High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe

Abstract

Antibody-drug conjugates (ADCs) represent a rapidly growing class of biotherapeutics that deliver drugs specifically to target cells by binding of the antibody component to surface receptors. The majority of ADCs require receptor internalization depending on intrinsic features of the specific ADC-antigen interaction. The development of potent ADCs would greatly benefit from the identification of efficiently internalizing antibodies at early stages of discovery. We developed a highly sensitive and rapid antibody internalization assay using an indirect Cypher5E label. The pH-activated CypHer5E label becomes fluorescent upon internalization into the acidic environment of endocytic organelles, whereas background fluorescence of noninternalized CypHer5E is minimal. The pH-dependency of the CypHer5E signal enables robust discrimination of antibody internalization from surface binding. The favorable signal-over-background ratio allows a homogeneous assay design with high-throughput fluorescence imaging in 384- and 1536-well formats. The biophysical readout of the primary internalization event substantially shortens incubation times compared to killing assays using toxin internalization. The assay was validated with tumor-relevant targets, including receptor tyrosine kinases (EGFR and HER2) and a class II cytokine receptor (TF) expressed by A431, AU565, and SKOV-3 cells and transient expression systems (CHO-S). Our method enables functional screening of large antibody libraries to identify therapeutic antibody candidates with internalization characteristics favorable for the development of ADCs.

Keywords: antibody internalization; antibody-drug conjugates; high-throughput screening; homogenous assay; pH-dependent dye.

Figure 1.

Dose response in direct and indirect CypHer5E antibody internalization assays correlates with target expression. (A) Table summarizing approximate surface copy numbers of EGFR, HER2, and tissue factor (TF) antigens expressed in molecules per cell detected on A431, SKOV-3, and AU565 cells used in this study. Antigen cell surface expression was quantified by flow cytometric QiFi analysis as described in the Materials and Methods section. (B) Direct assay format: cetuximab, HER2-153, and the negative control antibody huChromPure were conjugated with CypHer5E, resulting in the following dye to protein ratios (D/Ps): cetuximab-C5E D/P = 2.3, HER2-153-C5E D/P = 6.6, and huChromPure-C5E D/P = 6.1. A dose titration of the resulting CypHer5E (-C5E) conjugates was incubated with A431 and SKOV-3 cells. (C) Indirect assay format: unconjugated cetuximab, HER2-153, and the negative control antibody huChromPure were incubated with A431 and SKOV-3 cells in the presence of anti–human IgG Fab-CypHer5E conjugate (D/P = 3.8) at a final concentration of 192 ng/mL. The total cell-associated fluorescence (FL) intensities (± SD, n = 8) were measured on the 8200 Cellular Detection System after 6 h of incubation at room temperature. Representative data of two (A) and three (B) independent experiments are shown.

Figure 2.

Antibody-mediated endocytosis of a secondary anti–human IgG Fab-CypHer5E conjugate. (A) Indirect internalization assay performed at room temperature and 4 °C. A431 and SKOV-3 cells were incubated with cetuximab and HER2-153 in the presence of anti–human IgG Fab-CypHer5E conjugate. The total cell-associated fluorescence (FL) intensities (± SD, n = 4) were measured on the 8200 Cellular Detection System after 6 h of incubation. Representative data of n = 3 experiments is shown. (B) Colocalization of LysoTracker Red staining and internalized CypHer5E label. A431 cells were counterstained with the acidotropic LysoTracker Red probe prior to an indirect internalization assay at room temperature with cetuximab (anti–EGFR) in the presence of anti–human IgG Fab-CypHer5E conjugate. Subsequent imaging of live cells was performed with the Celigo Cytometer. I, brightfield image; II, LysoTracker Red (red); III, CypHer5E (green); IV, merge of II and III.

Figure 3.

Antigen and cell type–specific antibody internalization. Indirect internalization assays were performed with antibodies against EGFR, HER2, and TF for A431, SKOV-3, and AU565 cells representing cells with distinct target expression. The target expression is indicated in the left top corner of each particular chart in molecules per cell (SC: surface copy numbers). Cell-associated total fluorescence (FL) intensities (± SD, n = 8) were measured on the 8200 Cellular Detection System after 6 h of incubation at room temperature. The data shown are representative of at least two independent experiments, respectively.

Figure 4.

Antibody internalization by recombinant surface receptors. A431 cells (A) were subjected to dose-response internalization assays along with CHOs-EGFR cells transiently expressing human EGFR (B) and wild-type CHOs cells (C). A431 cells show significant surface expression of EGFR and minimal expression of HER2. A431, CHOs-EGFR, and CHOs cells were incubated with EGFR-specific antibodies (cetuximab, zalutumumab, panitumumab), a HER2-specific antibody (HER2-153), and a negative control antibody (huChromPure) in the presence of anti–human IgG Fab-CypHer5E conjugate. Total fluorescence (FL) intensities measured by the 8200 Cellular Detection System (± SD) were derived from duplicate wells.

Figure 5.

Time-resolved indirect CypHer5E internalization assay. A test panel of eight HER2-specific antibodies and a negative isotype control (huChromPure) were analyzed in an indirect CypHer5E internalization assay with AU565 cells using the 8200 Cellular Detection System macro-confocal scanner. The cellular total fluorescence (FL) intensities were assessed at the incubation times indicated. The average total intensity values of each eight wells are plotted. (A) CypHer5E internalization signals (Total FL) recorded at 0.5, 4.5, and 9.5 h of assay incubation, respectively. It should be noted that the presented graphs use different Y-axis scaling. (B) Increase of observed CypHer5E top fluorescence signals over time.

Figure 6.

Indirect antibody internalization assay in a 384-well and 1536-well format. (A) Summary of assay parameters obtained in a 384-well format with different imaging platforms. Indirect CypHer5E internalization assays were performed with A431, SKOV-3, and AU565 cells with target-specific positive control antibodies indicated. HuChromPure served as negative control antibody. The reactions were analyzed using either the 8200 Cellular Detection System (8200 CDS) or Celigo Cell Cytometer (Celigo). Assay parameters were calculated as described in in the Materials and Methods section based on octuplet assay points. LLOD, lower limit of detection; ULOD, upper limit of detection; S/B, signal-to-background ratio; S/N, signal-to-noise ratio. S/B, S/N, and Z factor were calculated at the ULOD. (B) Positive and negative control antibodies cetuximab, HER2-153, and huChromPure were incubated in either a 384-well format or a 1536-well format with A431 and AU565 cells in dose response in the presence of anti–human IgG Fab-CypHer5E conjugate. The cell-associated total fluorescence (FL) intensities (± SD, n = 8) were measured after 6 h of incubation at room temperature on the 8200 CDS macro confocal scanner.