ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody

Abstract

Purpose: Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response.

Experimental design: The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a 89Zr-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer.

Results: Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of 89Zr-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors.

Conclusions: The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.

Figure 1.

In vitro and in vivo characterization of the binding of muAR9.6 to ovarian cancer cells. A and B, Flow cytometry illustrating the strong binding of muAR9.6 to OVCAR3 cells as well as its marginal binding to SKOV3 cells. C, RBA showing the high (81.4% ± 2.1%) and blockable (11% ± 1%) binding of [⁸⁹Zr]Zr-DFO-muAR9.6 to MUC16-positive OVCAR3 cells as well as its low (15.2% ± 1.3%) binding to MUC16-negative SKOV3 cells. The inset shows cellular internalization of [⁸⁹Zr]Zr-DFO-muAR9.6 between 1 hour and 24 hours after adding to ∼1 million OVCAR3 cells. The blue bars represent the uptake of 10 ng of [⁸⁹Zr]Zr-DFO-muAR9.6 at each timepoint, whereas the red bars represent the blocked uptake of [⁸⁹Zr]Zr-DFO-muAR9.6 in the presence of a 1,000-fold excess of unlabeled muAR9.6. D, Immunostaining and fluorescence microscopy of OVCAR3 cells with muAR9.6 and FITC-labeled muAR9.6 revealing the membrane-bound localization of fluorescence. E, PET images acquired 5 days after the administration of the 1.2 ± 0.1 mg/kg [⁸⁹Zr]Zr-DFO-muAR9.6 (255 ± 49.5 μCi; 9.4 ± 1.8 MBq; 29.6 ± 2.0 μg) in female Nu/Nu mice bearing OVCAR3 and SKOV3 subcutaneous xenografts (n = 3 mice per tumor type). The differential uptake of the radioimmunoconjugate in the tumors (T) can be seen as well as accumulation in other tissue compartments, including the heart [H], liver [L], and bone [B]. The latter is the result of the accretion of free, osteophilic [⁸⁹Zr]Zr⁴⁺ released from the radioimmunoconjugate. Serial PET images are shown in Supplementary Figs. S7 and S8. F,Ex vivo biodistribution profile up to 5 days after the intravenous administration of 0.11 ± 0.02 mg/kg of [⁸⁹Zr]Zr-DFO-muAR9.6 (24 ± 1.4 μCi; 0.88 ± 0.05 MBq; 2.8 ± 0.5 μg) to female Nu/Nu mice bearing OVCAR3 and SKOV3 subcutaneous xenografts (n = 5 mice per tumor type). *, P ≤ 0.03; **, P ≤ 0.01; ***, P ≤ 0.0005. Detailed sets of %ID/g values are provided in Supplementary Tables S1 and S2. The maximum intensity projections have been scaled from 0% to 100%.

Figure 2.

ImmunoPET imaging and ex vivo biodistribution data delineate lymph node involvement in mice bearing OVCAR3 xenografts. A–D, Maximum intensity projection (MIP) PET-CT images (scaled 0–100) of mice with subcutaneous OVCAR3 xenografts acquired 5 days after the intravenous administration of 1.2 ± 0.1 mg/kg of [⁸⁹Zr]Zr-DFO-muAR9.6 (255 ± 49.5 μCi; 9.4 ± 1.8 MBq; 29.6 ± 2.0 μg) showing differing distributions of radioactivity. Heterogeneous patterns of uptake are evident in the tumor (T), lymph nodes (ALN = axillary lymph node; BLN = brachial lymph node; CLN = cervical lymph node; ILN = inguinal lymph node), blood (indicated by the heart, H), and liver (L). E,Ex vivo biodistribution data comparing the radioactivity concentrations (%ID/g) in the IALNs, CALNs, and subcutaneous tumors of the tumor-bearing mice whose biodistribution data were reported in Fig. 1F. F, Graph comparing the percentage of total injected dose (%ID) values for the IALN and CALN at 5 d p.i. in the OVCAR3 tumor-bearing mice whose biodistribution was shown in Fig. 1F. G,Ex vivo MIP PET image of the tumor, IALNs, and CALNs collected from an OVCAR3-bearing xenograft 7 days after the administration of 1.2 ± 0.1 mg/kg of [⁸⁹Zr]Zr-DFO-muAR9.6 (255 ± 49.5 μCi; 9.4 ± 1.8 MBq; 29.6 ± 2.0 μg). H, H&E-stained IALN from mouse depicted in Fig. 2D revealing no signs of overt infiltration by neoplastic cells. Detailed sets of %ID/g and %ID values are provided in Supplementary Tables S3 and S4. The maximum intensity projections have been scaled from 0% to 100%.

Figure 3.

ImmunoPET delineates the uptake of [⁸⁹Zr]Zr-DFO-muAR9.6 in orthotopic-turned-metastatic and PDX models of HGSOC. A, PET images (MIPs scaled 0–100) of female nude mice taken 6 weeks after surgical implantation of the left ovary with OVCAR3 cells. ImmunoPET imaging with 1 mg/kg of [⁸⁹Zr]Zr-DFO-muAR9.6 (210 ± 11.7 μCi; 7.8 ± 0.4 MBq; 24 μg) delineated the orthotopic tumor as well as a lymph node in the hepatic region. B, Periodic immunoPET imaging of a mouse bearing an orthotopic OVCAR3 xenograft showed disease progression from the primary site of tumor cell inoculation (left ovary) to the liver and distant sites including the mediastinal lymph nodes in the thorax and the lumbar aortic lymph node in the lower abdomen. In this experiment, the mouse was injected three times with [⁸⁹Zr]Zr-DFO-muAR9.6 (210 ± 11.7 μCi; 7.8 ± 0.4 MBq; 24 μg) at 6, 10, and 16 weeks after inoculation, and the images were collected 6 days after each administration of the radioimmunoconjugate. C, Cryosections of tumor tissue resected from three human patients with HGSOC showing strong immunoreactive staining with muAR9.6 relative to an isotype control IgG used for IHC. D, PET images of female NSG mice bearing three different types of subcutaneous PDX (T; n = 3 mice per PDX) injected with 1.3 mg/kg [⁸⁹Zr]Zr-DFO-muAR9.6 (248 ± 5.3 μCi; 9.2 ± 0.2 MBq; 33.0 ± 4.3 μg). E,Ex vivo biodistribution data collected 6 days after 0.12 ± 0.01 mg/kg (22.7 ± 1.2 μCi; 0.84 ± 0.04 MBq; 3.0 ± 0.3 μg) [⁸⁹Zr]Zr-DFO-muAR9.6 was administered intravenously to mice bearing three types of subcutaneous PDX (n = 4 mice per PDX). Detailed %ID/g values are provided in Supplementary Table S5. The maximum intensity projections have been scaled from 0% to 100%.

Figure 4.

ImmunoPET demonstrates the MUC16-dependent uptake of [⁸⁹Zr]Zr-DFO-muAR9.6 in xenograft models of pancreatic ductal adenocarcinoma (PDAC). A, Graph showing the differential binding of 3 ng of [⁸⁹Zr]Zr-DFO-muAR9.6 to five PDAC cell lines expressing different levels of MUC16. B, PET images of mice bearing subcutaneous PDAC xenografts acquired 6 days after the administration of 2.1 ± 0.4 mg/kg of [⁸⁹Zr]Zr-DFO-muAR9.6 (225 ± 22.1 μCi; 8.3 ± 0.8 MBq; 51.7 ± 9.8 μg). C,Ex vivo biodistribution data acquired from mice bearing subcutaneous PDAC xenografts 6 days after the intravenous injection of 0.3 ± 0.03 mg/kg [⁸⁹Zr]Zr-DFO-muAR9.6 (27.7 ± 0.9 μCi; 1.02 ± 0.03 MBq; 6.3 ± 0.8 μg; n = 4 mice per time point). On the graph, *, P ≤ 0.05. Detailed %ID/g values are provided in Supplementary Table S6. D, Representative bioluminescence (BLI) and PET images of female nude mice acquired 4 weeks after the surgical implantation of Luc-Capan-2 cells in the head of the pancreas. The immunoPET images were acquired 6 days after the administration of 1.57 mg/kg of [⁸⁹Zr]Zr-DFO-muAR9.6 (269.4 ± 5.4 μCi; 9.97 ± 0.2 MBq; 40 μg). The maximum intensity projections have been scaled from 0% to 100%.

Figure 5.

Humanized AR9.6 demonstrates robust in vitro, in vivo, and ex vivo binding to MUC16-expressing ovarian and pancreatic cancer cells. A, Humanization of muAR9.6 by CDR grafting. B,In vitro internalization data of [⁸⁹Zr]Zr-DFO-huAR9.6 in OVCAR3 cells at 1, 3, 6, and 24 hours. The blue bars represent the uptake of 10 ng of [⁸⁹Zr]Zr-DFO-huAR9.6 at each timepoint, whereas the red bars represent the blocked uptake of [⁸⁹Zr]Zr-DFO-huAR9.6 in the presence of a 1,000-fold excess of unlabeled huAR9.6. C,In vitro and ex vivo validation of the binding of huAR9.6 to MUC16^high (OVCAR3) and MUC16^neg (SKOV3) ovarian cancer cells using flow cytometry and immunohistochemical (IHC) staining of FFPE tumor sections. D,Ex vivo validation of the binding of huAR9.6 to FFPE sections from MUC16^high (S2-028), MUC16^med (BxPC-3), and MUC16^low (MIAPaCa-2) tumors. E and G, Serial PET-CT images of mice bearing subcutaneous S2–028 xenografts (E) and OVCAR3 xenografts (G; n = 3 mice per tumor xenograft) acquired after the intravenous administration of 2.14 mg/kg [⁸⁹Zr]Zr-DFO-huAR9.6 (250 μCi; 9.25 MBq; 53 μg) showing gradual accretion of radioactivity in the tumor (T) and the liver (L) as well as gradually declining activity concentrations in the blood (indicated by the heart [H]). F and H, Longitudinal ex vivo biodistribution data acquired after the i.v. injection of 0.25 mg/kg of [⁸⁹Zr]Zr-DFO-huAR9.6 (29 μCi; 1.07 MBq; 6.2 μg) in mice bearing subcutaneous S2-028 xenografts (F) and OVCAR3 xenografts (H; n = 4 mice per time point). In the graph shown in F; *, P = 0.0286. Detailed sets of %ID/g values are provided in Supplementary Tables S7 and S8.

Figure 6.

huAR9.6 can delineate lymph node involvement in a xenograft model of ovarian cancer. A, PET-CT image of a mouse bearing a subcutaneous OVCAR3 xenograft acquired 6 days after the intravenous injection of 2.14 mg/kg [⁸⁹Zr]Zr-DFO-huAR9.6 (250 μCi; 9.25 MBq; 53 μg) showing uptake of radioactivity in the tumor (T), liver (L), and ipsilateral lymph node chain. B, Pan-cytokeratin (pan-CK) IHC staining of the OVCAR3 tumor showing a pattern of CK expression that is characteristic of epithelial cancer cells. C–E, Pan-CK IHC staining of the PET-positive IALN showing (C) immunoreactive foci of neoplastic cells (red arrowheads) and lymphatic fluid (black arrows) in the subcapsular sinuses. D, The cortex of the LN showing CK positivity in star-shaped cells (green arrowheads) indicative of dendritic cells and (E) a cluster of neoplastic cells infiltrating the IALN (red arrows) (E); F, IHC staining of the OVCAR3 tumor with huAR9.6 showing membranous staining of the OVCAR3 cells. G–I, huAR9.6 IHC staining of the PET-positive IALN (G) showing the presence of a few neoplastic cells (red arrowheads). The inset shows the appearance of the huAR9.6-stained PET-positive but H&E-negative IALN, and the red box in the inset identifies the portion shown in the main image. H, The cortex and follicles of the LN showing positivity for huAR9.6 staining in star-shaped cells (yellow arrowheads) indicative of dendritic cells. I, A cluster of neoplastic cells draining into and infiltrating the medulla of the IALN (red arrows). The inset shows the architecture and appearance of the corresponding huAR9.6-stained PET-positive but H&E-negative IALN, and the red box in the inset identifies the portion shown in the main image.