Introduction
B-cell maturation antigen (BCMA), a cell-surface glycoprotein involved in B-cell maturation and differentiation, is overexpressed on multiple myeloma (MM) cells and associated with cell growth, survival, and immunosuppression (Kleber et al. 2021 ; Tai et al. 2016 ). Due to its wide expression in MM plasma cells, BCMA has emerged as an ideal target for MM therapies, and multiple BCMA-targeting drug classes have been developed, including antibody–drug conjugates (ADCs) (Tai and Anderson 2015 ). Belantamab mafodotin is a BCMA-targeting ADC composed of a humanized anti-BCMA IgG1 monoclonal antibody (mAb) conjugated to the microtubule inhibitor monomethyl auristatin F via a protease-resistant maleimidocaproyl linker (mcMMAF), with a drug-antibody ratio of four (Montes de Oca et al. 2021 ). Upon cell binding, belantamab mafodotin is rapidly internalized and cys-mcMMAF is proteolytically cleaved within endo-lysosomes allowing release of the drug, resulting in cell-cycle arrest and apoptosis; it also enhances antibody-dependent cellular cytotoxicity and phagocytosis (Montes de Oca et al. 2021 ; Tai et al. 2014 ).
Belantamab mafodotin has been evaluated as a monotherapy and in combination regimens in relapsed/refractory MM (RRMM) and is under investigation for other indications (ClinicalTrials.gov 2023 ; ClinicalTrials.gov 2024 ; Dimopoulos et al. 2024 , 2023 ; Hungria et al. 2024 ). Belantamab mafodotin combination regimens have shown significant progression-free survival benefits as early-line treatments for RRMM in phase 3 trials. In DREAMM-7 (belantamab mafodotin with bortezomib and dexamethasone versus daratumumab with bortezomib and dexamethasone), an overall survival benefit was also observed, and in DREAMM-8 (belantamab mafodotin with pomalidomide and dexamethasone [BPd] versus pomalidomide with bortezomib and dexamethasone), a trend for improved overall survival was observed with follow-up ongoing (Dimopoulos et al. 2025 , 2024 ; Hungria et al. 2025 , 2024 ). Based on results from DREAMM-7 and DREAMM-8, these regimens have been approved in multiple countries (GSK 2025 ).
Consistent with other ADCs with MMAF payloads, ocular adverse events associated with belantamab mafodotin have been reported, including corneal events, that require careful drug dose management (Farooq et al. 2020 ; Marquant et al. 2021 ; Popat et al. 2020 ). While the exact mechanism of corneal events associated with ADCs is unknown, a proposed mechanism is nonspecific uptake of the ADC in the basal epithelial layer of the cornea through macropinocytosis, which results in apoptosis and inflammation (Farooq et al. 2020 ; Marquant et al. 2021 ; Popat et al. 2020 ; Zhao et al. 2018 ).
This study investigated the mechanism of uptake and cytotoxicity with belantamab mafodotin in non-BCMA-expressing primary cells, including human corneal epithelial cells (HCEC) and renal proximal tubule cells (RPTEC), to understand the mechanism of toxicity leading to corneal events and to assess potential cytoprotective pathways.
Methods
Antibodies, compounds, and reagents
Belantamab mafodotin (ADC, GSK2857916A) and belantamab (mAb only, GSK2857914A) were obtained from Biopharm Analytical Sciences, GSK. 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), bovine serum albumin (BSA), imipramine, nystatin, phenoxybenzamine, wortmannin, chlorpromazine, levocabastine hydrochloride, desloratadine, fexofenadine hydrochloride, levocetirizine dihydrochloride, and ketotifen fumarate salt were obtained from Sigma. Human immunoglobulin solution (Flebogamma DIF) was obtained from Grifols. SV Total RNA isolation system, Caspase-Glo 3/7 assay was obtained from Promega. High-Capacity cDNA Reverse Transcription Kit was obtained from Applied Biosystems. Vivotag680 label was obtained from PerkinElmer. Alexa488 labelled dextran (10,000 molecular weight [MW] across experiments), Dextran-Cascade Blue, chicken-anti-mouse IgG AlexaFluor488, AlexaFluor488-human Transferrin, Hoechst nuclear stain and all siRNA constructs (AP2mu2 [catalog number 4392420, assay ID S3112], Arf6 [4390824, assay ID S1565], Graf1 [4392420, assay ID S23013], Caveolin-1 [4390824, assay ID S2446], CtBP1 [4390824, assay ID S3699], CDC42 [4392420, assay ID S2765], Flotillin-1 [4392420, assay ID S19915 ], Rabankyrin-5 [4392420, assay ID S28200 ], negative control siRNA [4390843]) were obtained from ThermoFisher. CellTracker and lipofectamine RNAiMAX were obtained from Invitrogen. Opti-MEM was obtained from Gibco. Mouse anti-alpha tubulin was obtained from Santa Cruz (sc-32293). Fluorescence mounting medium was obtained from DAKO Agilent. TaqMan Universal Mastermix was obtained from Applied Biosystems. Primers (assay ID Hs00171292_m1 for BCMA [catalog number 4331182]; Hs02786624_g1 for glyceraldehyde-3-phosphate dehydrogenase [GAPDH; 4331182]) used for PCR target and housekeeping genes were obtained from Applied Biosystems.
Cell lines and cell culture
HCEC, RPTEC, and NCI-H929 cells were obtained as described in Supplement Table S1 and cultured according to supplier’s instructions.
Characterization of HCEC and RPTEC
HCEC, RPTEC, and NCI-H929 (positive control) cells were analyzed for BCMA expression. Briefly, total RNA was extracted using the SV Total RNA isolation system according to the manufacturer’s instructions. Complementary DNA (cDNA) was prepared using the High-Capacity cDNA Reverse Transcription Kit, according to the manufacturer’s instruction. Quantitative polymerase chain reaction (qPCR) was performed for GAPDH (housekeeper control gene) and BCMA using 50 ng cDNA and TaqMan Universal Mastermix on a Quant Studio 7 Flex machine using the following thermal cycling: Step 1, 10 min at 25 °C; Step 2, 120 min at 37 °C; Step 3, 5 min at 85 °C; Step 4, end at 4 °C. Cycle Threshold values were analyzed to determine copy number using the following formula: 10 ([40−C2]/3.5) . Copy numbers were not normalized to GAPDH although their expression confirmed similar cDNA loading into the qPCR wells.
LC–MS/MS payload quantification in HCEC and RPTEC as a function of time
For bulk liquid-chromatography tandem mass spectrometry (LC–MS/MS) detection of ADC uptake and cys-mcMMAF release, HCEC and RPTEC were seeded at 50,000 cells/mL in T75 flasks. Cells were grown for 48 h then incubated with media containing 10 or 100 μg/mL of mAb or ADC, or vehicle control. Unless otherwise specified, cells were cultured in the growth medium noted in Supplement Table S1 at 37 °C/5% CO 2 . Cells were incubated for 1, 2, 4, 6, 12, 24, or 48 h, washed in phosphate buffered saline (PBS), and counted using a Countess (ThermoFisher Scientific); the cells were then pelleted and frozen in dry ice.
Preparation of analytical solutions of cys-mcMMAF, internal standard solution of [15N1 13C3 2H3] cys-mcMMAF, calibration standard, and samples are detailed in the Supplemental Methods (LC–MS/MS payload quantification in HCEC and RPTEC as a function of time subsection) . Ultra-high-performance LC–MS/MS was developed using the Xevo TQ-S. Method conditions are also described in the Supplemental Methods (LC–MS/MS conditions subsection).
ADC uptake by HCEC and RPTEC with or without macropinocytosis inhibitor EIPA
For fluorescent detection of ADC and mAb uptake, HCEC and RPTEC were seeded (0.5 × 10 5 HCEC/well and 1.0 × 10 5 RPTEC/well) in 24-well plates (IBIDI GMBH) compatible for microscopy (catalog number 82426) (Supplemental Methods, “Flow of uptake experiments” subsection). Seeding densities providing 70–80% confluence at treatment were established based on cell-specific proliferation rates. On the day of the assay, cells were incubated with either 10 μg/mL or 100 μg/mL of Vivotag680-labeled mAb or ADC, or vehicle control, in complete growth media for 20 or 44 h at 37 °C/5% CO 2 . Vivotag680-labeled ADC or mAb were then removed from cells, and Hoechst nuclear stain was added for 5 min in complete media. Media was removed and replaced with complete growth media, and fluorescence detected as described in the Supplemental Methods (Microscopy Analyses subsection).
To establish the role of macropinocytosis, cells were treated with EIPA (inhibitor of macropinocytosis) and then mAb and ADC uptake was detected fluorescently. The cytotoxicity of EIPA alone was assessed over a 3 and 48-h period in HCEC and RPTEC cells to identify sub-cytotoxic concentrations for planned mechanistic studies. EIPA (in dimethyl sulfoxide stock, A3085) was prepared in culture medium for a final concentration of 0–50 μM. Following treatment, plates were incubated for 30 min at room temperature (RT) in the dark. Caspase-Glo 3/7 assays were used as indicators of apoptosis, following manufacturer’s instructions and incubation at room temperature for 30 min in the dark. Luminescence was read on a FLUOstar Omega using Omega software, version 3.00 R2 (BMG LabTech). The sub-cytotoxic concentration of EIPA for use in uptake assays was determined to be 12.5 μM.
HCEC and RPTEC were seeded in 24-well plates compatible for microscopy (catalog number 82426) and cultured for 24 h. Cells were then pre-incubated with 12.5 μM EIPA, vehicle control, or media control for 30 min or 3 h. From each well, 800 μL of media was removed (leaving 200 μL to prevent drying) and mixed with Vivotag680-labeled mAb or ADC, or Alexa488-labeled dextran (a 10,000 MW molecule used as a positive control for pinocytosis), to obtain a solution of 100 μg/mL Vivotag680-labeled ADC or mAb, or 200 μg/mL Dextran-Alexa488 ( D22910 ) in presence of EIPA (12.5 µM) or control solution. The remaining 200 μL of media was removed from wells and replaced with 800 μL of the labeled mAb, ADC, or dextran solution. Cells were incubated for 1.5, 3, 4, or 47 h before removing the media and staining with Hoechst nuclear stain for 5 min in complete media. Media was removed and replaced with complete growth media, and fluorescence was detected as described in Supplemental Methods (Microscopy Analyses subsection).
ADC cytotoxic mechanism in HCEC and RPTEC
To assess the impact of ADC treatment on microtubule networks, HCEC were seeded in MatTek live cell dishes and cultured for 24 h. Cells were then treated with 100 µg/mL mAb or ADC, alongside a diluent control, in culture medium for 50 h before being fixed by incubating for 3 min in 100% methanol pre-chilled to −20 °C. For fluorescent antibody labeling of microtubules for confocal microscopy, cells were washed three times in PBS and incubated with blocking buffer (2% FBS, 2% BSA in PBS) for 30 min, then incubated with mouse anti-alpha tubulin diluted 1:2000 in blocking buffer for 1 h. Cells were washed three times in PBS and incubated with chicken-anti-mouse IgG AlexaFluor488 diluted 1:1000 in blocking buffer containing 1 µg/mL Hoechst nuclear stain. Cells were washed three times in PBS, and imaged in PBS using a Leica SP5 laser scanning confocal microscope and an Olympus IX73 widefield microscope.
The potential for ADC to induce apoptosis in HCEC and RPTEC was assessed using the Caspase-Glo 3/7 assay. HCEC or RPTEC were seeded in 96-well plates and cultured for 24 h before being treated with ADC at final concentrations of 0.1, 1, 3, 10, 30, 100, 300, and 577 μg/mL, or with media and vehicle control, in 100 μL of media, for 24, 48, or 72 h (Supplemental Methods, “Flow of caspase 3/7 assay to measure apoptosis” subsection).
The effect of EIPA on ADC cytotoxicity was assessed in RPTEC, and in HCEC in a separate experiment to evaluate a larger number of HCEC donor samples. HCEC or RPTEC were seeded in 96-well plates and cultured for 48 h for HCEC or 24 h for RPTEC (doubling times), then cells were pre-treated with EIPA at a final concentration of 3–12.5 μM for HCEC and 6–25 μM for RPTEC in media, or media control without EIPA. After 30 min, the pre-treatment was removed and replaced with ADC or mAb (1, 5, 10, 25, 50, and 100 μg/mL for HCEC; 10, 30, 50, 75, 150, and 300 μg/mL for RPTEC), in the concentrations of EIPA described previously, and media or vehicle control were added to the wells, for 48 h. Apoptosis was measured using the Caspase-Glo 3/7 assay as previously described.
ADC cytotoxicity and uptake in HCEC and RPTEC with endocytosis inhibitor treatment
The effect of endocytosis inhibitors on ADC uptake and cytotoxicity was assessed in HCEC and RPTEC using fluorescent microscopy. Cells were seeded in the 24-well plates compatible with microscopy (catalog number 82426). The following day, media was replaced with media containing inhibitor alone (wortmannin 400 nM, chlorpromazine 10 μM, nystatin 100 μM, imipramine 10 μM, phenoxybenzamine 10 μM) or untreated control, inhibitor plus dextran-AlexaFluor488 200 μg/mL, inhibitor plus mAb-Vivotag680 XL 100 μg/mL, or uptake inhibitor plus ADC-Vivotag680 XL 100 μg/mL. Co-dosing of uptake inhibitors was also evaluated, with cells treated with uptake inhibitor co-dosing alone (chlorpromazine 5 µM plus nystatin 50 µM plus imipramine 5 µM), uptake inhibitor co-dosing plus dextran-AlexaFluor488 200 μg/mL, mAb-Vivotag680 XL 100 μg/mL, or ADC-Vivotag680 XL 100 μg/mL. After a 48-h incubation, the media was replaced with fresh media containing Hoechst nuclear stain for 5 min. Media was replaced with growth media containing CellTracker, a fluorescent probe designed to freely pass through cell membranes, which was left for incubation during imaging. Fluorescence was detected as described in Supplemental Methods (Microscopy Analyses subsection).
To further evaluate ADC uptake in HCEC, key endocytic pathway proteins were depleted by siRNA, with uptake evaluated by flow cytometry. Methods for this assay are described in the Supplemental Methods (Effect of siRNA depletion of endocytic pathway proteins on ADC uptake subsection).
mAb and ADC uptake and cytotoxicity in HCEC and RPTEC treated with H1 inhibitor or human immunoglobulin solution
The effect of commercially available inhibitors of the histamine-1 (H1) receptor (expressed on corneal cells, which activates protein kinase C to induce macropinocytosis), as well as treatment consistent with human immunoglobulin solution, on the uptake of ADC by HCEC and RPTEC cells were evaluated via fluorescence microscopy. Cells were seeded in the 24-well plates compatible with microscopy (catalog number 82426). The following day, the media was exchanged with media containing H1 inhibitor alone (levocabastine 1.8 μM, levocetirizine 1.2 μM, desloratadine 2.2 μM, fexofenadine 34.1 μM, or ketotifen 0.52 μM), H1 inhibitor plus mAb-Vivotag680 XL 100 μg/mL, H1 inhibitor plus ADC-Vivotag680 XL 10 μg/mL or 100 μg/mL. The H1 inhibitor concentrations differed between the fluorescent microscopy and apoptosis endpoints to provide maximum opportunity for a measurable response for each endpoint; the highest concentrations without causing cytotoxicity were chosen. To assess human immunoglobulin solution effects, the day after cell seeding, the media was exchanged with media containing human immunoglobulin solution alone (8, 15, or 20 mg/mL) or untreated control, human immunoglobulin solution plus mAb-Vivotag680 XL 100 μg/mL, human immunoglobulin solution plus ADC-Vivotag680 XL 10 μg/mL or 100 μg/mL. After a 48-h incubation, media was replaced with fresh media containing Hoechst nuclear stain (1:5000) for 5 min. Media was replaced with growth media containing CellTracker (1:2000), which was left for incubation during imaging. Fluorescence was detected as described in Supplemental Methods (Microscopy Analyses subsection).
The effect of H1 inhibitors on caspase 3/7 in ADC-treated HCEC was evaluated. HCEC were seeded into 96-well plates and cultured for 48 h before being pre-treated with H1 inhibitors at sub-cytotoxic concentrations (levocabastine 180 μM, levocetirizine 150 μM, desloratadine 2.2 μM, fexofenadine 341 μM, ketotifen 52 μM) or untreated control for 30 min. The pre-treatment was replaced with H1 inhibitor plus ADC at 1, 5, 10, 25, 50, or 100 μg/mL for 48 h. Apoptosis was measured using the Caspase-Glo 3/7 assay.
The effect of human immunoglobulin solution treatment on apoptosis in ADC-treated HCEC cells was evaluated. HCEC cells were seeded into 96-well plates and cultured for 48 h before being pre-treated with human immunoglobulin solution (1, 4, 8, 15, or 20 mg/mL) or untreated control for 3 h. The pre-treatment was replaced with human immunoglobulin solution (at the same doses) plus ADC at 1, 5, 10, 25, 50, or 100 μg/mL for 48 h. Apoptosis was measured using the Caspase-Glo 3/7 assay.
Statistical analysis
All analyses employed the statistical programming language R, with RStudio. Additional details on statistical analysis are described in the Supplemental Methods (Statistical analysis subsection).
Results
BCMA expression was absent or negligible in HCEC and RPTEC
Compared with NCI-H929 cells, HCEC and RPTEC did not express meaningful levels of BCMA mRNA (Supplement Table S2 ), confirming that these cells were appropriate for evaluation of non-specific ADC uptake.
Characterization of ADC uptake and cytotoxicity
Uptake of ADC into HCEC and RPTEC was concentration and time-dependent
We evaluated uptake of Vivotag680-labeled ADC and mAb, both at 10 μg/mL and 100 μg/mL after 20 and 44 h of incubation (Fig. 1 ). In HCEC, uptake of ADC and mAb increased with concentration and time. Uptake of ADC and mAb was generally similar across donors at 20 h, but more variable at 44 h. There was a significant increase in ADC uptake from 10 μg/mL to 100 μg/mL at 20 h (+ 27.3%; p = 0.0054), with a more marked uptake at 44 h (+ 41.7%, p = 0.0002). Similar findings were observed for mAb, with increases in uptake from 10 μg/mL to 100 μg/mL at 20 h (+ 49.3%; p = 0.0001) and 44 h (+ 66.2%, p < 0.0001) (Supplement Table S3 and Supplement Fig. S1 ). ADC 100 μg/mL uptake was significantly lower than uptake of mAb 100 μg/mL at both 20 and 44 h (−27.9%, p = 0.0004 at 20 h; −18.9%, p = 0.0135 at 44 h). Fig. 1 Uptake of mAb and ADC by HCEC and RPTEC ( a ) and their effect on nuclei counts ( b ). HCEC and RPTEC were treated with Vivotag680-labeled ADC and mAb (10 or 100 µg/mL) for 20 and 44 h. Uptake was evaluated by compound intensity fluorescence. See Supplement Table S3 (uptake) and Supplement Table S4 (nuclei counts) for statistical comparisons. ADC, antibody–drug conjugate; Ctrl, control; hrs, hours; HCEC, human corneal epithelial cells; mAb, monoclonal antibody; min, minutes; RPTEC, renal proximal tubule epithelial cells
For RPTEC, ADC uptake significantly increased from 10 μg/mL to 100 μg/mL at 20 h (+ 30.2%, p = 0.0007) and 44 h (+ 27.1%, p = 0.0016), as did mAb uptake at 20 h (+ 78.4%, p < 0.0001) and 44 h (+ 97.0%, p < 0.0001). Uptake of ADC 100 μg/mL was significantly lower versus mAb 100 μg/mL at both time points (−35.0% at 20 h, p < 0.0001; −42.2% at 44 h, p < 0.0001) (Supplement Table S3 ).
LC–MS confirmed release of cys-mcMMAF with microtubule inhibition consistent with auristatin mechanism of action, and imaging showed ADC colocalization into lysosomes
LC–MS confirmed uptake of ADC 10 and 100 μg/mL and release of cys-mcMMAF in HCEC and RPTEC, with greater amounts of intracellular cys-mcMMAF in live HCEC versus RPTEC. At the final time point of 48 h, the HCEC concentration was on average 21.30 fg/cell for ADC 10 μg/mL (by donor: 26.00, 29.70, 8.21) and 102.72 fg/cell for ADC 100 μg/mL (by donor: 50.67, 153.63, and 103.87 fg/cell); RPTEC concentration was on average 10.99 fg/cell for ADC 10 μg/mL (by donor: 7.13, 4.49, 21.36) and 36.91 fg/cell for ADC 100 μg/mL (by donor: 22.14, 21.62, and 66.98 fg/cell). The intracellular concentration of cys-mcMMAF varied with time and ADC dose in both HCEC and RPTEC (Fig. 2 ), which is likely to be associated with donor variability. Fig. 2 LC–MS/MS quantification of cys-mcMMAF in HCEC ( a ) and RPTEC (donors) ( b ) treated with ADC 10 μg/mL and 100 μg/mL. ADC, antibody–drug conjugate; HCEC, human corneal epithelial cells; LC–MS/MS, liquid-chromatography tandem mass spectrometry; RPTEC, renal proximal tubule epithelial cells
Imaging with confocal microscopy showed that HCEC incubated with Vivotag680-labeled ADC 10 μg/mL for 24 h exhibited intracellular colocalization of ADC within a proportion of lysosomes (HCEC shown in Fig. 3 ). Manders colocalization coefficients further support colocalization into lysosomes, and the Pearson correlation coefficient indicated a moderate positive correlation between ADC and lysotracker signals (Supplement Fig. S2 ). Confocal imaging of HCEC after 50 h of incubation with 100 μg/mL of ADC demonstrated a breakdown and loss of organized microtubules (consistent with apoptosis), in contrast with untreated control cells and cells treated with 100 μg/mL mAb, which maintained dense microtubule networks (Fig. 4 ). Fig. 3 HCEC incubated with ADC exhibited co-localization of ADC within the lysosomes. HCEC were incubated with Vivotag680-labeled ADC for 24 h at 10 μg/mL. The lysosomes were labeled with lysotracker green, the nucleus is represented by the blue stain Hoechst, the treatment is in magenta, and the white depicts the co-localization between the compound and the lysosomes. ADC, antibody–drug conjugate; HCEC, human corneal epithelial cells Fig. 4 Microtubule stability in HCEC following live time course incubation with ADC, mAb, or untreated control. HCEC were incubated for 50 h with 100 µg/mL mAb, ADC, or left untreated (diluent control), fixed in methanol and labeled for alpha tubulin by immunofluorescence (green) and counterstained nucleus (blue). Confocal scale bars = 20 μm; a widefield image is displayed to demonstrate the effect on the wider cell population. Widefield scale bars = 100 μm. ADC, antibody–drug conjugate; HCEC, human corneal epithelial cells; mAb, monoclonal antibody
Cys-mcMMAF leads to increased caspase 3/7, likely through microtubule inhibition
The effect of ADC on caspase 3/7 activity in HCEC and RPTEC was explored over a range of ADC concentrations (0.1 to 577 μg/mL). In HCEC, caspase 3/7 was significantly increased with ADC ≥ 30 μg/mL (at 48 h [HCEC doubling time]; p = 0.003 at 30 μg/mL) and ≥ 100 μg/mL (at 48 [ p < 0.001] and 72 h [ p = 0.013]) compared with vehicle control (Supplement Fig. S3 ). The ADC minimum effective concentration (MEC) at 48 h was 54.4 μg/mL (95% CI 16.0–202.0). In RPTEC, caspase 3/7 was evaluated at 24 and 48 h based on a 24-h doubling time. At 24 h (1 donor), there was a trend for increased caspase 3/7 in RPTEC with ADC concentrations ≥ 300 μg/mL, which became significant at the 577 μg/mL concentration ( p = 0.001). Caspase 3/7 was significantly increased with ADC ≥ 100 μg/mL at 48 h ( p < 0.001 at 100 μg/mL) compared with vehicle control, and the MEC was 70.7 μg/mL (95% CI 32.2–100.3). The caspase-Glo 3/7 assay results were interpreted as evidence of apoptosis.
For cytotoxicity by nuclei count, there was a significant reduction in HCEC nuclei count with both ADC concentrations compared with untreated control. There was a larger drop in HCEC nuclei count for ADC 100 μg/mL at 20 h (−23.3% with ADC 10 μg/mL, p = 0.0026; −49.6% with ADC 100 μg/mL, p < 0.0001) and at 44 h (−50.9% with ADC 10 μg/mL, p < 0.0001; −75.7% with ADC 100 μg/mL, p < 0.0001) (Supplement Table S4 and Supplement Fig. S1 ). There was no significant reduction in nuclei count for HCEC incubated with mAb 10 μg/mL or 100 μg/mL compared with untreated control at 20 and 44 h. HCEC nuclei count was significantly reduced for ADC versus mAb for both doses at 20 and 44 h.
For cytotoxicity by nuclei count, RPTEC nuclei counts were significantly decreased with ADC 100 μg/mL versus untreated control at 20 h and, to a greater extent, at 44 h, and ADC 100 μg/mL significantly reduced nuclei counts versus mAb 100 μg/mL at both time points (Supplement Table S4 ).
HCECs exhibited greater ADC uptake and reduction in nuclei counts than RPTECs
In comparing results from HCEC and RPTEC by ADC concentrations and time points (Supplement Table S5 ), HCEC exhibited significantly greater uptake of ADC 100 μg/mL than RPTEC after 44 h incubation (+ 40.41, p = 0.0049). There were significantly greater reductions in HCEC nuclei counts than RPTEC nuclei counts when treated with ADC 10 μg/mL or 100 μg/mL at 20 h (−28.6% for 10 μg/mL, p = 0.0112; −44.6% for 100 μg/mL, p < 0.0001) and 44 h (−49.3% for 10 μg/mL, p < 0.0001; −62.3% for 100 μg/mL, p < 0.0001), while there were no significant differences in nuclei counts between HCEC and RPTEC when treated with mAb 10 μg/mL or 100 μg/mL.
Synopsis
Uptake of ADC was concentration- and time-dependent and higher in HCEC versus RPTEC, with evidence of localization within lysosomes. The ability of HCEC and RPTEC to catabolize and cleave ADC to release cys-mcMMAF was confirmed via LC–MS/MS. In HCEC, apoptosis and cytotoxicity likely occurred through extensive microtubule inhibition as a consequence of cys-mcMMAF. HCEC appeared more sensitive to ADC than RPTEC based on nuclei count and apoptosis assays.
Role of macropinocytosis in non-specific ADC uptake
EIPA at 12.5 μM significantly decreased ADC uptake in HCEC, with a modest, non-significant increase in nuclei counts versus ADC alone
The potential role of non-specific ADC uptake by macropinocytosis was further explored in HCEC and RPTEC using the sub-cytotoxic EIPA concentration of 12.5 μM (Supplement Fig. S4 ). Pre-treatments (30 min and 3 h) with EIPA 12.5 μM were followed by co-treatment with ADC, mAb, or dextran for 47 h. In HCEC, EIPA significantly reduced ADC uptake versus ADC 100 μg/mL alone (−19.4% with 30-min pre-treatment, p = 0.0345; −23.0% with 3-h pre-treatment, p = 0.0112) (Table 1 and Supplement Fig. S5 ). When combined with ADC, EIPA the reduction in nuclei counts was less compared to ADC alone, suggesting a potential protective effect, despite the lack of statistical significance (+ 237.9 cells with 30-min pre-treatment, p = 0.7288; + 91.5 cells with 3-h pre-treatment, p = 0.8939) (Table 1 and Supplement Fig. S6 ). For HCEC incubated with mAb 100 μg/mL or dextran 200 μg/mL, there was no significant change in the uptake of these molecules with EIPA. Change in nuclei counts were not significantly different with mAb or dextran alone compared with untreated control, but the addition of EIPA resulted in significant reductions versus mAb and dextran alone, respectively. In RPTEC, there was no significant change in the uptake of ADC, mAb, or dextran with the addition of EIPA, and no significant changes in nuclei counts with any of the treatments alone or with EIPA. Table 1 Effect of EIPA on uptake of mAb, ADC, and dextran in HCEC and RPTEC and nuclei counts HCEC RPTEC Uptake a % change 95% CI p -value % change 95% CI p -value mAb 100 µg/mL ± EIPA 12.5 µM mAb + DMSO vs mAb −12.74 −28.50 6.48 0.1751 2.82 −17.41 27.99 0.7893 mAb + EIPA 30min vs mAb −6.66 −23.51 13.91 0.4901 5.02 −15.64 30.73 0.6388 mAb + EIPA 3h vs mAb −10.63 −26.77 9.06 0.2622 4.63 −15.95 30.25 0.6640 ADC 100 µg/mL ± EIPA 12.5 µM ADC + DMSO vs ADC −5.90 −22.89 14.83 0.5420 5.61 −15.16 31.47 0.6010 ADC + EIPA 30min vs ADC −19.39 −33.94 −1.62 0.0345 2.33 −17.80 27.38 0.8249 ADC + EIPA 3h vs ADC −23.00 −36.90 −6.03 0.0112 3.48 −16.87 28.82 0.7422 Dextran 200 µg/mL ± EIPA 12.5 µM Dextran + DMSO vs dextran −3.72 −18.46 13.67 0.6402 −4.48 −22.20 17.26 0.6216 Dextran + EIPA 30min vs dextran 5.41 −10.72 24.45 0.5176 −0.93 −19.30 21.62 0.9192 Dextran + EIPA 3h vs dextran 2.18 −13.45 20.64 0.7900 −0.18 −18.69 22.55 0.9847 Nuclei counts b Change in count 95% CI p -value Change in count 95% CI p -value mAb 100 µg/mL ± EIPA 12.5 µM mAb ctrl : mAb vs untreated control 503.1 −793.0 1799.1 0.4368 −404.6 −1267.1 457.9 0.3477 mAb + DMSO vs mAb ctrl −182.5 −1538.1 1173.0 0.7902 −278.0 −1282.2 726.2 0.5833 mAb + EIPA 30min vs mAb ctrl −3773.7 −5129.2 −2418.1 < 0.0001 −251.7 −1255.9 752.5 0.6194 mAb + EIPA 3h vs mAb ctrl −3838.0 −5193.5 −2482.5 < 0.0001 −481.2 −1485.4 523.0 0.3432 ADC 100 µg/mL ± EIPA 12.5 µM ADC ctrl : ADC vs untreated control −5426.6 −6722.6 −4130.5 < 0.0001 −800.6 −1663.1 61.9 0.0679 ADC + DMSO vs ADC ctrl 403.0 −952.5 1758.5 0.5572 586.7 −417.5 1590.9 0.2485 ADC + EIPA 30min vs ADC ctrl 237.9 −1117.6 1593.5 0.7288 272.8 −731.4 1277.0 0.5904 ADC + EIPA 3h vs ADC ctrl 91.5 −1264.0 1447.1 0.8939 756.8 −247.4 1761.0 0.1377 Dextran 200 µg/mL ± EIPA 12.5 µM Dextran ctrl : dextran vs untreated control 980.4 −132.5 2093.3 0.0808 −411.7 −1114.2 290.8 0.2292 Dextran + DMSO vs dextran ctrl 217.0 −782.9 1217.0 0.6639 475.8 −252.7 1204.3 0.1924 Dextran + EIPA 30min vs dextran ctrl −3929.7 −4929.6 −2929.8 < 0.0001 −3.4 −731.9 725.0 0.9924 Dextran + EIPA 3h vs dextran ctrl −3538.0 −4537.9 −2538.1 < 0.0001 147.2 −581.3 875.7 0.6829 a Uptake at 47 h based on estimated changes in compound area fluorescence of Vivotag680-labeled mAb, ADC, or Alexa488-labeled dextran with or without EIPA co-treatment; additional data available in Supplement Fig. S5 b Estimated differences in nuclei counts of HCEC and RPTEC at 47 h; treatment with Vivotag680-labeled mAb, ADC, or Alexa488-labeled dextran with or without EIPA co-treatment; additional data available in Supplement Fig. S6 HCEC and RPTEC were pre-treated with EIPA for 30 min or 3 h; p -values for the test of no change relative to baseline ADC, antibody–drug conjugate; CI, confidence interval; Ctrl, control; DMSO, dimethyl sulfoxide; EIPA, 5-(N-Ethyl-N-isopropyl)amiloride; h, hours; HCEC, human corneal epithelial cells; mAb, monoclonal antibody; min, minutes; RPTEC, renal proximal tubule epithelial cells
Table 1
Effect of EIPA on uptake of mAb, ADC, and dextran in HCEC and RPTEC and nuclei counts
a Uptake at 47 h based on estimated changes in compound area fluorescence of Vivotag680-labeled mAb, ADC, or Alexa488-labeled dextran with or without EIPA co-treatment; additional data available in Supplement Fig. S5 b Estimated differences in nuclei counts of HCEC and RPTEC at 47 h; treatment with Vivotag680-labeled mAb, ADC, or Alexa488-labeled dextran with or without EIPA co-treatment; additional data available in Supplement Fig. S6 HCEC and RPTEC were pre-treated with EIPA for 30 min or 3 h; p -values for the test of no change relative to baseline ADC, antibody–drug conjugate; CI, confidence interval; Ctrl, control; DMSO, dimethyl sulfoxide; EIPA, 5-(N-Ethyl-N-isopropyl)amiloride; h, hours; HCEC, human corneal epithelial cells; mAb, monoclonal antibody; min, minutes; RPTEC, renal proximal tubule epithelial cells
| HCEC | RPTEC | |||||||
| Uptake a | % change | 95% CI | p -value | % change | 95% CI | p -value | ||
| mAb 100 µg/mL ± EIPA 12.5 µM | ||||||||
| mAb + DMSO vs mAb | −12.74 | −28.50 | 6.48 | 0.1751 | 2.82 | −17.41 | 27.99 | 0.7893 |
| mAb + EIPA 30min vs mAb | −6.66 | −23.51 | 13.91 | 0.4901 | 5.02 | −15.64 | 30.73 | 0.6388 |
| mAb + EIPA 3h vs mAb | −10.63 | −26.77 | 9.06 | 0.2622 | 4.63 | −15.95 | 30.25 | 0.6640 |
| ADC 100 µg/mL ± EIPA 12.5 µM | ||||||||
| ADC + DMSO vs ADC | −5.90 | −22.89 | 14.83 | 0.5420 | 5.61 | −15.16 | 31.47 | 0.6010 |
| ADC + EIPA 30min vs ADC | −19.39 | −33.94 | −1.62 | 0.0345 | 2.33 | −17.80 | 27.38 | 0.8249 |
| ADC + EIPA 3h vs ADC | −23.00 | −36.90 | −6.03 | 0.0112 | 3.48 | −16.87 | 28.82 | 0.7422 |
| Dextran 200 µg/mL ± EIPA 12.5 µM | ||||||||
| Dextran + DMSO vs dextran | −3.72 | −18.46 | 13.67 | 0.6402 | −4.48 | −22.20 | 17.26 | 0.6216 |
| Dextran + EIPA 30min vs dextran | 5.41 | −10.72 | 24.45 | 0.5176 | −0.93 | −19.30 | 21.62 | 0.9192 |
| Dextran + EIPA 3h vs dextran | 2.18 | −13.45 | 20.64 | 0.7900 | −0.18 | −18.69 | 22.55 | 0.9847 |
| Nuclei counts b | Change in count | 95% CI | p -value | Change in count | 95% CI | p -value | ||
| mAb 100 µg/mL ± EIPA 12.5 µM | ||||||||
| mAb ctrl : mAb vs untreated control | 503.1 | −793.0 | 1799.1 | 0.4368 | −404.6 | −1267.1 | 457.9 | 0.3477 |
| mAb + DMSO vs mAb ctrl | −182.5 | −1538.1 | 1173.0 | 0.7902 | −278.0 | −1282.2 | 726.2 | 0.5833 |
| mAb + EIPA 30min vs mAb ctrl | −3773.7 | −5129.2 | −2418.1 | < 0.0001 | −251.7 | −1255.9 | 752.5 | 0.6194 |
| mAb + EIPA 3h vs mAb ctrl | −3838.0 | −5193.5 | −2482.5 | < 0.0001 | −481.2 | −1485.4 | 523.0 | 0.3432 |
| ADC 100 µg/mL ± EIPA 12.5 µM | ||||||||
| ADC ctrl : ADC vs untreated control | −5426.6 | −6722.6 | −4130.5 | < 0.0001 | −800.6 | −1663.1 | 61.9 | 0.0679 |
| ADC + DMSO vs ADC ctrl | 403.0 | −952.5 | 1758.5 | 0.5572 | 586.7 | −417.5 | 1590.9 | 0.2485 |
| ADC + EIPA 30min vs ADC ctrl | 237.9 | −1117.6 | 1593.5 | 0.7288 | 272.8 | −731.4 | 1277.0 | 0.5904 |
| ADC + EIPA 3h vs ADC ctrl | 91.5 | −1264.0 | 1447.1 | 0.8939 | 756.8 | −247.4 | 1761.0 | 0.1377 |
| Dextran 200 µg/mL ± EIPA 12.5 µM | ||||||||
| Dextran ctrl : dextran vs untreated control | 980.4 | −132.5 | 2093.3 | 0.0808 | −411.7 | −1114.2 | 290.8 | 0.2292 |
| Dextran + DMSO vs dextran ctrl | 217.0 | −782.9 | 1217.0 | 0.6639 | 475.8 | −252.7 | 1204.3 | 0.1924 |
| Dextran + EIPA 30min vs dextran ctrl | −3929.7 | −4929.6 | −2929.8 | < 0.0001 | −3.4 | −731.9 | 725.0 | 0.9924 |
| Dextran + EIPA 3h vs dextran ctrl | −3538.0 | −4537.9 | −2538.1 | < 0.0001 | 147.2 | −581.3 | 875.7 | 0.6829 |
EIPA administration reduced ADC-mediated caspase 3/7 activity
To further assess the potential role of macropinocytosis in ADC cytotoxicity, caspase 3/7 activity was evaluated with EIPA co-treatment. Cells were pre- and co-treated with EIPA (range 3–12.5 μM in HCEC and 6–25 μg/mL in RPTEC) and ADC or mAb (range 1–100 μg/mL in HCEC and 10–300 μg/mL in RPTEC); concentrations were based on previous cytotoxicity findings. A statistically significant decrease in HCEC caspase 3/7 activity was observed with ADC 10 μg/mL with EIPA 12.5 μM compared with vehicle control with significant decreases also observed at higher ADC doses; in general, more robust decreases were observed with ADC 25–100 μg/mL and EIPA 3–12.5 μM (Fig. 5 ). Treatment with mAb with or without EIPA did not affect caspase 3/7 activity at any tested concentration in HCEC or RPTEC. Fig. 5 Effect of EIPA on ADC-mediated apoptosis in HCEC (a) and RPTEC (b) . HCEC and RPTEC were pre-treated with EIPA for 30 min and then co-treated with ADC plus EIPA or EIPA alone (control). Concentrations were 3–12 µM for EIPA and 1–100 µg for ADC. Apoptosis was measured using the Caspase 3/7 apoptosis assay (a luminescence test) after a 48-h co-treatment incubation period. EIPA effects were normalized for HCECs (i.e., dose response observed with EIPA only) but not for RPTEC (no dose response observed due to EIPA not affecting caspase endpoint in RPTEC), this is reflected in the y-axis labeling. Error bars represent 95% confidence intervals. Relative effect values below 1.0 indicate a reduction in the apoptosis marker. ADC, antibody–drug conjugate; EIPA, 5-(N-Ethyl-N-isopropyl)amiloride; h, hours; HCEC, human corneal epithelial cells; mAb, monoclonal antibody; RPTEC, renal proximal tubule epithelial cells
Synopsis
ADC and EIPA co-treatment led to moderate reduction in ADC uptake and less caspase 3/7 activity interpreted as reduced apoptosis. This indicates a role for macropinocytosis in the non-specific uptake of ADC. However, the modest reduction in uptake suggests involvement of other pathways in ADC uptake.
Multiple endocytic mechanisms may be involved in ADC uptake in HCEC and RPTEC
Co-treatment of ADC with nystatin or with chlorpromazine reduced ADC uptake in HCEC, and nystatin reduced cytotoxicity, while multiple endocytic inhibitors reduced uptake in RPTEC, but none reduced cytotoxicity
Given the moderate effects with EIPA, the potential role of endocytic pathways other than macropinocytosis was assessed using co-treatment of ADC, mAb, or dextran with endocytosis inhibitors (chlorpromazine [inhibits clathrin-mediated endocytosis], nystatin [inhibits caveolin-mediated endocytosis], or wortmannin, imipramine, and phenoxybenzamine [macropinocytosis inhibitors]) for 48 h. In HCEC, co-treatment of ADC 100 μg/mL with nystatin or with chlorpromazine significantly reduced ADC uptake by −18.5% and −20.4%, respectively, ( p = 0.0441 and 0.0202) compared with ADC alone (Table 2 ), but nystatin was the only compound to significantly reduce ADC cytotoxicity, with a + 109.8% greater nuclei count than ADC alone ( p = 0.0352) (Supplement Table S6 ). None of the compounds significantly affected mAb 100 μg/mL uptake or nuclei counts in HCEC. In RPTEC, intracellular ADC 100 μg/mL and mAb 100 μg/mL uptake were significantly reduced by all uptake inhibitors apart from wortmannin; none of the compounds had a significant effect on nuclei counts for RPTEC treated with ADC or mAb. Table 2 Effect of endocytosis inhibitors on uptake of mAb, ADC, and dextran in HCEC and RPTEC HCEC RPTEC % change 95% CI p -value % change 95% CI p -value mAb 100 µg/mL ± inhibitor mAb + wortmannin vs mAb −4.6 −21.9 16.6 0.9193 −26.8 −49.3 5.7 0.0976 mAb + chlorpromazine vs mAb −11.8 −27.8 7.7 0.3458 −40.1 −58.5 −13.5 0.0094 mAb + nystatin vs mAb −1.9 −19.7 19.9 0.9924 −51.5 −66.4 −29.9 0.0012 mAb + imipramine vs mAb 3.9 −15.0 26.9 0.9519 −41.8 −59.7 −16.0 0.0069 mAb + phenoxybenzamine vs mAb 10.4 −9.6 34.9 0.5538 −39.4 −58.0 −12.5 0.0108 mAb + co-dosing vs mAb −19.6 −28.1 −10.1 0.0008 −40.7 −56.4 −19.5 0.0196 ADC 100 µg/mL ± inhibitor ADC + wortmannin vs ADC −10.8 −27.0 8.9 0.4293 −26.8 −49.3 5.7 0.0975 ADC + chlorpromazine vs ADC −20.4 −34.8 −2.7 0.0202 −33.5 −53.9 −3.9 0.0310 ADC + nystatin vs ADC −18.5 −33.3 −0.4 0.0441 −49.2 −64.9 −26.7 0.0018 ADC + imipramine vs ADC −8.0 −24.7 12.4 0.6842 −35.7 −55.5 −7.2 0.0208 ADC + phenoxybenzamine vs ADC −8.2 −24.9 12.1 0.6652 −34.9 −54.9 −6.0 0.0239 ADC + co-dosing vs ADC −30.7 −38.1 −22.6 < 0.0001 −49.3 −62.7 −31.1 0.0123 Dextran 200 µg/mL ± inhibitor Dextran + wortmannin vs dextran −6.7 −23.6 14.0 0.7890 −20.8 −45.2 14.4 0.2500 Dextran + chlorpromazine vs dextran −14.3 −29.9 4.7 0.1829 −17.6 −43.0 19.0 0.3837 Dextran + nystatin vs dextran 10.6 −9.4 35.2 0.5386 −53.6 −67.9 −33.0 0.0008 Dextran + imipramine vs dextran 3.2 −15.6 26.1 0.9715 −0.6 −31.2 43.5 0.9999 Dextran + phenoxybenzamine vs dextran 1.1 −17.3 23.5 0.9983 4.9 −27.3 51.5 0.9685 Dextran + co-dosing vs dextran −1.6 −12.0 10 0.7582 −27.2 −46.4 −1.1 0.0471 Uptake at 48 h based on estimated changes in compound intensity fluorescence of Vivotag680-labeled mAb, ADC, or Alexa488-labeled dextran at 48 h with or without co-treatment with endocytosis inhibitors; p -values for the test of no change relative to baseline Inhibitor concentrations: wortmannin 400 nM, chlorpromazine 10 μM, nystatin 100 μM, imipramine 10 μM, phenoxybenzamine 10 μM Co-dosing with chlorpromazine 5 µM, nystatin 50 µM, and imipramine 5 µM ADC, antibody–drug conjugate; CI, confidence interval; HCEC, human corneal epithelial cells; mAb, monoclonal antibody; min, minutes; RPTEC, renal proximal tubule epithelial cells
Table 2
Effect of endocytosis inhibitors on uptake of mAb, ADC, and dextran in HCEC and RPTEC
Uptake at 48 h based on estimated changes in compound intensity fluorescence of Vivotag680-labeled mAb, ADC, or Alexa488-labeled dextran at 48 h with or without co-treatment with endocytosis inhibitors; p -values for the test of no change relative to baseline Inhibitor concentrations: wortmannin 400 nM, chlorpromazine 10 μM, nystatin 100 μM, imipramine 10 μM, phenoxybenzamine 10 μM Co-dosing with chlorpromazine 5 µM, nystatin 50 µM, and imipramine 5 µM ADC, antibody–drug conjugate; CI, confidence interval; HCEC, human corneal epithelial cells; mAb, monoclonal antibody; min, minutes; RPTEC, renal proximal tubule epithelial cells
| HCEC | RPTEC | |||||||
| % change | 95% CI | p -value | % change | 95% CI | p -value | |||
| mAb 100 µg/mL ± inhibitor | ||||||||
| mAb + wortmannin vs mAb | −4.6 | −21.9 | 16.6 | 0.9193 | −26.8 | −49.3 | 5.7 | 0.0976 |
| mAb + chlorpromazine vs mAb | −11.8 | −27.8 | 7.7 | 0.3458 | −40.1 | −58.5 | −13.5 | 0.0094 |
| mAb + nystatin vs mAb | −1.9 | −19.7 | 19.9 | 0.9924 | −51.5 | −66.4 | −29.9 | 0.0012 |
| mAb + imipramine vs mAb | 3.9 | −15.0 | 26.9 | 0.9519 | −41.8 | −59.7 | −16.0 | 0.0069 |
| mAb + phenoxybenzamine vs mAb | 10.4 | −9.6 | 34.9 | 0.5538 | −39.4 | −58.0 | −12.5 | 0.0108 |
| mAb + co-dosing vs mAb | −19.6 | −28.1 | −10.1 | 0.0008 | −40.7 | −56.4 | −19.5 | 0.0196 |
| ADC 100 µg/mL ± inhibitor | ||||||||
| ADC + wortmannin vs ADC | −10.8 | −27.0 | 8.9 | 0.4293 | −26.8 | −49.3 | 5.7 | 0.0975 |
| ADC + chlorpromazine vs ADC | −20.4 | −34.8 | −2.7 | 0.0202 | −33.5 | −53.9 | −3.9 | 0.0310 |
| ADC + nystatin vs ADC | −18.5 | −33.3 | −0.4 | 0.0441 | −49.2 | −64.9 | −26.7 | 0.0018 |
| ADC + imipramine vs ADC | −8.0 | −24.7 | 12.4 | 0.6842 | −35.7 | −55.5 | −7.2 | 0.0208 |
| ADC + phenoxybenzamine vs ADC | −8.2 | −24.9 | 12.1 | 0.6652 | −34.9 | −54.9 | −6.0 | 0.0239 |
| ADC + co-dosing vs ADC | −30.7 | −38.1 | −22.6 | < 0.0001 | −49.3 | −62.7 | −31.1 | 0.0123 |
| Dextran 200 µg/mL ± inhibitor | ||||||||
| Dextran + wortmannin vs dextran | −6.7 | −23.6 | 14.0 | 0.7890 | −20.8 | −45.2 | 14.4 | 0.2500 |
| Dextran + chlorpromazine vs dextran | −14.3 | −29.9 | 4.7 | 0.1829 | −17.6 | −43.0 | 19.0 | 0.3837 |
| Dextran + nystatin vs dextran | 10.6 | −9.4 | 35.2 | 0.5386 | −53.6 | −67.9 | −33.0 | 0.0008 |
| Dextran + imipramine vs dextran | 3.2 | −15.6 | 26.1 | 0.9715 | −0.6 | −31.2 | 43.5 | 0.9999 |
| Dextran + phenoxybenzamine vs dextran | 1.1 | −17.3 | 23.5 | 0.9983 | 4.9 | −27.3 | 51.5 | 0.9685 |
| Dextran + co-dosing vs dextran | −1.6 | −12.0 | 10 | 0.7582 | −27.2 | −46.4 | −1.1 | 0.0471 |
Co-treatment of ADC with a combination of chlorpromazine, nystatin, and imipramine led to significantly reduced uptake of ADC in HCEC and RPTEC and significantly greater nuclei counts in HCEC versus ADC alone
To further explore the effect of endocytosis inhibitors on ADC uptake and cytotoxicity, combined dosing of chlorpromazine, nystatin, and imipramine was performed with 48 h incubation. In HCEC, co-treatment with ADC 100 μg/mL significantly reduced uptake versus ADC alone (−30.7%, p < 0.0001) (Table 2 ) and was associated with significantly greater nuclei counts (+ 42.8%, p = 0.0024) (Supplement Table S6 ). Co-dosing also significantly reduced uptake of mAb 100 μg/mL (−19.6%, p = 0.0008) with no significant change in nuclei counts. Changes in dextran 200 μg/mL uptake with co-dosing were minimal (−1.6%, not statistically significant), but nuclei counts were significantly reduced (−22.9%, p = 0.0139). In RPTEC, co-dosing reduced uptake of ADC 100 μg/mL (−49.3%, p = 0.0123), mAb 100 μg/mL (−40.7%, p = 0.0196), and dextran 200 μg/mL (−27.2%, p = 0.0471) and significantly reduced nuclei counts for co-treatment with mAb (−60.2%, p = 0.0082) and dextran (−43.3%, p = 0.0260) but not ADC (+ 10.9%, p = 0.4641, not statistically significant).
No clear endocytic pathway mediating ADC uptake was found using siRNA depletion
Given the results with single-agent and co-dosing, siRNA analyses were performed to identify specific endocytic pathways with potential to affect ADC uptake. HCEC treated with siRNA, including siAP2mu2 (marker for clathrin-mediated endocytosis) and siCDC42 (involved in fluid phase endocytosis pathways), were incubated with Vivotag680-labeled ADC, mAb, transferrin-Alexa647, and dextran, and uptake was analyzed by flow cytometry (excluding ADC). With flow cytometry, a significant decrease in 10 nM transferrin uptake was seen in siAP2mu2 treated versus untreated cells and a significant decrease in 100 μg/mL dextran uptake was observed in siCDC42 treated cells after 1 h incubation, but there was no significant decrease in uptake of mAb 10 μg/mL with depletion of any endocytic protein (Supplement Fig. S7 ).
Synopsis
No individual receptor-mediated endocytic pathways were identified as being key to ADC uptake in HCEC, suggesting involvement of multiple and compensatory uptake mechanisms.
The uptake and cytoprotective effects of H1 inhibitors were inconsistent
H1 receptors are present on the surface of HCEC and have a potential role in inducing macropinocytosis through activation of protein kinase C (Mizuguchi et al. 2021 ; Tisdale et al. 2014 ). H1 receptor blockers have established long-term safety in humans and are available as over-the-counter topical eye drops. To determine if blocking H1 receptors could reduce ADC uptake, the effect of H1 inhibition was investigated with commercially available H1 inhibitors (ketotifen 0.52 μM, levocabastine 1.8 μM, desloratadine 2.2 μM, levocetirizine 1.2 μM and fexofenadine 34.1 μM). Co-treatment with H1 inhibitors and ADC or mAb showed inconsistent uptake and cytoprotective effects in HCEC and RPTEC. Full results from experiments of H1 inhibitors on uptake and cytotoxicity are presented in the Supplemental Results.
Human immunoglobulin solution demonstrated uptake inhibition and a cytoprotective effect
Human immunoglobulin solution was also explored for its potential to reduce ADC uptake and cytotoxicity through excess immunoglobulin competition. The effect of human immunoglobulin solution on ADC-mediated caspase 3/7 activity was evaluated in HCEC pre-treated with human immunoglobulin solution (1–20 mg/mL) and co-treated with ADC (1–100 μg/mL) for 48 h. Caspase 3/7 activity was significantly reduced in HCEC treated with ADC 50 μg/mL plus human immunoglobulin solution 20 mg/mL versus ADC alone (ratio 0.60 [95% CI 0.48–0.76], p < 0.0001), and ADC 100 μg/mL plus human immunoglobulin solution 15 mg/mL or 20 mg/mL (ratio 0.62 [95% CI 0.49–0.77], p < 0.0001 for 15 mg/mL; ratio 0.41 [95% CI 0.32–0.51], p < 0.0001 for 20 mg/mL) (Fig. 6 a). Fig. 6 Effect of human immunoglobulin solution on ADC-mediated apoptosis in HCEC ( a ): HCEC were treated with ADC alone or co-treated with ADC and human immunoglobulin solution. Concentrations were 1–100 µg/mL for ADC and 1–20 mg/mL for human immunoglobulin solution. HCEC were pre-treated with human immunoglobulin solution for 3 h. Apoptosis was measured using the Caspase-Glo 3/7 apoptosis assay (a luminescence test) at 48 h. P -values were adjusted upwards using the Benjamini Hochberg method, which controlled the false discovery rate. Effect of human immunoglobulin solution on uptake of mAb and ADC ( b ) and nuclei counts ( c ) in HCEC and RPTEC: HCEC and RPTEC were treated with ADC or mAb alone or co-treated with ADC and human immunoglobulin solution for 48 h. Concentrations were 10 and 100 µg/mL for ADC; 100 µg/mL for mAb; and 8, 15, and 20 mg/mL for human immunoglobulin solution. Uptake based on estimated changes in compound intensity fluorescence of Vivotag680-labeled mAb or ADC. Dunnett adjusted p -value for the test of no change from control: *** p < 0.001, ** p < 0.01, * p < 0.05. Error bars in panel a) represent 95% confidence intervals. ADC, antibody–drug conjugate; HCEC, human corneal epithelial cells; mAb, monoclonal antibody; RPTEC, renal proximal tubule epithelial cells
The effect of human immunoglobulin solution (8, 15, and 20 mg/mL) on ADC and mAb uptake and cytotoxicity was assessed. In HCEC, human immunoglobulin solution significantly inhibited uptake of ADC 10 μg/mL versus ADC alone (−30.1% with human immunoglobulin solution 15 mg/mL, p = 0.0028; −36.0% with human immunoglobulin solution 20 mg/mL, p = 0.0002), as well as ADC 100 μg/mL (−26.3 with human immunoglobulin solution 8 mg/mL, p = 0.0134; −34.1% with human immunoglobulin solution 15 mg/mL, p = 0.0004; and −37.8% with human immunoglobulin solution 20 mg/mL, p = 0.0001) (Fig. 6 b and Supplement Table S7 ). The reduction in uptake was associated with a reduction in cytotoxicity. Change in nuclei count versus control was −47.4% ( p = 0.0127) for ADC 100 μg/mL with human immunoglobulin solution 20 mg/mL but −70.8% ( p < 0.0001) for ADC alone, a 23.4% difference (Fig. 6 c and Supplement Table S8 ). All doses of human immunoglobulin solution significantly reduced mAb 100 μg/mL uptake, and there were no significant changes in nuclei count. Results for ADC were similar in RPTEC, with a reduction in uptake of −31.4% ( p < 0.0001) for ADC 100 μg/mL with human immunoglobulin solution 20 mg/mL versus ADC alone; there was also less cytotoxicity, with reductions in nuclei count versus control of −7.7% ( p = 0.5140) for ADC 100 μg/mL with human immunoglobulin solution 20 mg/mL and −36.0% ( p < 0.0001) for ADC alone, a 28.3% difference.
Synopsis
Human immunoglobulin solution reduced uptake of ADC and had cytoprotective effects (increased nuclei count and reduced caspase 3/7, interpreted as reduced apoptosis) over a range of concentrations.
Discussion
Belantamab mafodotin and other MMAF-conjugated ADCs are associated with corneal events, including keratopathy, blurred vision, dry eyes, and photophobia (Farooq et al. 2020 ; Popat et al. 2020 ). Corneal events with belantamab mafodotin are primarily mild to moderate in severity and typically resolve or improve with appropriate management, including dose holds and reductions after the initial dose, allowing patients to continue belantamab mafodotin treatment with no apparent loss of efficacy (Hungria et al. 2023 ; Mateos et al. 2025 ). Understanding the etiology of these corneal events could inform the development of effective supportive care prophylaxis and treatment.
This study characterized the uptake of the ADC belantamab mafodotin in human primary HCEC and RPTEC, cell lines that do not express BCMA, the target of belantamab mafodotin. Uptake was concentration- and time-dependent, and localization was observed within the endocytic pathway including lysosomes. In vitro experiments conducted in monolayers of HCEC and RPTEC indicated that ADC exposure results in significant concentration-dependent increases in apoptosis. There was a trend for HCEC to more readily uptake ADC than RPTEC, and levels of released payload were higher in HCEC; this may have led to the greater cytotoxic effects observed in HCEC versus RPTEC. As expected, some donor-to-donor variability in the uptake of ADC and mAb was observed; the number of donors was relatively small (n = 10) and a limitation of the study.
Treatment with the macropinocytosis inhibitor EIPA resulted in a modest but significant reduction (approximately 23%) in uptake of ADC into HCEC and reduced HCEC apoptosis. Reduction in uptake with EIPA suggests that ADC uptake in HCEC is driven in part by macropinocytosis, an actin-driven, clathrin-independent endocytic process that enables cells to internalize extracellular fluid and solutes through membrane ruffling and macropinosome formation (Ard et al. 2015 ); this is consistent with a previous report showing ADC uptake in HCEC via macropinocytosis (Zhao et al. 2018 ). In contrast, EIPA appeared to have no significant effect on ADC uptake into RPTEC. EIPA also did not significantly inhibit dextran uptake, a positive control for macropinocytosis, in HCEC or RPTEC, suggesting that the sub-cytotoxic concentration of EIPA was too low to effectively inhibit macropinocytosis of dextran; however, higher concentrations of EIPA could not be used as these were cytotoxic to HCEC. Nonetheless, mechanistic experiments in the same primary cells demonstrated that treatment with EIPA significantly reduced ADC-mediated apoptosis in HCEC and RPTEC, further supporting macropinocytosis as a mechanism of uptake.
Macropinocytosis in corneal and limbal epithelial cells is tightly regulated, integrating growth factor stimulation, cytoskeletal remodeling, and metabolic stress responses. Receptor tyrosine kinases (RTKs), such as EGFR and PDGFR, initiate this process via PI3K signaling, recruiting Akt and small GTPases (Rac1, Cdc42) crucial for membrane ruffling (Sugiyama et al. 2019 ). Specifically, PI3Kβ is required for growth factor-stimulated macropinocytosis, functioning with Rac1 during ruffle dynamics (Salloum et al. 2019 ; Swanson and King 2018 ), while Pak1 is essential for macropinosome closure (Ard et al. 2015 ; Salloum et al. 2019 ); their perturbation disrupts vesicle formation (Maekawa et al. 2014 ; Wu et al. 2024 ). Further regulation involves miR-103/107 in limbal epithelial cells, targeting scaffolding proteins like NEDD9 and SHC3 and macropinosome-associated effectors like ANKFY1 (Peng et al. 2017b ). Integration with mTOR, AMPK, PKC, and CDK5–dynamin signaling dictates cargo fate, aligning nutrient sensing with epithelial survival under stress (Nandi et al. 2017 ; Park et al. 2016 ; Peng et al. 2017b ). Physiologically, macropinocytosis may help corneal epithelial cells compensate for the nutrient-poor environment of the central stroma, while in limbal stem/progenitor cells, coordinated regulation of these pathways supports wound repair and tissue maintenance (Peng et al. 2017a ). Future work could combine advanced profiling and targeted interventions to causally link regulatory pathways involved in macropinocytosis, e.g., tyrosine kinase with downstream functional outcomes in corneal epithelial cells.
The contribution of other endocytic pathways was evaluated using co-treatment with wortmannin, chlorpromazine, nystatin, imipramine, and phenoxybenzamine. Co-treatment with nystatin or chlorpromazine, which target the caveolin and clathrin endocytic pathways, respectively, reduced ADC uptake into HCEC by approximately 20%, and nystatin reduced ADC-mediated cytotoxicity as measured by nuclei count. Although ADC uptake in RPTEC was reduced by all inhibitors tested except wortmannin, none of the compounds had a significant effect on ADC-mediated toxicity. The reduced uptake and cytotoxicity in HCEC suggest that uptake into HCEC may be mediated by multiple endocytic pathways, and co-dosing of chlorpromazine, nystatin, and imipramine with ADC also significantly reduced uptake and improved nuclei counts. However, when endocytic pathways were targeted by siRNA depletion in HCEC, no consistent effects were seen on ADC uptake, but interpretation of these results was limited due to low cell numbers caused by ADC cytotoxicity. Additionally, corneal cells are heterogeneous with varying cell profiles in each corneal layer, including expression of caveolin-1 in the limbus but minimal expression in the central cornea, and expression may be altered over multiple cell culture passages (passages 2–5) (Català et al. 2021 , 2023 ). Variable expression of endocytic pathways across different corneal cell types and during culture requires further investigation.
Although treatment with the H1 inhibitors ketotifen, fexofenadine, and desloratadine significantly reduced ADC-mediated apoptosis in HCEC in multiple donors, there was no significant cytoprotective effect of H1 inhibitors in a separate experiment measuring nuclei counts. The inconsistencies across results with the H1 inhibitors may reflect variation in H1 receptor stimulation in HCEC due to experimental conditions, which could have led to differences in induction of macropinocytosis through protein kinase C activation (Mizuguchi et al. 2021 ; Tisdale et al. 2014 ).
The effect of treatment of HCEC with human immunoglobulin solution to reduce uptake of ADC and ameliorate associated cytotoxicity was also evaluated, and demonstrated that pre-/co-treatment of human immunoglobulin solution with ADC significantly reduced ADC-mediated apoptosis in HCEC in a concentration-dependent manner. Furthermore, a −38.0% reduction of ADC uptake and reduced cytotoxicity in HCEC were observed with ADC 100 µg/mL and human immunoglobulin solution 20 mg/mL versus ADC alone. Uptake of ADCs into off-target cells via Fc-receptor mediated endocytosis is unlikely in HCECs, as these cells lack surface Fc gamma receptors (FCGR) (FCGR1A, FCGR2A, FCGR2B, FCGR3A, FCGR3B) (cell surface proteomics data, not shown). Moreover, the variance in ocular adverse events among marketed ADCs suggests limited involvement of FCGR uptake (Chen et al. 2024 ; Dy et al. 2024 ; Lindgren et al. 2024 ; Mao et al. 2024 ) and while mononuclear phagocytic uptake is high-capacity, it is saturable. Thus, human immunoglobulin solution likely competes with belantamab mafodotin to reduce uptake. Initial safety and efficacy of human immunoglobulin eye drops as a treatment for patients with dry eye disease was assessed in a pilot study, and the data highlighted their tolerability and support further clinical evaluation of human immunoglobulin eye drops for managing corneal events associated with MMAF-conjugated ADCs (Kwon et al. 2020 ).
The current study's reliance on in vitro primary cells is a limitation due to their inability to replicate the complex in vivo corneal microenvironment (e.g., cell–cell interactions, tear effects), necessitating careful interpretation. Furthermore, although belantamab mafodotin-induced ocular effects are observed in rat and rabbit studies, these models do not fully represent patient findings (Lee et al. 2025 ). Therefore, more human-representative models of ADC ocular effects (e.g., three-dimensional corneal organoids) are necessary for understanding the pathophysiology of ocular adverse events and developing mitigation strategies.
In conclusion, belantamab mafodotin undergoes HCEC and RPTEC uptake in a concentration- and time-dependent manner, despite a lack of BCMA expression on these cell types. Macropinocytosis plays a role in belantamab mafodotin uptake into these cells, where cys-mcMMAF is released resulting in apoptosis and cytotoxic effects. Other uptake mechanisms are likely to be involved, but additional pathways have yet to be determined. These in vitro data suggest a mechanism of uptake and toxicity leading to corneal events, but it will also be important to understand other mechanisms of belantamab mafodotin delivery to corneal tissues. Human immunoglobulin solution can competitively reduce the uptake and cytotoxicity of belantamab mafodotin in HCEC and should be explored further in clinical trials.
Supplementary Information
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Acknowledgements
Pamela Riester is acknowledged for her contribution to writing the report on Investigation of Uptake of Vivotag-680 Labelled GSK2857916A into Human Corneal Epithelial Cells and Renal Proximal Tubule Epithelial Cells and Renal Proximal Tubule Epithelial Cells. Mark Burman is acknowledged for his contribution to the in vitro apoptosis experimental work. David Berry is acknowledged for his contributions to the LC-MS/MS cys-mcMMAF quantification assay. Editorial support (in the form of writing assistance, including preparation of the draft manuscript under the direction and guidance of the authors, collating and incorporating authors’ comments for each draft, assembling tables and figures, grammatical editing and referencing) was provided by Alexus Rivas-John, PharmD, and Aithne Atkinson, PhD, at Fishawack Indicia Ltd, part of Avalere Health, and was funded by GSK.
Author contributions
Lucinda Weir, Carla Newman, and Chloe Taylor contributed to the research conception. All authors contributed to study design. Material preparation, data collection and/or analysis were performed by Chloe Taylor, Gurpreet Sohanpal, Carla Newman, Tanja Högg, James Kennedy, Eliot McKinley, Elena Koudouna, Edward Sayers, and Peter Watson. All authors were responsible for manuscript writing, review, and editing. All authors read and approved the final manuscript.
Funding
These studies were funded by GSK (studies N69460-01, N69460-02, N69460-04, N70440-40, N70440-42, N70440-44, N70440-45, N70517-1, N70517-02, N70517-03, N70517-6, N71801-15, ELN109309, ELN144763, ELN144764, ELN17985 , ELN20314 , ELN43728 , ELN46182 , ELN76344 , ELN76361 ). Drug linker technology licensed from Seagen Inc.; monoclonal antibody produced using POTELLIGENT Technology licensed from BioWa.
Data availability
Information about GSK’s data sharing commitments and access requests to anonymized individual participant data and associated documents can be requested for further research from https://www.gsk-studyregister.com/en/
Declarations
Ethical approval
Not required for this study as commercially available cell lines were investigated in vitro.
Conflicts of interest
CN, CT, GS, TH, JK, EM, and LW are employees of and hold financial equities in GSK. EK, EJS, ATJ, and PW report no conflicts of interest. Data were presented in part at the Society of Toxicology Annual Meeting and ToxExpo 2021.
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