Characterization of Belantamab Mafodotin-Induced Corneal Changes in Patients With Multiple Myeloma.

Introduction

Multiple myeloma (MM) accounts for approximately 10% of hematological cancers. 1 The investigational antibody-drug conjugate (ADC) belantamab mafodotin (GSK2857916) targets B-cell maturation antigen (BCMA) on malignant MM plasma cells. 2 , 3 It is an afucosylated humanized immunoglobulin G1 (IgG1) ADC comprising a monoclonal antibody conjugated to the microtubule inhibitor monomethyl auristatin-F (MMAF). Belantamab mafodotin internalization leads to cell cycle arrest, apoptosis, tumor lysis by antibody-dependent cellular toxicity, and phagocytosis. 2 Although not currently commercially available, 4 , 5 belantamab mafodotin combination regimens have demonstrated improvements in progression-free survival against standard care combinations in patients with relapsed or refractory multiple myeloma (RRMM) in phase 3 trials, including the DREAMM-7 (belantamab mafodotin with bortezomib and dexamethasone) and DREAMM-8 (belantamab mafodotin with pomalidomide and dexamethasone) trials. 6 , 7 , 8 A significant overall survival benefit has been confirmed for DREAMM-7, and a trend of improvement has been observed for DREAMM-8, with overall survival analysis ongoing. 6 , 7 , 8 , 9 These data supported new regulatory filings.

Corneal events, particularly microcystlike epithelial changes (pseudomicrocysts) are a well-described class effect of MMAF-containing ADCs. 10 , 11 , 12 , 13 , 14 , 15 ADCs are thought to reach the cornea via vascularized limbus 16 or tear film, where antigen-negative corneal epithelial cell death may occur through MMAF toxicity. 17 , 18 , 19 , 20 Although pseudomicrocyst development has not been replicated in belantamab mafodotin–treated preclinical models and confirmatory histology in patients is lacking, evidence suggests that belantamab mafodotin–driven pseudomicrocysts are partly due to ADC macropinocytosis by corneal epithelial cells. 16 , 21 , 22 Once inside cells, MMAF disrupts microtubule-dependent processes underlying migration, cell cycle progression, and differentiation of limbal cells. 22 , 23 Affected cells migrate toward the central cornea in various stages of apoptosis, and cells completing apoptosis slough off from the surface. 15 , 22 , 24 Pseudomicrocyst-associated changes may affect the cornea’s refractive power (eg, by causing an initial hyperopic shift, then a myopic shift with substantial astigmatism). 25 , 26 ADCs may also worsen dry eye diseases, with changes in tear film parameters occurring early in belantamab mafodotin exposure, 27 , 28 , 29 although this mechanism is incompletely understood.

This study aimed to characterize the mechanism and pathophysiology of pseudomicrocysts in patients with RRMM following treatment with belantamab mafodotin and to correlate clinical data with histopathological findings. We present a human tissue study of corneal epithelial changes associated with belantamab mafodotin treatment in patients with RRMM, with safety profile assessment of impression cytology (IC) or superficial keratectomy (SK) procedures. Additionally, animal studies were conducted to support regulatory submissions and to supplement clinical findings and characterize the potential etiology of corneal epithelial changes associated with belantamab mafodotin (see the eMethods 2 and eResults in Supplement 1 ); results from these studies are referred to in the Discussion.

Methods

Patient Selection and Study Design

Adults (≥18 years of age) with RRMM who were receiving or had received belantamab mafodotin during clinical trials or by prescription and were diagnosed by an ophthalmologist with symptomatic or asymptomatic pseudomicrocysts in 1 or more eye were eligible for inclusion (eFigure 1 in Supplement 1 ). Concomitant ophthalmic medication use related to allowable ophthalmic conditions was permissible. Excluded patients had superficial punctate keratopathy without evidence of pseudomicrocysts, decreased corneal sensation, or were affected by eye infections, active uveitis, or permanent legal blindness in the nonstudy eye. Patients with any condition potentially interfering with patient safety or participation, excess risk of delayed wound healing, or who were receiving concomitant medications that could affect the cornea were excluded. The study was conducted in accordance with the Declaration of Helsinki and the Good Clinical Practice guidelines, including institutional review board approval at each site. Patients provided signed written informed consent and did not receive a stipend or incentive to participate. Reporting guidelines for case series have been used in this report. 30

Patients were screened and had a confirmatory evaluation performed, including best-corrected visual acuity (BCVA) and slitlamp examination. Confirmatory anterior segment photography was required unless acceptable corneal images were available from an examination less than 14 days before IC or SK. Patients had 3 to 6 visits following their procedures, with additional visits required for delayed healing. A patient completed the study after finishing all phases, including the last visit. The study was conducted from March 26, 2020, to November 21, 2022, with procedures and initial histopathologic analysis from May 13, 2021, to September 12, 2022, and final analysis and reporting from May 19, 2021, to May 10, 2023.

Outcomes and End Points

The primary end point was evaluation of pathologic characteristics and composition of corneal epithelial changes obtained by IC or SK. Secondary end points were correlation of clinical data, including BCVA, symptoms, and grade and appearance of corneal changes on imaging, with histopathologic findings. The safety profile of IC and SK was assessed.

Exploratory end points were changes in BCVA, symptoms associated with reduced visual acuity or other ocular symptoms (Common Terminology Criteria for Adverse Events [CTCAE] version 5.0) from pre- to post-IC or SK procedure, and preprocedure concentrations of belantamab mafodotin and/or cysteine-maleimidocaproyl MMAF (cys-mcMMAF) in tear fluid and blood.

Procedures

The eye with more pseudomicrocysts was the study eye; if both eyes were affected equally, the worse-seeing eye was selected. Either IC or SK (eMethods 1 in Supplement 1 ) was performed by an ophthalmologist to collect corneal epithelial tissue with the most prominent changes, which was submitted for histopathologic evaluation.

Fixed tissue samples were processed in a central laboratory and analyzed by the study ophthalmic pathologist (V.L.). Tissue was examined microscopically, and morphology of corneal epithelium; presence or absence of apoptotic and mitotic cells, intraepithelial edema, bullae, intracellular and/or extracellular deposits, and inflammation; and presence of deeper corneal layers were recorded. When possible, immunohistochemical staining for IgG and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were performed to characterize intracellular deposits and apoptotic cells undetected by morphology.

Patients participated in tear fluid and blood pharmacokinetic analyses: tear fluid samples were collected prior to the IC or SK procedure to analyze the presence of belantamab mafodotin ADC and cys-mcMMAF, and blood collection was performed close to tear fluid sample collection to analyze plasma concentrations of belantamab mafodotin, total antibody, cys-mcMMAF, and soluble BCMA (sBCMA).

Safety assessments included adverse events (AEs) and serious AEs, along with details of duration, severity, causality, and outcomes.

Statistical Analyses of Clinical Data

Demographic characteristics, dosing levels and duration, and AEs were summarized using descriptive statistics. No formal statistical hypotheses were tested.

Results

Patient Disposition, Demographics, and Baseline Characteristics

Disposition

Nine of 16 screened patients were enrolled. All 9 patients completed either IC or SK procedures, and 6 had histologically evaluable corneal samples (4 from IC and 2 from SK, with 1 sample per patient; eTable 1 in Supplement 1 ).

Demographics and Baseline Characteristics

Demographic and baseline characteristics appeared consistent with the general RRMM population ( Table 1 ). 31 Briefly, of 9 patients included, median (range) patient age was 67.0 (57.0-81.0) years for those with samples obtained by IC and 68.0 (65.0-81.0) years for those with samples obtained by SK. Six patients (67%) were female. Belantamab mafodotin exposure ranged from 3.1 to 135.1 weeks. Concomitant ophthalmic medications (per standard of care) included preservative-free artificial tears, cyclosporine, brimonidine tartrate, and timolol maleate.

Histological Findings

Where available, Table 2 indicates time from the last dose of belantamab mafodotin and the number of doses the patient received prior to this study. For reference, in nonpathological studies, corneal cells are columnar, with high nuclear to cytoplasmic ratio at the basal layer, that then evolve to flattened, squamous cells with low nuclear to cytoplasmic ratio on the surface. When collected by IC, corneal cells are seen in sheets or small aggregates, with minimal cellular debris, inflammatory cells, and mitotic figures. 32 , 33

Case 1

The sample collected from the temporal region by IC revealed a membrane with adherent epithelial cells. Few apoptotic cells were observed morphologically. Eosinophilic intracytoplasmic inclusions (<10 μm) were present ( Figure 1 A). Lymphocytes were present; however, their location was not confirmed based on IC procedure limitations. There was no evidence of mitosis, intraepithelial edema, or extracellular deposits.

Case 2

The sample (location unknown) was collected by IC. Examination with slitlamp microscopy demonstrated pseudomicrocysts and punctate fluorescein staining (eFigure 1A and 1B in Supplement 1 ). Sheets of corneal epithelial cells were present, with some areas consisting of small aggregates or individual cells ( Figure 1 B). Multiple eosinophilic intracytoplasmic inclusions (<10 μm) were seen. A small number of cells exhibited amphophilic, mildly granular cytoplasm. No apoptotic cells were observed, and there was no evidence of mitosis, intraepithelial edema, extracellular deposits, or inflammation.

Case 3

The sample was collected from the temporal region by IC. Epithelial cells were observed as a continuous sheet, with some areas of small aggregates or individually scattered epithelial cells ( Figure 1 C). Few apoptotic cells and multiple eosinophilic intracytoplasmic inclusions (<10 μm) were seen. Few lymphocytes were observed. Although location could not be confirmed due to the nature of the procedure, some lymphocytes appeared to be embedded between epithelial cells, suggesting epithelial inflammation. There was no evidence of mitosis, intraepithelial edema, or extracellular deposits.

Case 4

The sample was collected from the superior or temporal area using IC and revealed a membrane with adherent epithelial cells in small aggregates or scattered individually ( Figure 1 D). Substantial debris was present, with few apoptotic cells observed. Eosinophilic intracytoplasmic inclusions (<10 μm) were seen. Lymphocytes were observed between epithelial cells. There was no evidence of mitosis, intraepithelial edema, or extracellular deposits.

Case 5

Tissue was collected by SK from the superior or temporal region (eFigure 3A in Supplement 1 ). Hematoxylin-eosin staining revealed a full-thickness epithelial biopsy, measuring 30 × 660 μm (thickness × length; eFigure 3C in Supplement 1 ). Apoptotic cells were observed based on morphology, with moderately more detected by TUNEL staining (eFigure 3D in Supplement 1 ). One mitotic figure was seen; however, this was within normal limits for corneal epithelium. Multiple eosinophilic intracytoplasmic inclusions (<10 μm) were seen near the basal layer, associated with fragmented nuclei. No intraepithelial edema, bullae, extracellular deposits, or inflammation was observed.

Case 6

The sample (location unknown) was collected by SK. Punctate fluorescein staining was observed in the cornea (eFigure 3B in Supplement 1 ). Multiple fragments of corneal epithelium were collected, with the largest measuring 30 × 3140 μm (thickness × length; Figure 2 A). Apoptotic cells were not morphologically observed; however, TUNEL staining revealed moderate amounts of apoptotic cells ( Figure 2 B). Multiple eosinophilic intracytoplasmic inclusions (<10 μm) associated with fragmented nuclei were seen near the basal layer. Some fragments of tissue contained basal cells with amphophilic, mildly granular cytoplasm ( Figure 2 C). Intracytoplasmic inclusions stained positive for IgG ( Figure 2 D). No mitosis, intraepithelial edema, bullae, extracellular deposits, or inflammation was observed.

Ocular Symptom Grading and Visual Acuity

Among those who had the IC procedure (n = 6), BCVA was only tested at visit 1, and there was no postprocedure comparison. Ocular symptom grading was only conducted at baseline of this study for these individuals (who received belantamab mafodotin before this study): 4 patients had grade 1 (cases 1, 2, 4, and 6), 1 patient had grade 2 (case 5), and 1 patient had grade 3 (case 3) ocular symptoms.

For individuals who underwent SK (n = 3), follow-up visits were conducted in addition to baseline measures. Two patients had improvement in BCVA and ocular symptom grade between first and follow-up visits; both had improvement of 1 line in BCVA (20/30 to 20/25 Snellen) and improvement in ocular symptom grading from CTCAE grade 2 to grade 1. The third patient had no change in either BCVA or ocular symptom grading (grade 1) at follow-up. Additional imaging was not available.

Pharmacokinetic Results

Tear fluid samples (7 patients) and blood samples (4 of these patients) were collected 6 to 112 days from last belantamab mafodotin dose ( Table 2 ). Antibody-drug conjugate was detected in tear fluid in 5 patients (4 with evaluable tissue samples) of 7; 2 patients with undetectable ADC had 51 and 112 days, respectively, between last belantamab mafodotin dose and sample collection. Tear fluid cys-mcMMAF was detected in 2 patients; these were among the patients with the shortest time to sample collection from belantamab mafodotin dosing (6 and 14 days, respectively).

Among 4 patients who had blood samples, plasma ADC was quantifiable in 3. Total antibody and ADC concentrations were lower or nonquantifiable with longer time intervals between belantamab mafodotin dosing and sampling. The individual with plasma ADC concentration below the lower limit of quantification had 112 days from the last dose to sample collection. Plasma total antibody and free sBCMA were quantifiable in all 4 patients, while plasma cys-mcMMAF was quantifiable in 1 patient with 14 days between dosing and sample collection.

Safety of IC and SK Procedures

No AEs or deaths were reported following IC or SK procedures, with no persistent epithelial defects or corneal ulcers within 2 weeks of the procedures. Corneal healing was observed in all patients.

Discussion

This case series study of corneal epithelial changes in 9 patients with RRMM who were treated with belantamab mafodotin and developed pseudomicrocysts showed that eosinophilic intracytoplasmic inclusions and/or basophilic granular cytoplasm were present, with samples from 5 patients demonstrating apoptosis and 3 having inflammation within the corneal epithelium. Pharmacokinetic analyses confirmed that patients’ tear fluid contained ADC and cys-mcMMAF. Total antibody and ADC plasma concentrations were lower with longer durations between dosing and sample collection, and cys-mcMMAF was detectable only in patients with shorter time between dosing and sampling. There were no AEs related to IC or SK, and all patients healed as expected, suggesting minimal to no effect on wound healing despite observed corneal changes. These results suggest that belantamab mafodotin reaches corneal epithelial cells and is associated with pseudomicrocyst formation.

Studies in animal models are summarized in the eMethods 2 and the eResults in Supplement 1 and showed presence of belantamab mafodotin and cys-mcMMAF in the eye and tear fluid. Corneal pathology post–belantamab mafodotin exposure, primarily apoptosis, was observed in the rat and rabbit models, consistent with clinical data reported here, but in these animal models there were some differences to the clinical findings—there was no evidence of pseudomicrocyst development, and in rats, there were no findings observed at ophthalmologic examination (using indirect ophthalmoscopy, slitlamp biomicroscopy, and fluorescein staining). Rats treated for 13 weeks also had increased mitoses in basal corneal epithelial cells that appeared arrested in anaphase, consistent with microtubular inhibition of the mitotic spindle; this cellular phenotype was not present in the human corneal epithelial samples examined. In contrast to clinical findings, the monkey model did not show notable changes during either ophthalmological or histopathological examinations, while the rat model showed histopathological findings, including mitoses not observed in patients, but did not show any notable ophthalmological changes. Since IC and SK do not capture deeper layers of the cornea reliably, evaluation for the presence or absence of increased mitoses in the basal layers of the epithelium is limited in these patients. Full-thickness assessment of the corneal epithelium over a broad area posttreatment would be required to establish this.

Although 5 of 7 tear fluid samples contained detectable amounts of ADCs and cys-mcMMAF was detected in tear samples from 2 patients, further study is required to confirm how belantamab mafodotin reaches the ocular surface. Animal model data indicate that belantamab mafodotin and cys-mcMMAF are detected in tear fluid. These findings suggest that the presence of belantamab mafodotin and cys-mcMMAF within the tear film could potentially contribute to or correlate with corneal toxicity. In addition, the clinical pattern of corneal changes, in which pseudomicrocysts are initially present in the corneal periphery, suggests that limbal vessels are a primary pathway by which ADCs enter the corneal epithelium.

Although pharmacokinetics data from animal models were limited and do not contribute to determining how much ADC reaches the corneal epithelium via plasma or tear fluid, the molar ratios of belantamab mafodotin to cys-mcMMAF in Dutch Belted rabbit samples indicate a considerably higher proportion of belantamab mafodotin than cys-mcMMAF in each matrix. When comparisons were permissible, the relative molar proportion of cys-mcMMAF was higher in rabbit tears than in plasma. 16 , 22 Free cys-mcMMAF has low cell membrane permeability but may be taken into corneal cells through macropinocytosis or other nonspecific endocytic mechanisms. 34 However, exposure data suggest contributions to intracellular levels of cys-mcMMAF from the tear compartment are probably not the primary mechanism of corneal toxicity. These findings could potentially assist future developments of prophylaxis or supportive care to mitigate corneal events associated with MMAF-containing ADCs.

Each patient who underwent either SK or IC procedures healed, with no evidence that BCVA was negatively impacted, although this was a small cohort. To our knowledge, this is the first evidence that performing these procedures on patients using ADCs results in minimal, if any, effect on corneal healing. Clinical findings corroborated with in vivo nonclinical data strengthen this study’s findings, although lack of pseudomicrocyst formation limited extrapolation from rat and rabbit models.

Limitations

This study’s main limitation is the small clinical population. Low patient enrollment and withdrawal of belantamab mafodotin from market following the result from the DREAMM-3 trial 4 , 5 limited the conclusions that can be drawn from small numbers of patients. Subsequent positive results from the DREAMM-7 and DREAMM-8 trials of belantamab mafodotin combination regimens are anticipated to support belantamab mafodotin reentering the market. 6 , 7 , 8 This study also did not permit correlation to each patient’s clinical response to belantamab mafodotin. Furthermore, as not all patients had evaluable specimens, tear fluid, and/or blood plasma collection, correlation of ADC pharmacokinetics findings with histopathological findings was limited. Clinical data confirmed the presence of ADC in tears and plasma, but we were unable to determine if there was a specific or more dominant pathologic route. Additionally, IC examines a single layer of cells detached from the corneal surface and is less invasive than SK; while IC can detect inflammation, it cannot further characterize or identify the location of the inflammation (tissue or tear film), thereby constraining the conclusions that may be drawn from IC samples. Animal models (rat and rabbit) suggested single cell apoptosis in the cornea with increased mitosis, but these models did not develop pseudomicrocysts and limit correlation with clinical findings.

Conclusions

In this case series study, intracytoplasmic inclusions were observed among corneal epithelial cells of patients with RRMM treated with belantamab mafodotin who developed pseudomicrocysts. The pattern of corneal changes suggested limbal vessels as a primary pathway for ADC to enter the cornea, but ADC was also detected in tear fluid. This study improves understanding of potential mechanisms behind pseudomicrocyst development with ADC treatment.

eMethods 1. Supplemental Methods: Ophthalmic Procedures

eTable 1. Patient Disposition

eFigure 1. Study Design

eFigure 2. Representative Images From IC Procedures

eFigure 3. Representative Images From SK Procedures for Case 5 (A–C) and Case 6 (D)

eMethods 2. Supplemental Methods: Animal Studies

eResults. Supplemental Results: Animal Studies In Vivo and Ex Vivo

eTable 2. Nano-Liquid Chromatography Mass Spectrometry of cys-mcMMAF in Various Tissues of Rats Exposed to a Single Intravenous Dose of Belantamab Mafodotin (30 mg/kg)

eTable 3. Molar Ratios of Belantamab Mafodotin ADC:cys-mcMMAF in Female Dutch Belted Rabbit Tears and Plasma Following Repeated Weekly Intravenous Dosing of Belantamab Mafodotin ADC

eTable 4. Incidence of Apoptosis, Increased Mitosis and Inflammation in the Cornea Following Belantamab Mafodotin Exposure in Animal Models

eFigure 4. Mean Radiant Efficiency of the Left and Right Eye Combined for Each Group of Rats With Fluorescent-Labeled Belantamab Mafodotin and the Control (GSK2857914 [Belantamab Antibody Without the Mafodotin Payload]) (A) and Left Versus Right Eye Correlation (B)

eFigure 5. Average Radiant Efficiency/cm2 of Tissue Taken at 24a and 48 Hours From Rats Dosed With Labeled Belantamab Mafodotin (30 mg/kg) or GSK2857914 (Belantamab Antibody Without the Mafodotin Payload; 30 mg/kg)

eFigure 6. Fluorescent-Labeled Belantamab Mafodotin Is Detected in Rat Tissue Following a Single Exposure

eFigure 7. Representative Images From a 3 Week Repeat Dose Toxicity Study (A, B, C) and From a Single Dose Toxicity Study (D, E)

eFigure 8. Images From Dutch Belted Rabbit Exposed to Repeat Dosing With Belantamab Mafodotin

eReferences.

Data Sharing Statement

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Associated Data

Supplementary Materials

eMethods 1. Supplemental Methods: Ophthalmic Procedures

eTable 1. Patient Disposition

eFigure 1. Study Design

eFigure 2. Representative Images From IC Procedures

eFigure 3. Representative Images From SK Procedures for Case 5 (A–C) and Case 6 (D)

eMethods 2. Supplemental Methods: Animal Studies

eResults. Supplemental Results: Animal Studies In Vivo and Ex Vivo

eTable 2. Nano-Liquid Chromatography Mass Spectrometry of cys-mcMMAF in Various Tissues of Rats Exposed to a Single Intravenous Dose of Belantamab Mafodotin (30 mg/kg)

eTable 3. Molar Ratios of Belantamab Mafodotin ADC:cys-mcMMAF in Female Dutch Belted Rabbit Tears and Plasma Following Repeated Weekly Intravenous Dosing of Belantamab Mafodotin ADC

eTable 4. Incidence of Apoptosis, Increased Mitosis and Inflammation in the Cornea Following Belantamab Mafodotin Exposure in Animal Models

eFigure 4. Mean Radiant Efficiency of the Left and Right Eye Combined for Each Group of Rats With Fluorescent-Labeled Belantamab Mafodotin and the Control (GSK2857914 [Belantamab Antibody Without the Mafodotin Payload]) (A) and Left Versus Right Eye Correlation (B)

eFigure 5. Average Radiant Efficiency/cm2 of Tissue Taken at 24a and 48 Hours From Rats Dosed With Labeled Belantamab Mafodotin (30 mg/kg) or GSK2857914 (Belantamab Antibody Without the Mafodotin Payload; 30 mg/kg)

eFigure 6. Fluorescent-Labeled Belantamab Mafodotin Is Detected in Rat Tissue Following a Single Exposure

eFigure 7. Representative Images From a 3 Week Repeat Dose Toxicity Study (A, B, C) and From a Single Dose Toxicity Study (D, E)

eFigure 8. Images From Dutch Belted Rabbit Exposed to Repeat Dosing With Belantamab Mafodotin

eReferences.

Data Sharing Statement