Nonclinical Characterization of Bexmarilimab, a Clever-1-Targeting Antibody for Supporting Immune Defense Against Cancers

Abstract

Common lymphatic endothelial and vascular endothelial receptor-1 (Clever-1) is a multifunctional type-1 transmembrane protein that plays an important role in immunosuppression against tumors. Clever-1 is highly expressed in a subset of human tumor-associated macrophages and associated with poor survival. In mice, Clever-1 supports tumor growth and metastasis formation, and its deficiency or blockage induces T-cell-dependent killing of cancer cells. Therefore, targeting Clever-1 could lead to T-cell activation and restoration of immune response also in patients with cancer. This is studied in an on-going clinical trial [Macrophage Antibody To INhibit immune Suppression (MATINS); NCT03733990] in patients with advanced solid tumors where bexmarilimab, a humanized IgG4 antibody against human Clever-1, shows promising safety and efficacy. Here, we report the humanization and nonclinical characterization of physicochemical properties, biological potency, and safety profile of bexmarilimab. Bexmarilimab showed high affinity to Clever-1 on KG-1 cells and bound to Clever-1 on the surface of classical and intermediate monocytes derived from healthy human blood. Bexmarilimab inhibited the internalization of its natural ligand acetylated low-density lipoprotein into KG-1 cells and increased TNFα secretion from macrophages but did not impair phagocytic clearance. Bexmarilimab did not induce significant cytokine release in human whole-blood cultures, did not contain nonsafe immunogenic glycans, or show any significant binding to human Fcγ receptors or complement pathway component C1q. In vivo, bexmarilimab showed dose-dependent duration of monocyte Clever-1 receptor occupancy in cynomolgus monkeys but did not induce a cytokine storm up to a dose of 100 mg/kg. In conclusion, these data support the clinical development of bexmarilimab for the restoration of immune response in cancers.

©2022 The Authors; Published by the American Association for Cancer Research.

Figures

Figure 1.

Characterization of bexmarilimab binding in vitro. A, Binding of biotinylated parental anti–Clever-1 antibody 3–372 to human Clever-1 in the presence of nonbiotinylated 3–372, chimeric anti–Clever-1 antibody and three composite antibodies (VH2/VK5, VH3/VK5, VH4/VK5) as determined by a competitive ELISA. IC50 values were calculated by three-parameter nonlinear curve fitting with 95% confidence intervals (CI). B, Bexmarilimab-recognized region of Clever-1. The orange boxes and blue closed circles indicate fasciclin domains and EGF-like domains, respectively. The arrowheads indicate relative positions of identified binding motifs, the core epitopes are shown in red. C, Bexmarilimab immunoreactivity (magenta) in human lymph node with CD31 (green) and Hoechst (blue) co-staining. Scale bar 40 μm. Arrowheads point to Clever-1 expression on lymphatic endothelial cells and the arrow points to a single CD31-negative cell. Binding of bexmarilimab to human CD14+ cells (D) and human KG-1 acute myelogenous leukemia cells (E) as determined by flow cytometry. F, Binding and quantitation of bexmarilimab and the isotype control to KG-1 cells transfected with a Clever-1–targeting shRNA as determined by flow cytometry. The wild-type histogram is shown in black, the Clever-1 knockdown histogram in red, and the isotype control binding in solid gray. APC, allophycocyanin. Receptor occupancy of bexmarilimab in classical (G) and intermediate monocytes (H), shown as change in fluorescence intensity of CD14+ cells that bind to labeled bexmarilimab or mAb 9–11. Representative data are shown for 1 of 3 donors. MFI, mean fluorescence intensity.

Figure 2.

Bexmarilimab binds to Clever-1 on monocytes and inhibits internalization of acLDL while inducing TNFα secretion. A, Internalized and surface-bound S. aureus particles in monocytes from healthy human donors after treatment with bexmarilimab, mAb 9–11, or their respective isotype controls. B, Internalization of acLDL into KG-1 cells via Clever-1 receptor in the presence of two batches of bexmarilimab, mAb 9–11, parental mouse antibody 3–372, and an isotype control as determined by a competition assay and flow cytometry. The dotted line indicates the level of internalization without a competitor compound present. C, Mean fold changes in TNFα secretion by primary human macrophages upon treatment with 0.1–50 μg/mL bexmarilimab compared with an isotype control. The macrophages were induced in vitro from CD14+ monocytes obtained from seven healthy donors. **, P < 0.01; NS, not significant compared with isotype control. The statistical analysis was performed using one-way repeated measures ANOVA followed by Dunnett test. D, Absolute levels of TNFα secretion by primary human macrophages (C) upon treatment with 0.1 to 50 μg/mL bexmarilimab or an isotype control. Each donor is shown separately. E, Whole blood from 3 healthy donors was incubated with bexmarilimab or Ig-control for 24 hours at concentrations of 1.33, 13.3, or 133 μg/mL. These correspond to the initial estimated concentrations after injection of the antibodies into a blood volume of 4.5 L at doses of 0.1, 1, or 10 mg/kg, respectively, in a 60-kg person. Receptor occupancy of bexmarilimab on human CD14+ cells was measured by flow cytometry. The binding of fluorescently labeled bexmarilimab on CD14+ cells after administration of nonlabeled bexmarilimab is shown in blue and the binding of fluorescently labeled 9–11 antibody is shown in grey. Data normalized to Ig-treated cells. F, Flow cytometry analysis of acLDL internalization in CD14+ cells after bexmarilimab treatment (E). The blood was spiked with 1 μg/mL of AF488-acLDL during bexmarilimab incubation. Data normalized to Ig-treated cells.

Figure 3.

Bexmarilimab does not contain immunogenic glycans or bind to human FcγRI or complement 1q. A, N-glycoform distribution on bexmarilimab as determined by quantitative N-glycan profiling. The analysis was performed in triplicate and glycans were assigned according to retention time. B, Binding of bexmarilimab and the positive control IgG rituximab to FcγRI as determined by SPR analysis. Colored lines correspond raw data, black lines correspond fitted data. C, Binding of bexmarilimab and rituximab to component C1q of the complement pathway as determined by ELISA; a representative graph of three experiments. s, seconds.

Figure 4.

Bexmarilimab does not induce cytokine release in whole blood. Release of cytokines (A) IL6 (A), IL10 (B), IFNγ (C), TNFα (D), and IL8 (E) upon bexmarilimab treatment of human whole blood as determined by Cytokine ScreenTM assay. Alemtuzumab and cetuximab were used as reference compounds. Each data point represents one donor (n = 21) and the graphs show the median, minimum, and maximum values. F, Cytokines induced by positive control [pokeweed mitogen (PWM)] compared with PBS. Statistical analysis was performed using one-way ANOVA followed by paired Student t test. ***, P < 0.001; ****, P < 0.0001 as compared with cetuximab.

Figure 5.

Bexmarilimab shows dose-dependent duration of receptor occupancy in vivo and only moderate increase in ALT levels in cynomolgus monkeys. Receptor occupancy of bexmarilimab in cynomolgus monkey monocytes after administration of a single i.v. dose of bexmarilimab at 3 mg/kg (A), 30 mg/kg (B), or 100 mg/kg (C; n  =  6 animals/dosing group). Change in MFI of CD14+ cells binding to fluorescently labeled bexmarilimab after administration of nonlabeled bexmarilimab is shown in blue and binding to fluorescently labeled 9–11 antibody is shown in orange. Concentration of the bexmarilimab in circulation (grey) was measured at the same time points as the receptor occupancy. D, Effect of bexmarilimab on ALT levels in cynomolgus monkeys on days 7 and 20 compared with predose levels. h, hours.