Toward in vitro-to-in vivo translation of monoclonal antibody pharmacokinetics: Application of a neonatal Fc receptor-mediated transcytosis assay to understand the interplaying clearance mechanisms

Figures & data

Figure 1. Basolateral to apical flux (Papp) of a ³H-IgG and FITC-dextran across rat and human FcRn over-expressing MDCK cells and EV cells. The highest Papp was observed in the cell line over-expressing FcRn and under pH 6.0/8.0, indicating a specific FcRn-mediated antibody transport. Data is shown as mean ± SD; n = 3 (separate wells).

Figure 2. Basolateral to apical flux (Papp) of FITC-labeled rat, mouse, human IgGs and chicken IgY across human FcRn and rat FcRn MDCK cells with a pH gradient (6.0/8.0). The inability of rodent IgG to bind to human FcRn was confirmed. Data is shown as mean ± SD; n = 3 (separate wells).

Figure 3. Transcytosis of different ³H-labeled IgG Fc mutants and non-binding control (chicken IgY) at pH 6.0/8.0 in human FcRn MDCK cells and EV MDCK cells as negative control. AAA, Wt, and YTE IgG clearly differed by their flux (Papp). Paired t-test P value (AAA vs. Wt IgG) = 0.024; P value (YTE vs. Wt IgG) = 0.041; P value (AAA vs. YTE IgG) = 0.002; Data is shown as mean ± SD; n = 3 (separate wells).

Figure 4. Effect of incubation time and donor/acceptor chamber pH on transcytosis of IgG mutants by human FcRn-over-expressing cells. The application of 5 h incubation time and a pH gradient of 6.0/7.4 differentiated the various molecules best. The fluxes were normalized to the flux of chicken IgY. Data is shown as mean ± SD; n  =  3 (separate wells).

Table 1. In vitro flux of IgG mutants across human and rat FcRn MDCK cells at pH 6.0/7.4.

Tested antibody	Flux across rat FcRn cells	Flux across human FcRn cells
IgY	1.0 ± 0.11	1.0 ± 0.03
AAA IgG	1.34 ± 0.26	0.70 ± 0.02
Wt IgG	2.72 ± 0.31	1.95 ± 0.12
YTE IgG	2.41 ± 0.38	3.11 ± 0.06

Only the YTE mutant showed a species difference in FcRn interaction leading to enhanced flux across human FcRn, but Wt-IgG-like flux across rat FcRn cells. The fluxes were normalized to the IgY flux obtained in the same experiment. Data is shown as mean ± SD; n  =  3 to 6 (separate wells).

Figure 5. Mean systemic clearance observed in rat after single mAb intravenous administration plotted against the reciprocal normalized flux obtained from the rat FcRn cellular transcytosis assay. IgY-normalized in vitro flux of 5 mAbs were inversely proportional with the corresponding in vivo CL values in rat. Data is shown as mean ± SD.

Table 2. Human in vitro and cynomolgus monkey in vivo data used for the in vitro-in vivo correlation

	In vivo data					In vitro data
Tested antibody	Description	Dose (mg/kg)	Terminal half life (h)	Total CL (mL/day/kg)	Number of animals	Wt IgG-normalizedin vitro fluxpH 6.0/7.4
mAb 1	hu IgG1, AAA mutant; FcRn non-binder	0.3	53	16.1	2	0.31 ± 0.01
mAb 2	hu IgG1, FcRn Wt binder	0.3	79	6.49	2	1.00 ± 0.09
mAb 3	hz IgG1, FcRn Wt binder	10	310 ± 57	3.12 ± 0.55	4	1.64 ± 0.23
mAb 4	hu IgG1, FcRn Wt binder	75	269	5.23	2	0.97 ± 0.07
mAb 5	hu IgG1, FcRn Wt binder	0.3	108	3.84	2	0.63 ± 0.09
mAb 6	hu IgG1, AAA; FcRn non-binder	0.3	32	15.4	2	0.25 ± 0.01
mAb 7	hu IgG1, FcRn Wt binder with FcRn-unrelated CL component	1	178	24.7^*	3	0.84 ± 0.1
mAb 8	chimeric IgG (hu Fc), FcRn Wt binder	5	NC^*	3.60^*	2	0.91 ± 0.1
mAb 9	chimeric IgG (hu Fc), FcRn Wt binder	0.8	380 ± 89	2.46 ± 0.35	3	0.95 ± 0.18
mAb 10	hu IgG1, enhanced FcRn-binding; positive charge patches	20	288	2.86	2	4.14 ± 0.82
mAb 11	hz IgG1, FcRn Wt binder (bevacizumab, Avastin)	5	Deng et al^20	3.97	Deng et al^20	0.83 ± 0.07
mAb 12	hu IgG1, FcRn Wt binder	20	235 ± 156	4.52 ± 1.07	3	1.27 ± 0.1
mAb 13	hu IgG1, enhanced FcRn-binding	20	137 ± 73	3.51 ± 0.8	3	3.42 ± 0.15
mAb 14	hz IgG1, FcRn Wt binder (trastuzumab, Herceptin)	5	Deng et al^20	5.52	Deng et al^20	1.77 ± 0.03
mAb 15	hu IgG4, FcRn Wt binder	2.5	355 ± 34	2.56 ± 0.33	3	1.10 ± 0.2
mAb 16	hu IgG1, FcRn Wt binder	10	59	9.81	2	1.03 ± 0.09
mAb 17	hz IgG1, FcRn Wt binder	10	194	5.88	2	1.06 ± 0.05
mAb 18	hz IgG1, FcRn Wt binder	0.15, 1.5, 15, 150	150^*	5.86^*	8	1.41 ± 0.06
mAb 19	hu IgG1, FcRn Wt binder	100	282	4.49	2	1.49 ± 0.26
mAb 20	hz IgG1, FcRn Wt binder	50	NC^*	3.34^*	3	2.13 ± 0.35
mAb 21	hu IgG1, FcRn Wt binder	1	218 ± 74.6	3.74 ± 0.32	4	1.07 ± 0.34
mAb 22	hz IgG1, FcRn Wt binder	30	240	4.61	2	0.95 ± 0.07
mAb 23	hz IgG1, FcRn Wt binder	30	94.2	3.57	2	1.11 ± 0.16
mAb 24	hu IgG1, FcRn Wt binder	150	242	8.16^*	6	2.75 ± 0.19

* PK parameters estimated by population approach, no SD applicable

mAb systemic clearance from non- compartmental (NCA) PK analysis and terminal half-life after intra-venous dose to monkeys is reported together with the respective mAb wt-normalized Papp from human cellular FcRn assay (pH 6.0/7.4). Data are given as mean values ± standard deviation in case of > 2 subjects or measurements.

hu: human; hz: humanized; Wt: wildtype mAb without mutations of the FcRn-binding sites; TMDD: target-mediated drug disposition.

Figure 6. (A) Correlation of reciprocal Wt IgG-normalized in vitro flux (pH gradient 6.0/7.4) of 7 mAbs across human FcRn MDCK cells with in vivo clearance in cynomolgus monkey. Data is plotted as mean values  ±  SD. Corresponding pairs of Fc mutant and Wt IgG are labeled with the same symbol (closed triangle, star, cross). The greyish area roughly indicates mAbs for which in vivo CL is hypothesized to be affected by “decelerating” processes, for mAbs in the white area “CL-accelerating” processes are thought to be involved. (B) Correlation of reciprocal Wt IgG-normalized in vitro flux (pH gradient 6.0/7.4) of 20 mAbs with wildtype FcRn binding ability across human FcRn MDCK cells with in vivo clearance in cynomolgus monkey. Data is plotted as mean values  ±  SD for CL and as reciprocal mean values of the Wt IgG-NF. The greyish area roughly indicates mAbs for which in vivo CL is hypothesized to be affected by “decelerating” processes, for mAbs in the white area “CL-accelerating” processes are thought to be involved.

Deng R, Iyer S, Theil FP, Mortensen DL, Fielder PJ, Prabhu S. Projecting human pharmacokinetics of therapeutic antibodies from nonclinical data What have we learned? Mabs-Austin 2011; 3:61-6; PMID:20962582; https://doi.org/10.4161/mabs.3.1.13799

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