Uterine serous papillary carcinomas overexpress human trophoblast-cell-surface marker (Trop-2) and are highly sensitive to immunotherapy with hRS7, a humanized anti-Trop-2 monoclonal antibody

Abstract

Background: Uterine serous papillary carcinoma (USPC) was an aggressive and chemotherapy resistant variant of endometrial cancer. The authors evaluated the expression of human trophoblast-cell-surface-marker (Trop-2) and the potential of hRS7, a humanized anti-Trop-2 monoclonal antibody, as a novel therapeutic strategy against USPC.

Methods: Trop-2 expression was evaluated by immunohistochemistry (IHC) in a total of 23 USPC. Six primary USPC cell lines were assessed by flow cytometry and real-time polymerase chain reaction (PCR) for Trop-2 expression. Sensitivity to hRS7 (Immunomedics, Inc.) antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity was tested in standard 5-hour ⁵¹Cr-release assays against primary USPC cell lines.

Results: Expression of Trop-2 was found in 15 of 23 (65%) of the tumor tissues tested by IHC and in 50% (3 of 6) of the USPC cell lines tested by real-time PCR and flow-cytometry (Trop-2 expression in USPC versus normal endometrial cells; P < .005). USPC cell lines overexpressing Trop-2, regardless of their intrinsic resistance to natural killer cytotoxicity, were highly sensitive to hRS7-mediated ADCC in vitro (range of killing, 28.2% to 64.4%) (P < .001). Negligible cytotoxicity against USPC was seen in the absence of hRS7 or in the presence of rituximab control antibody (range of killing, 1.1% to 12.4%). Incubation with interleukin-2 (50 IU/mL) in addition to hRS7 further increased the cytotoxic activity against USPC cell lines overexpressing Trop-2 (P = .008).

Conclusions: Trop-2 was highly expressed in uterine serous carcinoma at mRNA and protein levels. Primary USPC cell lines are highly sensitivity to hRS7-mediated cytotoxicity in vitro. hRS7 may represent a novel therapeutic agent for USPC refractory to standard treatment modalities.

Figure 1

Representative IHC localization analyses of Trop-2 in USPC specimens. Upper panel: normal endometrium negative for Trop-2. Lower panel: USPC specimen showing 3+ positivity for Trop-2 expression. Original magnification: x 200.

Figure 2

Flow cytometry histograms of primary 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 Trop-2. Rituximab (dashed line); hRS7 (solid black).

Figure 3

Representative cytotoxicity experiments (Effector to target: E/T; 25:1 and 50:1) using hRS7 against high Trop-2 expressing [i.e., USPC ARK-2 (upper panel), USPC ARK-3 (middle panel)] and low Trop-2 expressing USPC cell lines [i.e., USPC ARK-1 (lower panel)]. High levels of hRS7 induced cytotoxicity were evident against USPC ARK-2 and USPC ARK-3 primary cell lines expressing high levels of Trop-2. In contrast, negligible cytotoxicity was detectable against USPC ARK-1 (i.e., a low Trop-2 expressor cell line). In all cell lines tested, no significant cytotoxicity was detected in the absence of hRS7 or in the presence of Rituximab control mAb.

Figure 4

Representative effect of low doses of interleukin-2 (IL-2) in combination with hRS7 (2 μg/ml) on ADCC against USPC ARK-2 primary cell line (Effectors to target ratio 50 : 1). PBL from healthy donors were incubated for 5 hours in the presence of 100 IU/ml of IL-2. hRS7-mediated ADCC was significantly increased in the presence of low doses of IL-2. No significant increase in cytotoxicity was detected after 5 hours IL-2 treatment in the absence of hRS7 or in the presence of the Rituximab isotype control mAb.

Figure 5

Representative cytotoxicity experiments adding human serum (diluted 1:2) to hRS7 against USPC ARK-2 and USPC ARK-3 cell lines. USPC cell lines were treated with serum (with or without heat inactivation) in the presence or absence of the effector cells and hRS7 in a standard 5-hours ⁵¹Cr release assays. Incubation with physiological concentrations of IgG (i.e., heat-inactivated serum diluted 1:2) to PBL in the presence of hRS7 did not significantly reduce the degree of ADCC achieved in the presence of hRS7 against USPC ARK-2 (Upper Panel). Addition of untreated serum (diluted 1:2) to PBL in the presence of hRS7 consistently increased hRS7-mediated cytotoxicity against USPC-ARK-3 (p < 0.02) (lower panel).