hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor for immunotherapy of uterine serous papillary carcinoma

Abstract

Background: Uterine serous papillary adenocarcinoma (USPC) is a highly aggressive variant of endometrial cancer. Human immuno-conjugate molecule (hI-con1) is an antibody-like molecule targeted against tissue factor (TF), composed of two human Factor VII (fVII) as the targeting domain, fused to human immunoglobulin (Ig) G1 Fc as an effector domain. We evaluated hI-con1 potential activity against primary chemotherapy-resistant USPC cell lines expressing different levels of TF.

Methods: A total of 16 formalin-fixed, paraffin-embedded USPC samples were evaluated by immunohistochemistry (IHC) for TF expression. Six primary USPC cell lines, half of which overexpress the epidermal growth factor type II (HER2/neu) receptor at 3+ levels, were assessed by flow cytometry and real-time PCR for TF expression. Sensitivity to hI-con1-dependent cell-mediated cytotoxicity (IDCC) was evaluated in 5-hour-chromium release assays. Finally, to investigate the effect of interleukin-2 (IL-2) on IDCC, 5-h (51)Cr assays were also conducted in the presence of low doses of IL-2 (i.e., 50-100 IU ml(-1)).

Results: Cytoplasmic and/or membrane TF expression was observed in all 16 (100%) USPC samples tested by IHC, but not in normal endometrium. High expression of TF was found in 50% (three out of six) of the USPC cell lines tested by real-time PCR and flow cytometry when compared with normal endometrial cells (NECs; P<0.001). Uterine serous papillary adenocarcinoma cell lines overexpressing TF, regardless of their high or low HER2/neu expression, were highly sensitive to IDCC (mean killing+/-s.d., 65.6+/-3.7%, range 57.5-77.0%, P<0.001), although negligible cytotoxicity against USPC was seen in the absence of hI-con1 or in the presence of Rituximab control antibody. The addition of low doses of IL-2 further increased the cytotoxic effect induced by hI-con1 against chemotherapy-resistant USPC.

Conclusion: hI-con1 induces strong cytotoxicity against primary chemotherapy-resistant USPC cell lines overexpressing TF. The hI-con1 may represent a novel therapeutic agent for the treatment of patients harbouring advanced, recurrent and/or metastatic USPC refractory to standard treatment modalities.

Figure 1

Representative immunohistochemistry localisation analyses of tissue factor (TF) in uterine serous papillary adenocarcinoma (USPC) specimens. Upper left panel: normal endometrium, which is negative for TF. Lower left panel: USPC specimen showing cytoplasmic expression of TF. Upper right panel: USPC specimen showing membrane expression of TF. Lower right panel: USPC specimen showing both cytoplasmic and membrane expressions of TF. Original magnification: × 200.

Figure 2

Flow cytometry histograms of primary uterine serous papillary adenocarcinoma (USPC) cell lines showing high (USPC-ARK-2, USPC-ARK-3 and USPC-ARK-6) and low (USPC-ARK-1, USPC-ARK-4 and USPC-ARK-5) expression of tissue factor. Dashed line represents isotype and solid black represents human immuno-conjugate molecule.

Figure 3

Representative cytotoxicity experiments using human immuno-conjugate molecule (hI-con1) against primary uterine serous papillary adenocarcinoma (USPC) with high vs low tissue factor (TF) expression. Upper panels: high TF USPC cell lines. Lower panels: low TF USPC cell lines. Negligible cytotoxicity was detected in the absence of hI-con1 or in the presence of rituximab control monoclonal antibody.

Figure 4

Effect of low doses of interleukin-2 (IL-2) in combination with human immuno-conjugate molecule (hI-con1; 30 μg ml⁻¹) on antibody-dependent cell-mediated cytotoxicity against USPC-ARK-2, USPC-ARK-3 and USPC-ARK-6 primary cell lines (effectors to target ratio 25 : 1). Peripheral blood lymphocytes (PBLs) from healthy donors were incubated for 4 h in the presence of 100 IU ml⁻¹ of IL-2. The hI-con1-mediated antibody-dependent cell-mediated cytotoxicity was significantly increased in the presence of low doses of IL-2. No significant increase in cytotoxicity was detected after 4-h IL-2 treatment in the absence of hI-con1 or in the presence of the rituximab isotype control monoclonal antibody.