Background
Uterine serous carcinoma is an aggressive form of endometrial cancer that carries an extremely poor prognosis. Solitomab is a novel bispecific single-chain antibody construct that targets epithelial cell adhesion molecule on tumor cells and also contains a CD3 binding region. We evaluated the expression levels of epithelial cell adhesion molecule and the in vitro activity of solitomab against primary uterine serous carcinoma cell lines in vitro and ex-vivo in the ascites of patients with uterine serous carcinoma.
Objective
The purpose of this study was to determine the frequency of expression of epithelial cell adhesion molecule on uterine serous carcinoma cell lines and the ability of solitomab to modulate immune responses (T-cell proliferation, activation, cytokine production, and tumor killing) to tumor cells when it is combined with lymphocytes and epithelial cell adhesion molecule–positive cell lines or epithelial cell adhesion molecule–positive ascitic fluid in vitro.
Study Design
Epithelial cell adhesion molecule expression was evaluated by flow cytometry in a total of 14 primary uterine serous carcinoma cell lines. Sensitivity to solitomab-dependent cellular-cytotoxicity was tested against a panel of primary uterine serous carcinoma cell lines that express different levels of epithelial cell adhesion molecule in standard 4-hour chromium release assays. The proliferative activity, activation, cytokine secretion (ie, type I vs type II), and cytotoxicity of solitomab in autologous tumor-associated T cells in the ascitic fluid of patients with uterine serous carcinoma was also evaluated by carboxyfluorescein succinimidyl ester and flow-cytometry assays. Differences in epithelial cell adhesion molecule expression, solitomab-dependent cellular-cytotoxicity levels were analyzed with the use of an unpaired t test. T-cell activation marker increase and cytokine release were analyzed by a paired t test.
Results
Surface expression of epithelial cell adhesion molecule was found in 85.7% (12 of 14) of the uterine serous carcinoma cell lines that were tested by flow cytometry. Epithelial cell adhesion molecule–positive cell lines were found resistant to natural killer cells or T-cell–mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean killing ± standard of the mean, 2.7% ± 3.1% after incubation of epithelial cell adhesion molecule–positive cell lines with control bispecific antibody construct). In contrast, after incubation with solitomab, epithelial cell adhesion molecule–positive uterine serous carcinoma cells became highly sensitive to T-cell cytotoxicity (mean killing, 25.7% ± 4.5%; P < .0001) by peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with epithelial cell adhesion molecule that expressed malignant cells in ascites with solitomab resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (ie, CD25 and HLA-DR), and a reduction in number of viable uterine serous carcinoma cells in ascites ( P < .001).
Conclusion
Solitomab induces robust immunologic responses in vitro that result in increased T-cell activation, proliferation, production of cytokines, and direct killing of tumor cells. These findings suggest that solitomab may represent a novel, potentially effective agent for the treatment of recurrent/metastatic and/or chemo-resistant uterine serous carcinoma–overexpressing epithelial cell adhesion molecule.
Cancer of the uterine corpus is the most prevalent gynecologic tumor in women, with an estimated 54,870 cases and 10,170 deaths in the United States in 2015. Most cancers of the uterus are early-stage, low-grade, and endometrioid tumors (ie, type I). These neoplasms frequently are diagnosed in younger women, are associated with a history of hyperestrogenism as the main risk factor, and typically have a favorable prognosis with appropriate therapy. In contrast, type II endometrial cancers are poorly differentiated tumors, often with uterine serous papillary carcinoma (USC) or clear cell histologic evidence. USC accounts for a disproportionately large number of endometrial cancer deaths, despite the fact that it is found in only 10% of women with newly diagnosed endometrial cancer.
With the goal of the identification of novel diagnostic and therapeutic molecular markers against this deadly variant of endometrial cancer, our group recently has evaluated the genetic landscape and gene expression profiling of a large number of USCs. This study demonstrated epithelial cell adhesion molecule (EpCAM, also known as TROP-1 or TACSTD1), a calcium-independent hemophilic cell adhesion molecule of 39-42 kd, as one of the top differentially expressed genes in USC. The EpCAM protein consists of an extracellular domain with two epidermal growth factor-like repeats, a transmembrane domain, and a short cytoplasmic domain of 26 amino acids. The physiologic function of EpCAM is to promote cell adhesion through low level expression on the basolateral and intercellular surface of simple, pseudostratified, and transitional epithelia, which includes most epithelial tissues in the female genital tract. EpCAM is a multifunctional protein and takes part in mediating cell signaling, cell migration, proliferation, and differentiation. Importantly, because of its relatively low expression in normal tissues and high expression on the cell surface of multiple human carcinomas, EpCAM represents an attractive target for immunotherapy.
Solitomab (MT110, AMG 110; Amgen Research Munich GmbH, Munich, Germany) is an EpCAM/CD3-bispecific single-chain antibody construct in development for the treatment of patients with multiple solid tumors that express EpCAM (CD326). Solitomab mechanism of action is based on the targeting of the EpCAM tumor antigen on tumor cells and the recruitment and activation of T-effector cells via CD3 binding. Solitomab has shown remarkable antitumor activity in preclinical ovarian tumor xenograft models because of the ability to engage resting polyclonal CD8+ and CD4+ T cells for highly potent redirected lysis of target tumor cells.
In this study, we report the first evaluation of the in vitro activity of solitomab against multiple primary USC cell lines and unmanipulated malignant tumor cells that were collected from the ascites of patients who condition harbored recurrent-chemotherapy resistant USC. Our results demonstrate impressive solitomab antitumor activity against USC cell lines and tumor cells that were isolated from the ascites of patients with USC.
Methods
Patients and sample processing
All patients signed an informed consent form according to institutional guidelines; approval for this in vitro study was obtained from the institutional review board. A total of 14 primary USC cell lines were established after sterile processing of surgical biopsy samples, as described previously. Ascitic fluid samples were collected from two additional patients with cytologically confirmed USC recurrence at the time of a therapeutic paracentesis performed at the time of progression after multiple lines of salvage chemotherapy. Patient characteristics of all USC cell lines and the ascitic fluid effusate are described in Table . Primary USC cell lines and freshly collected tumor cells that were floating in the ascitic fluid were tested for presence of EpCAM-positive uterine cancer cells by flow cytometry, as described later.
| Tumor cell line designation | Histologic finding | Binding of epithelial cell adhesion molecule monoclonal antibody | |
|---|---|---|---|
| Cells, % | Mean fluorescence intensity | ||
| USPC-ARK-1 | Pure | 100 | 89 |
| USPC-ARK-2 | Pure | 100 | 124 |
| USPC-ARK-3 | Pure | 100 | 171 |
| USPC-ARK-4 | Mixed | 100 | 78 |
| USPC-ARK-5 | Pure | 100 | 114 |
| USPC-ARK-6 | Mixed | 100 | 86 |
| USPC-ARK-7 | Pure | 100 | 16 |
| USPC-ARK-8 | Pure | 100 | 454 |
| USPC-ARK-11 | Mixed | 20 | 14 |
| USPC-ARK-19 | Pure | 100 | 337 |
| USPC-ARK-20 | Mixed | 100 | 393 |
| USPC-ARK-21 | Pure | 100 | 573 |
| USPC-ARK-22 | Pure | 100 | 1222 |
| USPC-ARK-24 | Mixed | 54 | 60 |
| Ascites T0 | |||
| USPC-ARK-19 | Pure | 100 | 1127 |
| USPC-ARK-22 | Pure | 100 | 827 |
Ex vivo therapy of malignant ascitic fluid samples
Malignant ascites from two patients with USC were analyzed after ex vivo treatment with solitomab or a control bispecific antibody. The malignant ascites were plated in duplicate in 6-well flat microtiter plate. The ascites was treated with the bispecific antibody construct, solitomab at a concentration of 1μg/mL for 5 days. As a control condition, the ascites were treated with control bispecific T-cell–engaging antibody construct (BiTE) huMEC14 also at a concentration of 1μg/mL. The effect of solitomab on the malignant ascites tumor cells was assessed by observation of induction of morphologic changes and extent of cytotoxicity and for evidence of T-cell activation and induction of cytokine release, as described later.
Flow cytometry
Characterization of EpCAM expression in malignant ascitic cells before treatment was performed by fluorescence-activated cell sorting (FACS) analysis. The anti-human EpCAM-PE antibody clone 1B7 (eBioscience Inc, San Diego, CA) was used for flow cytometry studies. The immunoglobulin G1-PE antibody (BD Biosciences, San Jose, CA) was used as antibody isotype control for the anti-EpCAM antibody. The detection of the immune cell fractions was determined by the use of anti–CD8-PE and anti–CD4-PE antibodies. Activation of immune cells was detected by anti-CD25 and anti–HLA-DR antibody. Analysis was conducted with FACScalibur flow cytometer with Cell Quest software (Becton Dickinson Labware, Franklin Lakes, NJ).
T-cell stimulation assay
Solitomab-induced T-cell activation was measured by the detection of CD25 protein surface expression and HLA-DR expression on CD8 + and CD4 + T cells by FACS. Solitomab-mediated stimulation of T cells was calculated according to the following formula: percentage of CD8 + /CD25 + expression = [number of CD8 + /CD25 + cells/total number of CD8 + cells] × 100. Similarly, using the same equation the number of CD8 + /HLA-DR + , CD4 + /CD25 + and CD4 + / HLA-DR + expression was calculated.
Cytokine analysis
The level of solitomab-dependent cytokine induction was compared with the corresponding value of percentage of cytokine release in the control nonspecific antibody control wells. This was performed by treating the solitomab and control nonspecific antibody wells (at a concentration of 1μg/mL of solitomab) with phorbol myristate acetate and ionomycin followed by a 3-hour incubation period to allow for lymphocyte stimulation. Brefeldin A was added, and a further incubation for 3 hours occurred to enhance intracellular cytokine-staining signals. Cytokine analysis of the supernatants was performed by FACS analysis after adding anti–CD8-FITC antibody for surface staining followed by fixation, permeabilization, and intracellular staining with anti–interleukin-4-PE antibody and anti–interferon (IFN) gamma-PE antibody. Solitomab-mediated release of each of these cytokines was calculated according to the following exemplary formula: percentage of CD8 + / IFN gamma–containing cells = [number of CD8 + / IFN gamma cells/ total number of CD8 + cells] × 100. Similar calculations were performed for CD4 + T cells (ie, gated CD3+/CD8− T cells).
Proliferation assay of tumor-associated T lymphocytes (TAL) after the addition of EpCAM BiTE by carboxyfluorescein succinimidyl ester (CFSE) cell proliferation
Briefly, cells were harvested from ascites samples and washed twice with phosphate-buffered saline solution and immediately stained with CFSE (CellTrace CFSE Cell Proliferation Kit, Invitrogen, Carlsbad, CA) at a working concentration of 10 μmol/L. The CFSE-labeled cells were plated and cultured in the presence of control BiTE huMEC14 or solitomab for 5 days. Cells were collected and labeled with CD8 and CD4 (Becton Dickinson Labware) and analyzed on a flow cytometer (FacsCalibur; Becton Dickinson Labware) with the use of Cell Quest software. The percentage and numbers of CD8+ and CD4+ T cells in the control BiTE wells vs solitomab-treated wells were calculated after analysis by flow cytometry to determine TAL proliferation that was induced in this coculture system.
The standard 4-hour chromium ( 51 Cr) release assay was used to measure the cytotoxic reactivity of Ficoll-Hypaque–separated peripheral blood lymphocytes (PBLs) from several healthy donors against 5 representative USC cell lines at effector to target ratios (E:T) of 10:1 and 20:1. The release of 51 Cr from target cells was measured as evidence of tumor cell lysis after exposure of the tumor cells to a concentration of 1μg/mL of solitomab. The negative control conditions were the incubation of target cells alone or with PBL without BiTE antibody construct. As a positive control condition, 1% sodium dodecyl sulfate was used to achieve complete lysis of target cells. The control BiTE huMEC14 at 1μg/mL was used as the negative control for solitomab in this bioassay. The control BiTE antibody construct shared the CD3 binding arm with solitomab but otherwise recognizes an herbicide as an irrelevant antigen instead of recognizing EpCAM. The percentage cytotoxicity of solitomab was calculated by the following formula: % cytotoxicity = 100 × (E–S)/(T–S), where E is the experimental release, S is the spontaneous release by effector cells, T is the maximum release by target cells lysed with 1% sodium dodecyl sulfate.
Statistics
Differences in EpCAM expression by flow cytometry were analyzed by the unpaired t test. The unpaired t test was used to evaluate the differences in solitomab-dependent cellular-cytotoxicity (ADCC) levels in primary tumor cell lines that were treated with solitomab vs control BiTE huMEC14. T-cell activation marker increase and cytokine release were analyzed by the paired t test. Statistical analysis was performed with SPSS software (version 18; SPSS Inc, Chicago, IL). A probability value of <.05 was considered to be the level of statistical significance.
Results
EpCAM levels in primary USC cell lines and ascitic samples
We used flow cytometry to obtain highly sensitive measurements of EpCAM surface expression in 14 primary USC lines (2 established from patients whose condition harbored chemotherapy-resistant disease) and in 2 ascitic fluid samples that were collected through paracentesis that was performed at the time of tumor progression after multiple regimens of chemotherapy. Table depicts the characteristics of the patients from which the samples were collected and shows the percentage of EpCAM-positive cells and corresponding mean fluorescence intensity in primary USC cell lines and ascitic fluid samples by flow cytometry. Among the USC primary cell lines, 12 of 14 (85.7%) were found to express EpCAM in 100% of the tumor cells ( Table ; Figure 1 ). EpCAM expression levels were highest in uterine serous papillary adenocarcinoma (USPC)–ARK-22 specimens (both in the primary cell line that was tested after multiple passages in vitro and in the freshly collected tumor cells floating in patient’s ascites that were collected at the time of tumor progression; Table ). In contrast, lower EpCAM expression was found in USPC-ARK-4, -7, -11, and -24.
USC primary cell lines are resistant to natural killer (NK) cell activity but sensitive to solitomab-mediated T-cell cytotoxicity
Standard 4-hour 51 Cr release assay cytotoxicity titration was performed with solitomab (EpCAM BiTE) from doses of 0-2.5 μg/mL in several experiments that were aimed at determining the optimum dose of solitomab against a representative USC. From these experiments the 1 μg/mL concentration emerged as the optimum concentration for tumor killing ( Figure 2 , upper panel). Next, 5 representative primary uterine cancer cell lines were evaluated for their sensitivity to NK and T cells. These USC cell lines were exposed to PBLs that were collected from multiple healthy donors in several cytotoxicity assays.
