Characterization of zolbetuximab in pancreatic cancer models

Abstract

In healthy tissue, the tight junction protein Claudin 18.2 (CLDN18.2) is present only in the gastric mucosa. Upon malignant transformation of gastric epithelial tissue, perturbations in cell polarity lead to cell surface exposure of CLDN18.2 epitopes. Moreover, CLDN18.2 is aberrantly expressed in malignancies of several other organs, such as pancreatic cancer (PC). A monoclonal antibody, zolbetuximab (formerly known as IMAB362), has been generated against CLDN18.2. In a phase 2 clinical trial (FAST: NCT01630083), zolbetuximab in conjunction with chemotherapy prolonged overall and progression-free survival over chemotherapy alone and improved quality of life. In this study, the mechanism of action and antitumor activity of zolbetuximab were investigated using nonclinical PC models. Zolbetuximab bound specifically and with strong affinity to human PC cells that expressed CLDN18.2 on the cell surface. In ex vivo systems using immune effector cells and serum from healthy donors, zolbetuximab induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), resulting in the lysis of cultured human PC cells. The amplitude of ADCC and CDC directly correlated with cell surface CLDN18.2 levels. The chemotherapeutic agent gemcitabine upregulated CLDN18.2 expression in cultured human PC cells and enhanced zolbetuximab-induced ADCC. In mouse xenograft tumors derived from human PC cell lines, including gemcitabine-refractory ones, zolbetuximab slowed tumor growth, benefited survival, and attenuated metastases development. The results presented here validate CLDN18.2 as a targetable biomarker in PC and support extension of the clinical development of zolbetuximab to patients with CLDN18.2-expressing PC.

Keywords: ADCC; Claudin 18.2; IMAB362; antibody-dependent cellular cytotoxicity; complement-dependent cytotoxicity; immunotherapy; monoclonal antibody; pancreatic cancer; targeted therapy; zolbetuximab.

Figure 1.

Expression of CLDN18.2 in human PC cell lines.

1. (a) Typical image of CLDN18.2⁺ pancreatic ductal adenocarcinoma. Staining was performed with 43-14A antibody. Magnification: 200x.

2. (b) Transcript (qRT-PCR) and (b) protein (western blot) levels of CLDN18.2 in PC cell lines with endogenous (light red bars) and transduced (dark red bars) CLDN18.2 expression. qRT-PCR data are mean ± SD of 1–9 independent measurements. Cell lines with a relative expression level above 1 × 10⁵ were considered CLDN18.2⁺ (dotted line in a). Gray bars represent non-PC cell lines and controls.

3. (c) Detection of CLDN18 protein in pancreatic cancer cell lysates. Western blot analysis was performed using a CLDN18 antibody detecting the C-terminal of CLDN18.1 and CLDN18.2 (C-term, Zymed) and a loading control antibody detecting β-actin. Lysates of SKBR-3 cells were used as negative control, whereas lysates of HEK293 cells stably transfected with CLDN18.2 (HEK293-p740) were used as positive control. BxPC-3 (e) and BxPC-3 (a) represent BxPC-3 cell lines from ECACC and ATCC, respectively.

4. (d) Effect of treatment with Gem or GemOx on CLDN18.2 mRNA expression. mRNA was isolated from DAN-G cells (untreated, treated with Gem [1 ng/mL] or GemOx [Gem 1 ng/mL + Ox 10 ng/mL] for 2 days) or Patu 8988S cells (untreated or treated with Gem [10 ng/mL] or GemOx [Gem 10 ng/mL + Ox 100 ng/mL] for 3 days). RNA was reverse transcribed to cDNA and CLDN18.2 transcript levels analyzed by qRT-PCR. Expression levels are depicted relative to the housekeeping gene HPRT.

5. (e) Effect of treatment with Gem or GemOx on CLDN18 protein expression. CLDN18 protein expression was analyzed in total cell lysates of untreated, Gem- (1 ng/mL) or GemOx- (Gem 10 ng/mL + Ox 100 ng/mL) treated DAN-G, or Patu 8988S cells by Western blot and detected with the Zymed C-term polyclonal antibody. Actin served as loading control.

6. (f) Influence of Gem, and 5-FU on surface expression of CLDN18.2 in Patu 8988S pancreatic cancer cells. CLDN18.2 expression was detected using flow cytometry with zolbetuximab as primary antibody and anti-hu-IgG-APC as secondary antibody. Histogram shows CLDN18.2 expression on Patu 8988S cells treated for 3 days with DMSO (gray line), 10 ng/mL Gem, 500 ng/mL 5-FU, 100 ng/mL PTX, or 500 ng/mL Ox (red line).

Abbreviations: 5-FU, 5-fluorouracil; APC, allophycocyanin; CLDN, claudin; FITC, fluorescein isothiocyanate; Gem, gemcita-bine; GemOx, gemcitabine in combination with oxaliplatin; HPRT, hypoxanthin-guanine-phosphoriboseyltransferase; MFI, mean fluorescence intensity; n.d., not detectable; NTC, non-template control; PC, pancreatic cancer; PTX, paclitaxel; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation.

Figure 2.

Zolbetuximab induces cytotoxicity against human PC cells. (a) Binding dynamics of zolbetuximab (FITC-conjugated) to cell-surface CLDN18.2 on human PC cells by flow cytometry.(b) Density of the zolbetuximab epitope (zolbetuximab molecules bound per cell) on human PC cells. Data are depicted as zolbetuximab molecules bound per cell.(c) Specific lysis of CLDN18.2-expressing cell lines (left panel, endogenous; right panel, transduced) with negative control (MIA PaCa-2) by zolbetuximab-induced ADCC. Data are (a) mean of two or (b) mean ± SD of 3–5 independent donors per cell line.(d) Zolbetuximab-induced ADCC EC₅₀ in DAN-G cells with or without 1 ng/mL Gem pretreatment. Data are mean ± SD of 10 independent donors. P value was calculated with an unpaired t-test. (e) Specific lysis of CLDN18.2-expressing cells (left panel, endogenous; right panel, transduced) by zolbetuximab-induced CDC. Healthy human serum pool served as a complement source. Data are mean ± SD of triplicate.Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; EC₅₀, half-maximal effective concentration; SD, standard deviation.

Figure 3.

Zolbetuximab alone and in combination with gemcitabine exhibits in vivo antitumor activity in mouse xenograft models. (a) Tumor growth kinetics of BxPC-3~ CLDN18.2 xenografts. The size of SC tumors were measured twice weekly. BxPC-3~ CLDN18.2 xenograft tumors were inoculated by SC injection of 8.5 × 10⁶ BxPC-3~ CLDN18.2 cells into the flanks of 10 female Hsd:Athymic Nude-Foxn1^nu mice per treatment group. On Day 3 after tumor cell injection, treatments were initiated with Gem (100 mg/kg IP) and were continued weekly for 6 weeks. 24 h after every injection of Gem, 200 μg zolbetuximab or saline control treatments were applied IV into the tail vein. Zolbetuximab treatment was continued semi-weekly with alternating IP and IV injections until mice were euthanized. Data are mean ± SEM. *P < .05; **P < .01 based on Tukey’s multiple comparisons test.(b) Tumor growth kinetics of MIA PaCa-2~ CLDN18.2 xenografts (left). The size of tumors was measured twice weekly. Data are mean ± SEM. Kaplan–Meier survival estimates (right). Mia PaCa2~ CLDN18.2 xenograft tumors were inoculated by SC injection of 5 × 10⁶ MIA PaCa-2~ CLDN18.2 cells into the flank of 10 female Hsd:Athymic Nude-Foxn1^nu mice per treatment group. On Day 4 after tumor cell injection, treatment was initiated with Gem (50 mg/kg IP) and was continued weekly for 6 weeks. 24 h after injection of Gem, 200 μg zolbetuximab or control treatments were applied IV into the tail vein. Zolbetuximab treatment was continued semi-weekly with alternating IP and IV injections until mice were euthanized (left). *P < .05 (zolbetuximab vs Gem, zolbetuximab+ Gem vs saline); **P < .01 (zolbetuximab+ Gem vs Gem) based on Dunn’s multiple comparisons test (right). *P < .05 (zolbetuximab+ Gem vs Gem) based on log-rank test.(c) IHC analysis of CLDN18.2⁺ tumor cells in MIA PaCa-2~ CLDN18.2 xenografts after treatment with zolbetuximab, Gem, or both. Percentage of CLDN18.2⁺ tumor cells in MIA PaCa-2~ CLDN18.2 xenografts (upper panel). Data points represent individual measurements with the horizontal line representing mean ± SD. ***P < .001 based on one-way ANOVA followed by a Tukey’s multiple comparisons test. CLDN18.2 protein expression in zolbetuximab treated and untreated MIA PaCa-2~ CLDN18.2 xenografts (lower panel). FFPE tissue sections of (a–c) isotype-treated control and (d–f) zolbetuximab-treated mice bearing MIA PaCa-2~ CLDN18.2 xenografts (a, d). 1× overview, magnification 100× (b, e) and 200× (c, f). Staining was performed with Zymed anti-CLDN18 antibody.Abbreviations: ANOVA, analysis of variance; FFPE, formalin-fixed paraffin-embedded; Gem, gemcitabine; IHC, immunohistochemistry; IP, intraperitoneal; IV, intravenous; SC, subcutaneous; SD, standard deviation; SEM, standard error of the mean.

Figure 4.

Zolbetuximab alone and in combination with gemcitabine prevents lung metastasis formation in IV mouse xenograft models. Mice were inoculated by IV injection of 2 × 10⁶ (a) SUIT-2~ CLDN18.2 or (b) Patu 8988S human PC cells into the tail vein of female Hsd:Athymic Nude-Foxn1^nu mice. Alternating IV/IP injections with 200 µg zolbetuximab or isotype (with 100 mg/kg IP gemcitabine in combination studies) were initiated on Day 3 post-graft or 2 weeks after tumor injection and given twice per week. Mice were euthanized at different time points, or after the animals in the control group showed clear physiologic signs of metastatic disease.(a) Human DNA content with zolbetuximab (200 μg) or isotype.(b) Human DNA content (left panel) and MHC-I on lung surface (right panel) with or without zolbetuximab + Gem (zolbetuximab 200 μg + Gem 100 mg/kg semi-weekly for 4 weeks or with 200 μg isotype control antibody + 100 mg/kg Gem semi-weekly). Data are mean ± SEM. **P < .01based on Mann–Whitney U-test (two-tailed). MHC-I staining was performed with anti-human MHC-I (EPR1394Y) antibody.Abbreviations: Gem, gemcitabine; IHC, immunohistochemistry; IP, intraperitoneal; IV, intravenous; MHC, major histocompatibility complex; SEM, standard error of the mean.