Expression of Tissue factor in Adenocarcinoma and Squamous Cell Carcinoma of the Uterine Cervix: Implications for immunotherapy with hI-con1, a factor VII-IgGF_cchimeric protein targeting tissue factor

Despite aggressive screening programs, cervical cancer remains an important public health issue. In the United States about 12,200 new cases of cervical cancer as well as 4,210 deaths from cervical cancer are estimated for 2010 [1]. Although cervical cancer is, to a large extent, a preventable disease, it remains an important health problem for women, especially in underserved and minority groups in industrially developed nations and women in developing countries without established screening programs. While early stage cervical cancer can be cured by radical surgery or radiotherapy with equal effectiveness [2], pelvic radiation represents the standard therapy for the treatment of locally advanced disease. Despite technological advances, however, up to 35% of patients overall will develop recurrent disease, for which treatment results are poor [3]. A deeper understanding of the molecular basis of cervical cancer has the potential to significantly refine the diagnosis and management of these tumors and may eventually lead to the development of novel, more specific, and more effective treatments for prevention of disease progression following first-line therapy.

Angiogenesis, the formation of new vessels from pre-existing vasculature, is known to represent a critical step in the development, progression and metastatic process of human solid tumors. Tissue factor (TF), a transmembrane receptor for coagulation factor VII/VIIa (fVII), is aberrantly expressed in human cancers and on endothelial cells within the tumor vasculature [4, 5]. Importantly, tumor cells characterized by a high production of TF and vascular endothelial growth factor (VEGF), a crucial initiator of angiogenesis, are known to generate solid tumors characterized by intense vascularity and highly aggressive behavior [6]. Consistent with this view, several studies have shown that VEGF is overexpressed and secreted in a variety of human tumors including cervical carcinomas [7] and an elevated expression of VEGF is correlated clinically with cervical cancer metastasis and poor patient survival [8]. While a direct regulation of VEGF expression in human tumor cells by the cytoplasmic tail of TF has been previously demonstrated [7], recent studies indicate that type-2 proteinase activated receptor (PAR-2) is intimately involved in TF-mediated signaling and angiogenesis [9]. These data suggest a potential direct role for TF in tumor growth [9].

hI-con1™ (Iconic Therapeutics, Inc.; Atlanta, GA) is a previously characterized immuno-conjugate molecule developed against TF [10–12]. It is composed of two identical protein chains consisting of human fVII as the targeting domain fused to human IgG₁ F_c as the effector domain; the two chains are held together by the disulfide bonds normally present in IgG. The hI-con1 is designed to bind to TF with far higher affinity and specificity than can be achieved with an anti-TF antibody. Indeed, the hI-con1 has several important advantages over monoclonal antibodies for targeting TF including: 1) The K_d for fVII domain binding to TF is about 10^-12 M [13], in contrast to anti-TF antibodies that have a K_d in the range of 10^-8 to 10^-9 M for TF [14], and 2) the hI-con1 is produced by recombinant DNA technology, allowing a completely human hI-con1 to be made for future clinical trials. Because binding of fVII to TF could induce disseminated intravascular coagulation, a potentially lethal vascular disease, an amino acid substitution was introduced into the fVII domain of the hI-con1 (Lys 341 to Ala) to inhibit initiation of the coagulation pathway without reducing the strong affinity for TF [10, 15]. The human F_c domain of the hI-con1 may thus potentially activate powerful cytolytic responses mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) against both TF-expressing tumor cells and tumor vascular endothelial cells that bind the hI-con1 molecule.

To our knowledge, no studies have specifically described the expression of tissue factor in cervical cancer. Therefore, in this report we investigated the expression of TF at mRNA and protein levels in multiple primary cervical tumor cell lines and evaluated for the first time the in vitro potential of hI-con1 as a novel immunotherapeutic agent against biologically aggressive cervical cancer cell lines overexpressing TF.

The management of disseminated carcinoma of the cervix that is no longer amenable to control with surgery or radiation therapy has not improved significantly with the advent of modern chemotherapy [2, 3]. The one-year survival rate remains between 10 and 15 percent [2, 3]. Thus, the development of novel, target-specific and effective modalities against cervical cancer refractory to standard treatments remains desperately needed.

We have evaluated Tissue Factor (TF) expression by qRT-PCR, IHC and flow cytometry in cervical tissue samples and multiple primary cervical cancer cell lines with squamous- and adenocarcinoma histology, some of which were derived from recurrent chemo-radiation resistant tumors. In addition, we have tested the in vitro activity of hI-con1, a previously characterized immuno-conjugate molecule developed against TF [10–12], as a novel therapy against multiple primary cervical cancer cell lines in vitro. We found both squamous- and adenocarcinoma cell lines to overexpress TF when compared to normal cervical keratinocytes. We speculate that TF expression may be a common and important event in malignant transformation of the cervix into biologically aggressive squamous and adenocarcinoma cervical cancers. Furthermore, both cell lines available to this study established from recurrent/metastatic tumors were found to express extremely high levels of TF. These data are consistent with previous reports in other human solid tumors showing that a high production of TF and vascular endothelial growth factor (VEGF), generate solid tumors characterized by highly aggressive biologic behavior [6]. In this regard, TF is known to be involved in pathological angiogenesis and is abnormally overexpressed in multiple human tumors and in tumor vascular endothelial cells but not on normal quiescent vascular endothelial cells [5, 6]. Although TF, as a cell surface receptor, is physiologically expressed on extravascular cells of many organs and in the adventitial layer of the blood vessel wall, it is sequestered by coagulation factor VII (fVII), a natural ligand for TF, at these sites by the tight endothelial cell layer of the normal vasculature [5, 6]. Thus, pathologically expressed TF may provide a target for the development of novel cancer therapies effective not only against tumor cells but also tumor blood vessels [10–12, 18].

To our knowledge, our results are the first to describe self-production of TF-coagulation factor VII complex by primary cervical cancer cell lines. These data are however consistent with multiple previous reports demonstrating an association between cervical cancer and deep-vein thrombosis and/or pulmonary thromboembolism (i.e., Trousseau syndrome) [19–22], a pathologic state previously reported to be associated with TF overexpression [23]. Importantly, primary cervical cancer cell lines overexpressing TF, regardless of their histology or chemo-radiation therapy resistance, were found to be highly sensitive to hI-con1-mediated cytotoxicity in vitro.

TF is known to play an important role in tumor metastatic process, possibly by inducing the coating of the tumor cell with fibrin that would trap the cells in the microvasculature, thereby aiding metastases [24–26]. Recently, however, a possible direct role for TF in tumor growth has also been suggested by studies showing a dramatically reduced tumor growth in mice where a selective reduction in TF was achieved using small interfering RNA [26]. Of great interest, in these studies, the reduction of TF expression did not affect growth of the tumor cells in vitro, suggesting that TF-mediated enhancement of tumor growth requires a factor present in vivo that is not present when cells are grown in vitro [26]. A potential candidate to explain these findings is therefore fVIIa, which would form a TF:fVIIa complex on the surface of tumor cells in vivo leading to activation of type-2 proteinase activated receptor (PAR2) dependent signaling [9]. These findings combined with our results suggest that TF overexpression may potentially provide an additional growth advantage to biologically aggressive cervical cancers in vivo.

The potential cytotoxic activity of hI-con1 against human melanoma and prostate tumor cells has previously been demonstrated by Hu et al. [10–12]. In a recent study we extended their results by evaluating the cytotoxic potential of hI-con1 against multiple high grade, biologically aggressive, Type II endometrial cancer cell lines [17]. We found all endometrial carcinomas that showed high TF expression, regardless of their high or low HER2/neu expression, were highly susceptible to IDCC in the presence of effector cells [17]. In the current study we expanded our research work with hI-con1 to multiple primary cervical carcinoma cell lines with squamous and adenocarcinoma histology. It is worth noting that although these cell lines were resistant to natural killer cytotoxic activity, IDCC resulted in killing of up to 50% of tumor cells in 5-hour ⁵¹Cr release assays. Taken together, these in vitro results strongly suggest that TF may provide a novel target for the treatment of resistant/residual cervical disease and the destruction of their tumor vasculature that should result in hI-con1-induced lysis of tumor cells as well as tumor endothelial cells in vivo. Consistent with this hypothesis and the central role of angiogenesis in cervical cancer development and progression, bevacizumab, a humanized antibody able to bind and inactivate VEGF, has been recently reported to shrink cervical tumors and delay progression without appreciable toxicity. On the basis of these encouraging results, bevacizumab is currently being studied in a Gynecologic Oncology Group (GOG) phase III trial [27].

For effective cytotoxicity, the effector cells must be able to interact with the immune-conjugate at the target site in vivo, even in the presence of high concentrations of human IgG. In this study we showed that hI-con1-dependent cytotoxicity against cervical cancer cell lines was not inhibited by high concentrations (up to 50%) of human serum. These data, therefore, suggest that in the presence of effector PBL, human serum may not significantly decrease IDCC against cervical cancer cell lines. Moreover, these results indicate that the binding of hI-con1 to the F_c receptor on mononuclear effector cells is likely to occur in vivo.

The degree of ADCC in vitro and in vivo is known to be mainly dependent on the number and function of NK cells [28]. NK cells are considered to be the best suited lymphocytes for ADCC because they carry F_cγRIII receptors, which are activator molecules, and not F_cγRIIb receptors, which inhibit ADCC [29]. NK cells also carry receptors for interleukin-2 (IL-2), a cytokine which induces expansion of the NK cell population and enhances their function [29–31]. Therefore, low dose IL-2 administered in the outpatient setting has been previously used to enhance cancer patients' immune response to monoclonal antibodies with little toxicity [30, 31]. These findings are particularly interesting because in previous studies we have found that a major barrier to the successful development of therapeutic immunization of advanced cervical cancer patients harboring disease refractory to radiation and chemotherapy was represented by a profound state of immunosuppression present in the majority of these patients [32]. This anergic state, reported by other investigators who have also evaluated the immunocompetence of cervical cancer patients [33], has been attributed to the compounded negative effects of tumor-induced immunomodulation with those resulting from radiation and chemotherapy [34, 35]. Importantly, our in vitro experiments reveal a significant increase of hI-con1-induced cytotoxicity after the brief incubation of PBL and tumor cells with IL-2 compared to the cytotoxicity induced by hI-con1 in the absence of IL-2. IL-2 seems to therefore enhance the cytotoxic potential of the effector cells. The administration of low doses of IL-2 might therefore be a valid therapeutic option in order to increase IDCC in heavily pretreated cervical carcinoma patients.

In conclusion, high expression of TF in a large number of cervical carcinomas makes hI-con1 an attractive agent for immunotherapy of recurrent/refractory cervical cancer. In this study, we have demonstrated significant hI-con1-mediated killing of multiple primary cervical cancer cell lines regardless of their squamous or adenocarcinoma histology. hI-con1 might therefore represent a novel treatment of this aggressive disease and, potentially, multiple other human tumors overexpressing TF.