A glypican-1-targeted antibody-drug conjugate exhibits potent tumor growth inhibition in glypican-1-positive pancreatic cancer and esophageal squamous cell carcinoma

Abstract

An antibody-drug conjugate (ADC) is a promising therapeutic modality because selective and effective delivery of an anti-cancer drug is achieved by drug-conjugated antibody-targeting cancer antigen. Glypican 1 (GPC1) is highly expressed in malignant tumors, including pancreatic ductal adenocarcinoma (PDAC) and esophageal squamous cell carcinoma (ESCC). Herein, we describe the usefulness of GPC1-targeting ADC. Humanized anti-GPC1 antibody (clone T2) was developed and conjugated with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PABC) linkers (humanized GPC1-ADC[MMAE]). Humanized GPC1-ADC(MMAE) inhibited the growth of GPC1-positive PDAC and ESCC cell lines via inducing cycle arrest in the G2/M phase and apoptosis in vitro. The binding activity of humanized GPC1-ADC(MMAE) with GPC1 was comparable with that of the unconjugated anti-GPC1 antibody. The humanized GPC1-ADC(MMAE) was effective in GPC1-positive BxPC-3 subcutaneously xenografted mice but not in GPC1-negative BxPC-3-GPC1-KO xenografted mice. To assess the bystander killing activity of the humanized GPC1-ADC(MMAE), a mixture of GPC1-positive BxPC-3 and GPC1-negative BxPC-3-GPC1-KO-Luc cells were subcutaneously inoculated, and a heterogenous GPC1-expressing tumor model was developed. The humanized GPC1-ADC(MMAE) inhibited the tumor growth and decreased the luciferase signal, measured with an in vivo imaging system (IVIS), which suggests that the suppression of the BxPC-3-GPC1-KO-Luc population. The humanized GPC1-ADC(MMAE) also inhibited the established liver metastases of BxPC-3 cells and significantly improved the overall survival of the mice. It exhibited a potent antitumor effect on the GPC1-positive PDAC and ESCC patient-derived xenograft (PDX) models. Our preclinical data demonstrate that GPC1 is a promising therapeutic target for ADC.

Keywords: Antibody-drug conjugate; Esophageal squamous cell carcinoma; Glypican-1; Pancreatic cancer.

Fig. 1

Structure, characterization, and internalization activity of the humanized GPC1-ADC A, Flow cytometric analysis of GPC1 expression. The shaded histogram profile indicates the isotype control, and the open histogram indicates the humanized anti-GPC1 antibody-staining results. B, Structure of the humanized GPC1-ADC consisting of the humanized anti-GPC1 antibody (clone T2) conjugated to the MMAE payload. C, BxPC-3 cells are incubated with unconjugated humanized anti-GPC1 mAb (blue closed circles) or humanized GPC1-ADC (red closed triangles). The mean fluorescence intensity at various concentrations is shown. D, Time-course analysis of the internalization activity of the antibody or ADC in BxPC-3 cells. The left panel shows unconjugated humanized anti-GPC1 mAb (clone T2); and the right panel, humanized GPC1-ADC. E, GPC1-ADC internalizes and locates in the lysosomes of BxPC-3 cells. Cell surface and intracellular GPC1 visualized on fluorescence microscopy. Green indicates the humanized anti-GPC1 antibody (clone T2) or humanized GPC1-ADC; red, the lysosomal marker LAMP-1; and blue, 4′,6-diamidino-2-phenylindole-stained DNA. Scale bar: 10 mm. (Color version of figure is available online)

Fig. 2

In vitro cell growth inhibitory activity of ADC A, Cancer cells were treated with humanized GPC1-ADC(MMAE) or human IgG4-ADC (a control-ADC[MMAE]) for 144 h. The humanized GPC1-ADC(MMAE) significantly inhibited the growth of the GPC1-positive cancer cell lines compared with the control-ADC(MMAE). In the GPC1-negative BxPC-3-GPC1 and TE8-GPC1 KO cell lines, neither treatment had any inhibitory effect. B, Induction of G2/M phase cell cycle arrest in BxPC-3, KP-2, PK-8, TE-8, TE-14, and KYSE70 cells treated with humanized GPC1-ADC(MMAE). Cells were treated with either 16 nM control-ADC(MMAE) or 16 nM humanized GPC1-ADC(MMAE). After 24 h, cell cycle analysis was performed by flow cytometry with propidium iodide DNA staining. **P < 0.01, by one-way ANOVA, followed by Dunnett's post hoc test. C, Induction of apoptosis in BxPC-3, KP-2, PK-8, TE-8, TE-14, and KYSE70 cells treated with 16 nM control-ADC(MMAE) or 16 nM humanized GPC1-ADC(MMAE). Caspase 3/7 activation was monitored using the Caspase Glo 3/7 assay system at 48 h after addition of ADCs. Caspase 3/7 activation relative to untreated cells was detected in these cells treated with increasing concentrations of humanized GPC1–ADC. *P < 0.05 and **P < 0.01, by Student's t test. D, Western blot analysis of proteins involved in the apoptosis pathways. BxPC-3 and TE-8 cells were treated with either 16 nM control-ADC(MMAE) or 16 nM humanized GPC1-ADC(MMAE). After 48 h, cells were harvested and lysed. The protein level of poly ADP-ribose polymerase (PARP), cleaved PARP, and Cleaved Caspase-3 was detected by western blot; β-actin was used as a loading control.

Fig. 3

Antitumor activity of the humanized GPC1-ADC(MMAE) in vivo A, Representative images of IHC staining for GPC1 in xenografted tumor tissues from BxPC-3, BxPC-3-GPC1-KO-Luc#15, and the co-inoculation of BxPC-3 and BxPC-3-GPC1-KO-Luc#15. Scale bar: 100 mm. B, Antitumor efficacy of the humanized GPC1-ADC(MMAE) in the BxPC-3 xenograft model (each n = 7 per group). Each point on the graph represents the average tumor volume. The changes in the relative body weight are represented. C, Antitumor efficacy of humanized GPC1-ADC(MMAE) in the BxPC-3-GPC1-KO-Luc#15 xenograft model (each n = 7 per group). Each point on the graph represents the average tumor volume. Changes in the relative body weight are also represented. D, Bystander killing effect of the humanized GPC1-ADC(MMAE) in the co-inoculated conditions in vivo. The time-course of the luciferase activity was detected by bioluminescence imaging (BLI) after the intraperitoneal administration of the substrate. The imaging data of the luciferase activity are shown in D. E, Tumor volumes are shown in the left panel. Quantification of the tumor burden from the BxPC-3-GPC1-KO-Luc#15 is shown in the middle panel. Changes in relative body weight are depicted in the right panel. F, BxPC-3-GPC1-KO cell viability after treatment with the conditioned medium from the humanized GPC1-ADC-treated BxPC-3 cells. G, Conditioned media generated by exposing the BxPC-3 cells with the control-ADC or humanized GPC1-ADC for 48 h. After 144 h, the cell viability was determined.

Fig. 4

Antitumor activity of the humanized GPC1-ADC(MMAE) in the pancreatic cancer liver metastasis xenograft model A, Representative images of IHC staining for GPC1 in tumor tissues in the matched primary and liver metastases of a patient with PDAC. Scale bar: 100 mm. B, Representative images of IHC staining for GPC1 in BxPC-3-Luc#2 pancreatic cancer liver metastases after intrasplenic injection of tumor cells are shown. Scale bar: 100 mm. C, The humanized GPC1-ADC(MMAE) inhibits the BxPC-3-Luc#2 pancreatic cancer liver metastases in the mice after intrasplenic injection of tumor cells. BLI was used to monitor the BxPC-3-Luc#2 pancreatic cancer liver metastases in the mice after intrasplenic injection of cancer cells. BLI was used 7 to 14 days post-inoculation to randomize the mice into vehicle (n = 13) or 10-mg/kg humanized GPC1-ADC(MMAE) treatment groups (n = 10). Note the decrease in bioluminescence in the humanized GPC1-ADC(MMAE)-treated group at 49 days post-inoculation. D, Quantification of the tumor burden from the BxPC-3-Luc#2 liver metastasis study shown in C. Changes in relative body weight. The error bars denote the SEM. E, Kaplan-Meier survival analysis of the BxPC-3-Luc#2 liver metastasis study shown in C. Log-rank analysis: p < 0.0001, humanized GPC1-ADC(MMAE) versus vehicle.

Fig. 5

Antitumor activity of the humanized GPC1-ADC(MMAE) in the pancreatic cancer and ESCC PDX models A-E, Antitumor efficacy of the humanized GPC1-ADC(MMAE) in the PK565, PK175, KPK1, ESCC2, and ESCC14 PDX models (n = 7–8 per group). Representative images of IHC staining for GPC1 in xenografted tumor tissues from untreated mice are inserted. Scale bar: 100 mm. The tumor-bearing mice were given PBS, control-ADC(MMAE) (10 mg/kg), or humanized GPC1-ADC(MMAE) (1, 3, and 10 mg/kg) intravenously on days 0, 7, 14, and 21. Each point on the graph represents the average tumor volume. Changes in body weight are also represented. F, Humanized GPC1-ADC causes mitotic arrest in vivo. Animals bearing the PK565 tumor xenografts are given a single dose of PBS, control-ADC(MMAE) (10 mg/kg), or humanized GPC1-ADC(MMAE) (10 mg/kg). After 24 h, the tumors were harvested and stained with an anti-phospho-histone H3 (Ser10) antibody to detect mitotic cells. Scale bar: 100 mm. G Phospho-histone H3 (Ser10) staining was assessed as the ratio of mitotic cells to the total number of tumor cells in 4 fields (magnification, x 200). **P < 0.01, one-way ANOVA, followed by Dunnett's post hoc test.