Cellular Binding and Internalization Assay for an Anti-FcγRIIB Antibody Using Human Liver Non-parenchymal Cells

Abstract

A cellular assay for evaluating the binding and internalization of biologics using primary human liver sinusoidal endothelial cells (LSEC) is not readily available, since human LSEC generally lose their receptor expression and internalization activity during the purifying processes and cell culturing. Here, we propose a novel cell-based assay using human liver non-parenchymal cells (NPC) as an alternative method using LSEC. To identify the LSEC population, NPC were stained with CD31 and CD45, and analyzed by flow cytometry. The expression of Fc gamma receptor IIB (FcγRIIB), one of the LSEC markers was detected in the CD31-positive and the CD45-negative fractions. The concentration-dependent binding and internalization of the anti-FcγRIIB antibody was also quantified in the LSEC fraction in human NPC. Saturated binding and internalization curves were obtained for the anti-FcγRIIB antibody. In the case of the negative control antibody, however, binding and internalization were negligible. The findings reported here indicate that cell-based assays using fresh human liver NPC will be useful for evaluating the binding and internalization of biologics as well as for determining pharmacokinetic parameters.

INTRODUCTION

The liver sinusoidal endothelial cell (LSEC) is type of liver non-parenchymal cells (NPC) that account for approximately 40% of cells in liver tissue.^1,2) These cells play a major role in clearing a wide variety of macromolecules such as antibodies, antibody-antigen complexes (immune complex), lipids, and oligonucleotides as well as viruses and pathogens via specific receptors that are located on the cell membrane.^1,3) It should be noted here that LSEC that express Fc gamma receptor IIB (FcγRIIB) in NPC mainly involves the elimination of an antibody and an immune complex via their Fc regions. Hence, the binding affinity of the Fc region against FcγRIIB can have a large impact on their elimination. Therefore, evaluating the binding and internalization of an antibody in that cell is indispensable in terms of understanding the pharmacokinetic properties of an antibody.

In general, LSEC is isolated by differential centrifugation, magnetic cell separation, and flow cytometry. Immortalized cells have also been reported recently.⁴⁾ However, evaluating the binding and internalization of such large molecules in LSEC is generally difficult, especially in primary fresh human cell (Table 1). One reason for this is that in human LSEC, biological activity including receptor expression and cellular internalization is minimal. While isolated human LSEC is commercially available, detailed information concerning the isolation method and the validation by functional assays for the characteristics of LSEC, including the evaluation of binding and the internalization of biologics is not available. It is noteworthy that most such cells lack typical receptor expression and internalization activity. It is plausible that co-culturing LSEC with hepatocytes and/or liver NPC per se, or humoral factors derived from these cells is needed for the homeostasis of LSEC to be maintained.^5,6) Therefore, an alternative method for obtaining an LSEC fraction in which protein expression and internalization activity are maintained is important in terms of analyzing the binding and internalization of macromolecules. While a flow cytometry-based method involving the use of mouse and monkey NPC has been reported previously,⁷⁾ information regarding human cells has not.

Table 1. Comparison of Technical Aspects of LSEC and Liver NPC for the in Vitro Assay

	LSEC	Liver NPC
Cell character	Single cell	Various cells including LSEC
Preparation	Required isolation and purification from liver NPC by the differential centrifugation and/or the magnetic cell separation	Just only necessary to remove parenchymal cell from liver suspension
Procurement of primary fresh human cell	Difficult to obtain primary human cell that keeps biological activity including protein expression and uptake	Relatively easy to get primary human cell

Here, we propose an approach for identifying human LSEC, and a method for determining the cellular binding and/or uptake-associating parameters from fresh human liver NPC. NPC are prepared by simply removing parenchymal cells from liver tissue. NPC were stained with antibodies against CD31 and CD45,^8,9) and the LSEC marker-expressing cell population was then identified by the flow cytometry. Additionally, the expression of FcγRIIB, a known LSEC marker, was confirmed.^3,8,9) The concentration dependent binding and internalization of an anti-FcγRIIB antibody (clone 2B6¹⁰⁾) was kinetically analyzed to determine the binding-related parameters, KD (the binding affinity) and B_max (the maximum binding), and internalization-related parameters, K_m (the Michaelis–Menten constant) and V_max (the maximum velocity).

MATERIALS AND METHODS

Human Liver NPC

Human liver NPC were purchased from Sekisui XenoTech, LLC (CP-21-002 R&D; Kansas City, KS, U.S.A.). The NPC were prepared by Sekisui Xenotech, LLC. Donor information is shown in Table 2. In a typical experiment, the liver was perfused with a collagenase solution, and the tissue was massaged to disperse the cells. The resulting suspension was centrifuged to remove the parenchymal cells, and the supernatant that includes the NPC, was collected.

Table 2. Donor Information

Donor number	H1478	H1485	H1486
Race	Caucasian	Caucasian	Hispanic
Age	43	45	23
Gender	Male	Male	Male
Height (cm)	170	188	163
Weight (kg)	62.9	85.8	39
Time interval between aortic cross-clamp and receipt of liver	23 h 35 min	14 h 34 min	23 h

Binding and Internalization Assay

For the binding and internalization assay of the anti-FcγRIIB antibody (clone 2B6, TAB-036WM, Creative Biolabs, Shirley, NY, U.S.A.), the antibody was labeled with Alexa Fluor 488 by a labeling kit (A10235, Life Technologies, Carlsbad, CA, U.S.A.) according to the manufacturer’s protocol. The NPC (2 × 10⁵ cells/50 μL/well) were incubated with 50 μL of the antibody at concentrations of 0.003–10 μg/mL at 4 °C for 60 min (for binding) and 37 °C for 10 min (for internalization) in a hepatocyte culture medium (CC-3198, LONZA, Basel, Switzerland) containing 2% bovine serum albumin (A9418, Sigma-Aldrich, St. Louis, MO, U.S.A.), followed by washing with phosphate-buffered saline containing fetal bovine serum (FBS, 174012, Nichirei, Tokyo, Japan) three times on ice. To identify the LSEC fraction, cells were stained with an anti-CD31 antibody-VioBlue (100-fold diluted, 130-110-674, Miltenyi Biotec, Bergisch Gladbach, Germany) and an anti-CD45 antibody-APC (100-fold diluted, 130-110-633, Miltenyi), and analyzed by FACS CantoII (Becton Dickinson, Franklin Lakes, NJ, U.S.A.). Approximately 30000 events were recorded by following conditions: flow rate, 3 μL/s; sample volume, 120 μL; washing volume, 200 μL. Alexa Fluor 488-labeled calibration beads (488 A, Bangs Laboratories, Fishers, IN, U.S.A.) were used to quantify the cellular amount of the antibody on each experiment, and the detected fluorescence intensity was converted to the amount of antibody using methodology in a previous report.⁷⁾ One drop of the bead solution was diluted by 400 μL of FBS-containing phosphate-buffered saline (PBS), and 10000 events were measured by same conditions as were used for the cellular samples. The concentration-dependent binding and internalization data was also analyzed with the receptor-ligand binding equation and the Michaelis–Menten equation, as described in a previous report.⁷⁾

RESULTS

Identification of FcγRIIB-Expressing LSEC in Human Liver NPC

Human liver NPC were analyzed by flow cytometry after staining with CD31 and CD45. The representative results of experiments using three donors are shown in Fig. 1. Figure 1a shows a plot of the forward scatter (FSC) and side scatter (SSC). As shown in the rectangular area, the fraction that satisfies the middle to high FSC and middle to low SSC was gated to remove the debris and dead cells. After this area was expanded by CD31 (VioBlue) and CD45 (APC), three populations, referred to P1, P2, and P3, were identified in the stained samples as shown in Fig. 1b, while P2 was not detected in the unstained control sample. The P2 fraction (CD31 high/CD45 low) showed a high FcγRIIB expression (Fig. 1c). On the other hand, the P1 (CD31 low/CD45 high) and the P3 (CD31 low/CD45 low) fractions expressed only minor amounts of FcγRIIB. These data indicate that the P2 population is FcγRIIB-expressing LSEC.

Fig. 1. Gating of the Cell Populations in Human Liver NPC and the Expression of FcγRIIB

(a) Dot plot of FSC and SSC. (b) Dot plot of CD31 (Pacific Blue) and CD45 (APC). CD31 low/CD45 high, CD31 high/CD45 low, and CD31 low/CD45 low population ware referred to as P1, P2, and P3, respectively in the unstained and stained samples. (c) The profile for the expression of FcγRIIB (FITC) in each population. Red indicates an anti-FcγRIIB antibody and blue indicates a negative control antibody. Representative results from experiments using three donors are shown.

Binding and Internalization Assay in Human Liver NPC

A titration assay was performed to confirm whether the fraction P2 can be used to evaluate the binding and uptake of the macromolecules. As described above, an Alexa Fluor 488-labeled anti-FcγRIIB antibody was used as a model macromolecule. To evaluate cellular binding, NPC were incubated with antibodies at 4 °C for 60 min, and the concentration-dependent binding in the P2 fraction was plotted in Fig. 2a. The Anti-FcγRIIB antibody showed saturable binding curve, while the binding of a negative control (mouse immunoglobulin G (IgG)) antibody, whose Fc does not bind to human FcγRIIB, was under the detection limit. The data were analyzed with the receptor-ligand binding equation to estimate the KD and B_max (Table 3). To reveal the cellular internalization, NPC were incubated at 37 °C for 10 min, and the uptake velocity was plotted in Fig. 2b. The Anti-FcγRIIB antibody showed concentration-dependent saturation, while the internalization was, in turn, not found in the case of the negative control antibody. The Michaelis–Menten equation was used to calculate the internalization-related parameters, K_m and V_max (Table 3).

Fig. 2. The Binding and Internalization of the Anti-FcγRIIB Antibody in the P2 Fraction

(a) The concentration-dependent cellular binding of the anti-FcγRIIB antibody. NPC were incubated with the antibody at the concentrations of 0.003–10 μg/mL at 4 °C for 60 min, and after staining with CD31 and CD45, the cells were analyzed by flow cytometer. (b) The concentration-dependent cellular internalization of the anti-FcγRIIB antibody. NPC were incubated with the antibody at concentrations of 0.003–10 μg/mL at 37 °C for 10 min, and the cells were analyzed by flow cytometry. Each point indicates the mean ± standard deviation (S.D.) (n = 3). Information on the three donors is included in Materials and Methods.

Table 3. Parameters for the Binding and Internalization of the Anti-FcγRIIB Antibody

Parameter	Unit	Estimated Value	CV (%)
KD	nM	0.579	16.7
Bmax	pmol/2 × 105 cell	0.189	3.27
Km	nM	0.709	11.2
Vmax	pmol/min/2 × 105 cell	0.0221	2.26

Binding affinity (KD), maximum binding amount (B_max), Michaelis–Menten constant (K_m), and maximum internalization velocity (V_max) were estimated by fitting analysis.

DISCUSSION

In this research, we report on the development of a novel flow cytometry-based assay for evaluating the binding and uptake of an antibody using fresh human liver NPC. The above results suggest that human NPC would be useful for revealing detailed information concerning the binding and internalization of the antibody against FcγRIIB. To expand the application of this method, antibodies against other receptors, and other types of macromolecules such as an immune complex and an oligonucleotide should be studied in future. This human NPC assay is expected to be used for predicting human pharmacokinetic profiles of an antibody, although validation of the in vitro parameters will be needed to confirm the accuracy of prediction. However, further studies will be needed to identify the specific markers to permit the LSEC population to be separated from other populations more cleanly, and to compare the assay using NPC to the one using isolated and/or immortalized LSEC.

In conclusion, we demonstrate herein that the human liver NPC assay using flow cytometry is an attractive tool for evaluating the binding and internalization of macromolecules. Assays using human NPC are expected to be used for revealing binding and internalization for predicting the pharmacokinetics of biologics in drug discovery.

Acknowledgments

We wish to thank Dr. M. S. Feather for his helpful advice in writing the English manuscript.

Conflict of Interest

Y.N. and K.O. are employees of Chugai Pharmaceutical Co., Ltd.

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