A Novel Bispecific Antibody for EpCAM-Directed Inhibition of the CD73/Adenosine Immune Checkpoint in Ovarian Cancer

Abstract

PD-1/PD-L1-inhibiting antibodies have shown disappointing efficacy in patients with refractory ovarian cancer (OC). Apparently, OC cells exploit nonoverlapping immunosuppressive mechanisms to evade the immune system. In this respect, the CD73-adenosine inhibitory immune checkpoint is of particular interest, as it rapidly converts pro-inflammatory ATP released from cancer cells to immunosuppressive adenosine (ADO). Moreover, cancer-cell-produced ADO is known to form a highly immunosuppressive extra-tumoral 'halo' that chronically inhibits the anticancer activity of various immune effector cells. Thus far, conventional CD73-blocking antibodies such as oleclumab show limited clinical efficacy, probably due to the fact that it indiscriminately binds to and blocks CD73 on a massive surplus of normal cells. To address this issue, we constructed a novel bispecific antibody (bsAb) CD73xEpCAM that inhibits CD73 expressed on the OC cell surface in an EpCAM-directed manner. Importantly, bsAb CD73xEpCAM showed potent capacity to inhibit the CD73 enzyme activity in an EpCAM-directed manner and restore the cytotoxic activity of ADO-suppressed anticancer T cells. Additionally, treatment with bsAb CD73xEpCAM potently inhibited the proliferative capacity of OC cells and enhanced their sensitivity to cisplatin, doxorubicin, 5FU, and ionizing radiation. BsAb CD73xEpCAM may be useful in the development of tumor-directed immunotherapeutic approaches to overcome the CD73-mediated immunosuppression in patients with refractory OC.

Keywords: CD73; EpCAM; adenosine; bispecific antibody; ovarian cancer.

Figure 1

BsAb CD73xEpCAM has dual binding specificity for CD73 and EpCAM. (A) Competitive binding assay in which bsAb CD73xEpCAM (1 μg/mL) was pretreated with excess amounts of soluble CD73 (sCD73), soluble EpCAM (sEpCAM), or a combination thereof (10 μg) and then evaluated for binding to OvCAR3 cancer cells. (B) Dose-dependent binding of bsAb CD73xEpCAM to L37 and L37.EpCAM. (C) Binding of bsAb CD73xEpCAM (1 μg/mL) (or controls) to parental OvCAR3and corresponding CD73-KO and EpCAM-KO variants thereof. (D) Residual binding of bsAb CD73xEpCAM (1μg/mL) (or controls) to OvCAR3 cancer cells at indicated time points after 30 s of incubation and subsequent washing with PBS. (E) Residual CD73 membrane presence on OvCAR3 cells after treatment with bsAb CD73xEpCAM (0.01–10 µg/mL) (or controls) for 5 h. All experiments were analyzed by flow cytometry. All graphs represent mean ± SD.

Figure 2

BsAb CD73xEpCAM potently inhibits the enzyme activity of CD73 in an EpCAM-directed manner. (A) Kinetics of inhibition of OvCAR3-exposed CD73 enzyme activity by bsAb CD73xEpCAM (1 μg/mL) (or controls). Inhibition of CD73 enzyme activity by treatment (15 min) with bsAb CD73xEpCAM (1 μg/mL) (or controls) of (B) CD73^pos/EpCAM^pos cancer cell lines, (C) CD73^pos/EpCAM^neg cancer cell lines, and (D) primary-OC-patient-derived carcinoma cells. (E) Competitive CD73 enzyme inhibition assay on OvCAR3 cells after treatment (15 min) with bsAb CD73xEpCAM in the presence of excess amounts of soluble EpCAM (sEpCAM). (F) Dose-dependent inhibition of CD73 enzyme activity by bsAb CD73xEpCAM (0.01–10 µg/mL) exposed on parental OvCAR3 cells versus OvCAR3 EpCAM-KO cells. CD73-mediated hydrolysis of AMP to ADO was evaluated using a colorimetric malachite green-based Pi assay. All graphs represent mean ± SD. Statistical analysis in (B,D) (group-mean) was performed using unpaired t-test (* p < 0.05, ** p < 0.01).

Figure 3

BsAb CD73xEpCAM restores the anticancer activities of ADO-suppressed T cells. (A) Representative images of activated CFSE-FarRed-labeled PBMCs treated (15 min) (or not) with bsAb CD73xEpCAM (1 µg/mL) (or controls), washed, and then cultured in medium supplemented with AMP at 37 °C for 7 d. Scale bar = 400 µm. (B) Cluster size (µm²/image) of activated proliferating T cell quantified using live cell imaging by taking pictures at 4× magnification at 37 °C every 6 h. (C) Percentage of AnnexinV-PI^pos (apoptotic) OvCAR3 cancer cells treated (15 min) with bsAb CD73xEpCAM (1 µg/mL) (or controls), washed, and then cultured in medium supplemented with AMP at 37 °C for 24 h. Subsequently, PBMCs were redirected to kill OvCAR3 cancer cells at increasing effector (E) to target (T) cell ratios. (D) IFN-γ levels excreted in culture supernatant (C) were measured by ELISA. All graphs represent mean ± SD. Ole = oleclumab in graphs B and C. Statistical analysis in D was performed using unpaired t-test (** p < 0.01).

Figure 4

BsAb CD73xEpCAM inhibits the proliferative capacity of EpCAM-expressing cancer cells. Proliferation of (A) OvCAR3 and (B) primary-OC-patient-derived carcinoma cells after treatment with bsAb CD73xEpCAM (or controls) (1 μg/mL), using the RTCA xCELLigence instrument. The readout is indicated as cell-index, an arbitrary unit for attachment of adherent cells and cell proliferation measured at 37 °C every 15 min. (C) Inhibition of colony formation by OvCAR3 after treatment (15 min) with bsAb CD73xEpCAM (0.1–2 μg/mL) (or controls) and cultured at 37 °C for 14 d. (D) Table with IC50 values (μg/mL) calculated for graph (C). All graphs represent mean ± SD.

Figure 5

BsAb CD73xEpCAM sensitizes OvCAR3 cancer cells towards chemo- and radiotherapy. OvCAR3 cell confluence after pretreatment (15 min) with (A) bsAb CD73xEpCAM (1 μg/mL) (or controls) and subsequent treatment with (B) cisplatin (1 µg/mL), (C) doxorubicin (200 nM), or (D) 5FU (15 μg/mL). Cell confluence was evaluated using live cell imaging technology by taking pictures at 4× magnification every 4 h for 4 d. (E) Percentage of OvCAR3 cell colonies after pretreatment (15 min) with bsAb CD73xEpCAM (1 µg/mL) (or controls) and subsequent treatment with 5FU (15 µg/mL), cisplatin (1 µg/mL), or doxorubicin (200 nM) at 37 °C for 14 d. (F) Percentage of OvCAR3 cell colonies after pretreatment (15 min) with bsAb CD73xEpCAM (1 µg/mL), irradiated (or not) with 2 Gy and subsequently cultured at 37 °C for 14 d. All graphs represent mean ± SD. Statistical analysis in E (group-mean) and F was performed using unpaired t-test (* p < 0.05).