Objective
We evaluated the expression of human trophoblast cell-surface marker (Trop-2) and the potential of hRS7, a humanized monoclonal anti-Trop-2 antibody, against treatment-refractory cervical cancer.
Study Design
Trop-2 expression was evaluated by immunohistochemistry, real-time polymerase chain reaction, and flow cytometry. Sensitivity to hRS7 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity was tested in 5-hour chromium release assays. The effect of interleukin (IL)-2 on hRS7 ADCC was also investigated.
Results
Membrane Trop-2 expression was observed in 8 of 8 (100%) of the cancer samples tested by immunohistochemistry, but not in normal cervix. High messenger RNA expression by real-time polymerase chain reaction and high Trop-2 surface expression by flow cytometry were detected in 80% of cervical cancers (4 of 5 cell lines). Although these tumors were resistant to natural killer cell–dependent cytotoxicity in vitro (mean killing, 6.0%), Trop-2-positive cell lines showed high sensitivity to hRS7 ADCC (range of killing, 30.6–73.2%). Incubation with IL-2 further increased the level of cytotoxicity against Trop-2-positive tumors.
Conclusion
hRS7 may represent a novel treatment option for patients with cervical cancer refractory to conventional treatment modalities.
Cancer of the uterine cervix is a significant public health issue as it is the fourth leading cause of cancer death in women worldwide, with about 529,800 new cases and 275,100 deaths each year. In the United States, 12,200 new cases of cervical cancer and 4210 deaths were estimated for 2010. Cervical cancers caught in earlier stages can be treated by radical surgery or radiotherapy with equal effectiveness. However, despite screening programs, many patients still present with advanced-stage disease. For patients with locally advanced disease, pelvic radiation represents the standard of therapy. However, up to 35% of patients will develop recurrent disease for which treatment results are poor. Thus, there is a large need to develop targeted therapies for cervical cancer patients who have failed chemotherapy, radiation, and/or surgery.
Human trophoblast cell-surface marker (Trop-2) (also termed TACSTD2, GA733-1, M1S1, and EGP-1) is a surface glycoprotein originally identified in human placental trophoblastic tissue and subsequently reported to be highly expressed by various types of human carcinomas, but rarely in normal adult tissues. The biological function of Trop-2 is unclear, although recently it has been speculated that it exerts its action via activation of the extracellular-signal-regulated kinases /mitogen-activated protein kinases pathway. However, its overexpression has been found to correlate with invasive behavior and poor prognosis in various human carcinomas. Due to the overexpression of Trop-2 by tumor cells and its location on the cell surface, it is an attractive target for cancer immunotherapy, as recently demonstrated by our group against a rare but highly aggressive variant of endometrial cancer (ie, uterine serous carcinoma).
hRS7 is a humanized IgG1 monoclonal antibody (MAb) developed against Trop-2 using complementary-determining region and transfection techniques of the murine RS7-3G11 antibody (Immunomedics Inc, Morris Plains, NJ). RS7-3G11 was shown to be rapidly internalized by target cells, so hRS7 was initially tested labeled with a radioiodinated, diethylenetriaminepentaacetic acid-appended peptide, designated immunomedics’ residualizing peptide 4 ( 131 I-IMP-R4) to evaluate its effectiveness in preclinical radioimmunotherapy studies on breast cancer xenograft models. These results suggested that hRS7 may be promising as a carrier for radiometabolic therapy after labeling with suitable radionuclides. Nevertheless, our group has recently shown that hRS7 also induces antibody-dependent cell-mediated cytotoxicity (ADCC) against biologically aggressive (ie, type II) endometrial carcinoma cell lines overexpressing Trop-2. To our knowledge, however, the potential ability of hRS7 in inducing ADCC against primary cervical carcinoma cell lines has not been previously studied.
We, therefore, studied the expression of Trop-2 in multiple cervical carcinoma cell lines and investigated the in vitro potential of hRS7 as an innovative immunotherapeutic agent against cervical cancer cell lines overexpressing Trop-2.
Materials and Methods
Trop-2 immunostaining of formalin-fixed cervical cancer tissues
Formalin-fixed, paraffin-embedded tissue blocks from 8 patients harboring stage Ib (6 patients), stage II (1 patient), and stage IIIb (1 patient) cervical carcinomas (ie, 5 squamous and 3 adenocarcinomas) and 5 normal cervical control tissues obtained from similar-age women were evaluated by standard immunohistochemical (IHC) staining for Trop-2 surface expression. Specimens were reviewed by a surgical pathologist (N.B.). Briefly, IHC stains were performed on 4-μm-thick sections of formalin-fixed, paraffin-embedded tissue as previously described. The purified goat polyclonal antibody against the recombinant human Trop-2 extracellular domain (R&D Systems Inc, Minneapolis, MN) (diluted 1:100) was applied for 1 hour. A secondary biotinylated antigoat antibody (Vector Laboratories, Burlingame, CA) (diluted 1:250) and the streptavidin-biotin complex (StreptABComplex/horseradish peroxidase; Dako, Carpinteria, CA) were applied, then 3’3-diaminobenzidine (Dako) was used as chromogen and the sections were counterstained by hematoxylin (Dako). Cases with <10% membranous staining in tumor cells were considered negative for Trop-2 expression. The intensity of membranous immunoreactivity for Trop-2 in tumor cells was subjectively scored as follow: (a) 0, negative; (b) 1+, weak membrane staining; (c) 2+, medium staining; and (d) 3+, strong membrane staining. Appropriate negative and positive controls were performed with each case.
Establishment of primary cervical cancer cell lines
Primary cervical tumor cell lines from 5 patients were established after sterile processing of fresh tumor biopsies collected at the time of primary surgery under approval of the institutional review board. Tumors were staged according to the International Federation of Gynecology and Obstetrics staging system. Source-patient characteristics of these 5 cell lines are described in Table 1 .
| Cell line | Age at diagnosis, y | FIGO stage | Histology | HPV status | mRNA copy no. |
|---|---|---|---|---|---|
| CVX-SCC-1 | 42 | Recurrence | Squamous cell | 18 | 120.35 |
| CVX-SCC-2 | 40 | IB | Squamous cell | 16 | 3035.66 |
| ADX-1 | 25 | IB | Adenocarcinoma | 18 | 351.28 |
| ADX-2 | 33 | IB | Adenosquamous | 18 | 9.4 |
| ADX-3 | 33 | IB | Adenosquamous | 18 | 117.56 |
Quantitative real-time polymerase chain reaction in primary cell lines
RNA isolation from the 5 primary cervical cancer cell lines ( Table 1 ) was performed using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Since Trop-2 is an intronless gene, all RNA samples were treated with Turbo DNase enzyme (Turbo DNA-free kit; Applied Biosystems, Foster City, CA) to remove any contaminating DNA that was present. Quantitative real-time (qRT)-polymerase chain reaction (PCR) was performed in duplicate by using a primer set and probe specific for the Trop-2 (ie, Trop2-EX56) with a 7500 real-time PCR system using the manufacturer’s recommended protocol (Applied Biosystems). The comparative threshold cycle ( C T ) method (Applied Biosystems) was used to determine gene expression in each sample relative to the value observed in the lowest nonmalignant cervical keratinocyte cell sample, using glyceraldehyde-3-phosphate dehydrogenase (assay ID Hs99999905_m1) RNA as internal control.
Flow cytometry
The humanized anti-Trop-2 MAb hRS7 (Immunomedics Inc) was used for flow cytometry studies. Briefly, 5 primary cervical cancer cell lines established from the above-described patients were treated with 2 μg/mL of hRS7. The chimeric anti-CD20 MAb rituximab (Rituxan; Genentech, San Francisco, CA) at the dose of 2.5 μg/mL was used as negative control. A goat antihuman fragment antigen binding immunoglobulin (BioSource International, Camarillo, CA) was used as a secondary reagent. Analysis was conducted with a FACScan, using Cell Quest software (Beckton Dickinson, Franklin Lakes, NJ).
Tests for ADCC
A standard 5-hour chromium ( 51 Cr) release assay was performed to measure the cytotoxic reactivity of Ficoll-Paque PLUS (GE Healthcare, Kings Park, NY) separated peripheral blood lymphocytes (PBLs) obtained from several healthy donors against all 5 cervical cancer cell lines. The release of 51 Cr from the target cells was measured as evidence of tumor cell lysis after exposure of tumor cells to 2 μg/mL of hRS7. This dose was used because previous hRS7 dose-titration experiments demonstrated that killing of cervical cancer cells plateaued at a hRS7 concentration of 2 μg/mL (data not shown). Controls included the incubation of target cells alone or with PBL or MAb separately. The chimeric anti-CD20 MAb rituximab was used as a negative control for hRS7 in all bioassays. ADCC was calculated as the percentage of killing of target cells observed with hRS7 plus effector cells compared with 51 Cr release from target cells incubated alone.
Interleukin-2 enhancement of ADCC
To investigate the effect of interleukin (IL)-2 on hRS7-mediated ADCC, effector PBLs were incubated for 5 hours at 37°C at a final concentration of IL-2 (Aldesleukin; Chiron Therapeutics, Emeryville, CA) ranging from 50-100 IU/mL in 96-well microtiter plates. Target cells were primary cervical cancer cell lines exposed to 2 μg/mL of hRS7, whereas controls included the incubation of target cells alone or with PBL in the presence or absence of IL-2 or MAb, respectively. Rituximab was used as a control MAb. ADCC was calculated as the percentage of killing of target cells observed with MAb plus effector PBL, as compared with target cells incubated alone.
Tests for complement-mediated target cell lysis and γ-globulin inhibition
A standard 5-hour 51 Cr release assay identical to those performed for ADCC assays was used. However, human serum was added as a source of complement to test for complement-mediated target cell lysis. To test for the possible inhibition of ADCC against cervical cancer cell lines by physiological human serum concentrations of γ-globulin, human serum diluted 1:2 was added in the presence or absence of effector PBL. The effect of heat-inactivated human serum (56°C for 60 minutes) was also tested in the presence of effector PBL. Controls included the incubation of target cells alone or with either lymphocytes or MAb separately. Rituximab was used as a control MAb.
Statistical analysis
qRT-PCR data were evaluated using unequal variance t test for cervical carcinoma vs normal difference. Differences in Trop-2 expression by flow cytometry were analyzed by the unpaired t test, and a P value of < .05 among samples was considered to be significant. Kruskal-Wallis test and χ 2 analysis were used to evaluate differences in hRS7-induced ADCC levels in primary tumor cell lines. Statistical analysis was performed using software (PASW Statistics, version 18; SPSS, Chicago, IL).
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