The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies

Figures & data

Table 1. General description of the IgGs

Compounds	mAb Isotype	CDR Charge Patch Engineered	Increased FcRn Affinity	TMD In Cynomolgus Monkeys
A	IgG1	+	−	−
A-V308P	IgG1	+	+	−
B	IgG1	−	−	−
B-V308P	IgG1	−	+	−
C	IgG4	+	−	+
C-V308P	IgG4	+	+	+
D	IgG4	−	−	+
D-V308P	IgG4	−	+	+

* All the molecules are humanized IgGs. The ‘+’ and ‘−’ signs indicate the presence and absence of a characteristic, respectively.

Table 2. Biophysical and cynomolgus monkey FcRn binding properties of the IgGs

				Tm (°C)
Compounds	Cynomolgus Monkey FcRn Kd (nM) at pH 6	Heparin Binding at 100 μg/mL (OD450-OD630)	pI	CH2	CH3	Fab
A	105 ± 5	0.034 ± 0.0019	8.8	67	83	70
A-V308P	2.3 ± 0.2	0.039 ± 0.0012	8.6	68	83	72
B	115 ± 3	0.134 ± 0.0061	9.1	71	83	72
B-V308P	2.1 ± 0.6	0.116 ± 0.0015	9.0	70	82	70
C	93 ± 4	0.410 ± 0.0151	9.2	69	72	76
C-V308P	1.6 ± 0.8	0.426 ± 0.0016	9.1	71	72	74
D	121 ± 15	3.899 ± 0.0018	9.5	69	73	76
D-V308P	0.9 ± 0.2	3.961 ± 0.0015	9.5	71	70	78

Figure 1. CDR charge balancing reduces non-specific binding to heparin. Non-specific binding of (A) A and B and (B) C and D to heparin-coated plates.

Figure 2. CDR charge balancing improves the cynomolgus monkey pharmacokinetics of mAbs. Pharmacokinetic profiles of (A) mAbs A and B and (B) C and D. mAbs A and C are CDR charge balanced from mAbs B and D, respectively. Data are the mean of 2 animals/time point for mAb D. Data are the mean ± SD (standard deviation) for 4 animals/time point for mAb C and 3 animals/time point for mAbs A and B. The pharmacokinetics were assessed following a single IV dose of 2 mg/kg for mAbs A and B and 10 mg/kg for mAb C and D.

Table 3. Cynomolgus monkey pharmacokinetic parameters of the IgGs

Antibodies	Dose (mg/kg)	Cmax (μg/mL)	AUC0-inf (hr*μg/mL)	CL (mL/hr/kg)	T1/2 (hr)
A	2 (n = 3)	32 + 3	4091 + 929	0.51 + 0.12	397 + 213
A-V308P	2 (n = 3)	38 + 3	10583 + 1880	0.19 + 0.03	443 + 18
B	2 (n = 3)	17 + 8	1148 + 339	1.87 + 0.67	145 + 33
B-V308P	2 (n = 3)	10 + 2	2864 +238	1.02 + 0.58	431 + 241
C	10 (n = 4)	226 + 22	5431 + 747	1.86 + 0.28	132 + 44
C-V308P	10 (n = 3)	245 + 27	6762 + 1151	1.51 + 0.26	77 + 34
D	10 (n = 2)	114	1059	9.70	161
D-V308P	10 (n = 3)	119 + 23	1437 + 178	7.02 + 0.82	132 + 78

C_max, maximal observed serum concentration; AUC_0-inf, area under the serum concentration curve from time zero extrapolated to infinite time; CL, clearance; T_1/2, elimination half-life.

Figure 3. The V308P Fc variant shows no improvements in the cynomolgus monkey pharmacokinetics for a mAb with charge-based NSB, but does improve the clearance of a mAb with no charge-related NSB. Mean pharmacokinetic profiles of (A) mAbs A and A-V308P, which had no charge-associated NSB, and (B) B and B-V308P, which showed binding to heparin in vitro. Individual animal pharmacokinetic profiles of (C) mAbs A and A-V308P and (D) B and B-V308P. Data are the mean ± SD (standard deviation) for 3 animals/time point for all the mAbs. The pharmacokinetics were assessed following a single IV dose of 2 mg/kg for each mAb.

Figure 4. The V308P Fc variant shows no improvements in the cynomolgus monkey pharmacokinetics for mAb with and without charge-based NSB in the presence of a TMD clearance component. Mean pharmacokinetic profiles of (A) mAbs C and C-V308P, which had no charge-associated NSB but bind target antigen, and (B) D and D-V308P, which also bind target antigen, as well as showed binding to heparin in vitro. Individual animal pharmacokinetic profiles of (C) mAbs C and C-V308P and (D) D and D-V308P. Data are the mean ± SD (standard deviation) for 3 animals/time point for all the V308P Fc variant mAbs and 4 animals/time point for mAb C. Data are the mean of 2 animals/time point for mAb D. The pharmacokinetics were assessed following a single IV dose of 10 mg/kg for each mAb.