Preclinical assessment of a novel human antibody VH domain targeting mesothelin as an antibody-drug conjugate

Abstract

Mesothelin (MSLN) has been a validated tumor-associated antigen target for several solid tumors for over a decade, making it an attractive option for therapeutic interventions. Novel antibodies with high affinity and better therapeutic properties are needed. In the current study, we have isolated and characterized a novel heavy chain variable (VH) domain 3C9 from a large-size human immunoglobulin VH domain library. 3C9 exhibited high affinity (KD [dissociation constant] <3 nM) and binding specificity in a membrane proteome array (MPA). In a mouse xenograft model, 3C9 fused to human IgG1 Fc was detected at tumor sites as early as 8 h post-infusion and remained at the site for over 10 days. Furthermore, 3C9 fused to a human Fc domain drug conjugate effectively inhibited MSLN-positive tumor growth in a mouse xenograft model. The X-ray crystal structure of full-length MSLN in complex with 3C9 reveals interaction of the 3C9 domains with two distinctive residue patches on the MSLN surface. This newly discovered VH antibody domain has a high potential as a therapeutic candidate for MSLN-expressing cancers.

Keywords: ADC; MSLN; VH domain; in vivo distribution; library; structure.

Graphical abstract

Figure 1

Characterization of 3C9 (A) SEC data of both VH and VH-Fc 3C9. Aggregation-resistant VH and VH-Fc ab8 were used as controls. (B and C) ELISA of VH/VH-Fc binders binding to human MSLN. (D) 3C9 did not show any binding to 293F cells, but it exhibited positive binding when tested on 293F cells that were transiently transfected with MSLN. 3C9 was labeled in red, while the isotype control was labeled in blue. (E) Individual cell lines were analyzed by flow cytometry after staining with either 50 nM VH-Fc 3C9 (yellow) or 50 nM IgG1 m912 (red). The isotype control was labeled in blue. (F) SPR of 3C9 VH and the VH-Fc forms. (G) Lack of non-specific binding measured by a membrane proteome array (MPA). Antibody domain 3C9 was fused to human Fc protein for examination by flow cytometry. VH-Fc 3C9 (20 μg/mL) was tested in a membrane proteome array against 6,000 different human membrane proteins.

Figure 2

Structure of MSLN-3C9 complex, determined by crystallography (A) MSLN (coordinate shown from residue 300 to 582, purple) complexed with two VH 3C9 domains, VH_1 and VH_2 (green and blue, respectively). (B) The same structure, viewed from different orientations, highlighting swapping of the C-terminal beta-strands in two VH 3C9 domains. (C) Map of VH_1 (green) and VH_2 (blue) interaction sites on MSLN (purple) with highlight of the locations of Y346 and Y374 (orange). (D) Side chain interaction of MSLN with VH_1 (green) with highlight of Y374 (orange). (E) Side chain interaction of MSLN with VH_2 (blue) with highlight of Y346 (orange). Expanded figures for (A) are shown in Figures S1 and S2; extended figures for (B) and (C) are shown in Figure S3; extended figures for (D) and (E) are shown in Figure S4.

Figure 3

3C9 does not compete with MORAb-009 (A and B) Surface presentation of MORab-009 (ice blue, PDB 7UED), 3C9 VH_1 (green), and VH_2 (blue) on MSLN (purple, ribbon) are shown from two different orientations. (C) VH 3C9 and MORAb-009 have no competition in competing ELISA.

Figure 4

Pharmacokinetics of VH-Fc 3C9-YF750 following i.p. administration and imaging at the indicated time points (A) Schematic view of the mice injection and data collection. (B) PBS-YF750 group, VH-Fc 3C9-YF750 group, and IgG1 m912-YF750 group; nude mice (n = 5) were administrated VH-Fc 3C9-YF750 or PBS-YF750 by the i.p. route. The fluorescence intensity at the tumor location (red dashed line circle) of mice shown in (B) was measured at the indicated time points. To confirm and pinpoint the location of the tumor cells, the substrate was injected i.p. at 10 d to measure the signal of luciferase from AsPC-1-luciferase cells. To confirm the antibody enriched in the tumor, the tumor was dissected at 14 dpi for YF750 measurement. (C) Total fluorescence in tumor (ph/s). Data are represented as mean ± SEM.

Figure 5

In vitro cell viability assays MSLN-specific antibody-drug conjugates (ADCs) were evaluated using in vitro cell viability assays on various human cell lines expressing MSLN, including pancreas adenocarcinoma (AsPC-1), mesothelioma (NCI-H2452), epithelioid carcinomas (PANC-1), and ovarian adenocarcinomas (SK-OV-3). Negative control cell lines, namely epidermoid carcinoma (A-431) and Ewing’s sarcoma (A-673), were used for comparison. EC50 values were determined for the cytotoxicity against AsPC-1 and NCI-H2452 cells.

Figure 6

In vivo study of VH-Fc 3C9 ADC (A) Schematic view of the mice injection and data collection. (B) Relative luciferase signals of tumor in control group and ADC groups. p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. (C) Fluorescence measurements of control group and ADC group. (D) Body weight measurement of control group and ADC groups. (E) Survival rate measurement.

Figure 7

Induction of cell-cycle arrest and apoptosis by VH-Fc 3C9 MMAE in MSLN-positive tumor cells (A) Flow cytometric analysis of DNA content distribution. The histogram displays live cells stained with Vybrant DyeCycle Violet stain, with clear labeling of G1 and G2 phase histogram peaks. These results are representative of two independent experiments. (B) Quantitative assessment of the G0/G1 and G2/M phases in AsPC-1 cells and NCI-H2452 cells upon treatment, indicating the effects of VH-Fc 3C9 MMAE. (C) Detection of Caspase-3 or -7 activity. Data shown represent results from two independent experiments and are presented as mean values ± standard error of the mean (SEM). Statistical analysis was conducted using a two-way repeated-measures ANOVA test, with significance levels denoted as follows: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.