution at steady state, WT = wild type