Efficacy of Bispecific Antibody Targeting EpCAM and CD3 for Immunotherapy in Ovarian Cancer Ascites: An Experimental Study

Abstract

Objective

This study aimed to explore the value of M701, targeting epithelial cell adhesion molecule (EpCAM) and CD3, in the immunotherapy of ovarian cancer ascites by the in vitro assay.

Methods

The expression of EpCAM in ovarian cancer tissues was analyzed by databases. The EpCAM expression and immune cell infiltration in different foci of ovarian cancer were detected by 8-channel flow cytometry. The toxic effect of M701 on OVCAR3 was tested using the in vitro cytotoxicity assay. The 3D cell culture and drug intervention experiments were performed to evaluate the therapeutic effect of M701 in ovarian cancer specimens. Flow cytometry was used to examine the effect of M701 on the binding of immune cells to tumor cells and the activation capacity of T cells.

Results

The results of the bioinformatic analysis showed that the expression of EpCAM in ovarian cancer tissue was significantly higher than that in normal ovarian tissue. The 8-channel flow cytometry of clinical samples showed that the EpCAM expression and lymphocyte infiltration were significantly heterogeneous among ovarian cancer patients and lesions at different sites. The in vitro experiment results showed that M701 had a significant killing effect on OVCAR3 cells. M701 also obviously killed primary tumor cells derived from some patients with ovarian cancer ascites. M701 could mediate the binding of CD3⁺ T cells to EpCAM⁺ tumor cells and induce T cell activation in a dose-dependent manner.

Conclusion

M701 showed significant inhibitory activity on tumor cells derived from ovarian cancer ascites, which had a promising application in immunotherapy for patients with ovarian cancer ascites.

Keywords

ovarian cancer / ascites / EpCAM / immunotherapy / bispecific antibody

References

[1] SungH, FerlayJ, SiegelRL, et al.. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2021, 71(3): 209-249

[2] ReidBM, PermuthJB, SellersTA. Epidemiology of ovarian cancer: a review. Cancer Biol Med, 2017, 14(1): 9-32

[3] FerrissJS, JavaJJ, BookmanMA, et al.. Ascites predicts treatment benefit of bevacizumab in front-line therapy of advanced epithelial ovarian, fallopian tube and peritoneal cancers: An NRG Oncology/GOG study. Gynecol Oncol, 2015, 139(1): 17-22

[4] BaertT, FerreroA, SehouliJ, et al.. The systemic treatment of recurrent ovarian cancer revisited. Ann Oncol, 2021, 32(6): 710-725

[5] den OudenJE, ZamanGJR, DylusJ, et al.. Chemotherapy sensitivity testing on ovarian cancer cells isolated from malignant ascites. Oncotarget, 2020, 11(49): 4570-4581

[6] KippsE, TanDSP, KayeSB. Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research. Nat Rev Cancer, 2013, 13(4): 273-282

[7] GallegoA, MendiolaM, HernandoB, et al.. Prognostic markers of inflammation in endometrioid and clear cell ovarian cancer. Int J Gynecol Cancer, 2022, 32(8): 1009-1016

[8] JlassiA, ManaiM, MorjenM, et al.. VISTA+/CD8+status correlates with favorable prognosis in Epithelial ovarian cancer. PLoS One, 2023, 18(3): e0278849-e

[9] UkitaM, HamanishiJ, YoshitomiH, et al.. CXCL13-producing CD4+T cells accumulate in the early phase of tertiary lymphoid structures in ovarian cancer. JCI Insight, 2022, 7(12): e157215

[10] AmblardE, SoumelisV. Context-Dependent Effects Explain Divergent Prognostic Roles of Tregs in Cancer. Cancers, 2022, 14(12): 2991

[11] BhandaruM, RotteA. Monoclonal Antibodies for the Treatment of Melanoma: Present and Future Strategies. Methods Mol Biol, 2019, 1904: 83-108

[12] WudhikarnK, KingAC, GeyerMB, et al.. Outcomes of relapsed B-cell acute lymphoblastic leukemia after sequential treatment with blinatumomab and inotuzumab. Blood Adv, 2022, 6(5): 1432-1443

[13] RibasA, WolchokJD. Cancer immunotherapy using checkpoint blockade. Science, 2018, 359(6382): 1350-1355

[14] RosenbergSA, RestifoNP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science, 2015, 348(6230): 62-68

[15] NiL, LuJ. Interferon gamma in cancer immunotherapy. Cancer Med, 2018, 7(9): 4509-4516

[16] MorandS, DevanaboyinaM, StaatsH, et al.. Ovarian Cancer Immunotherapy and Personalized Medicine. Int J Mol Sci, 2021, 22(12): 6532

[17] MashhadiSMY, KazemimaneshM, ArashkiaA, et al.. Shedding light on the EpCAM: An overview. J Cell Physiol, 2019, 234(8): 12569-12580

[18] PatriarcaC, MacchiRM, MarschnerAK, et al.. Epithelial cell adhesion molecule expression (CD326) in cancer: A short review. Cancer Treat Rev, 2012, 38(1): 68-75

[19] Herreros-PomaresA, Aguilar-GallardoC, Calabuig-FarinasS, et al.. EpCAM duality becomes this molecule in a new Dr. Jekyll and Mr. Hyde tale. Crit Rev Oncol Hematol, 2018, 126: 52-63

[20] SeeberA, UntergasserG, SpizzoG, et al.. Predominant expression of truncated EpCAM is associated with a more aggressive phenotype and predicts poor overall survival in colorectal cancer. Int J Cancer, 2016, 139(3): 657-663

[21] MassonerP, ThommT, MackB, et al.. EpCAM is overexpressed in local and metastatic prostate cancer, suppressed by chemotherapy and modulated by MET-associated miRNA-200c/205. Br J Cancer, 2014, 111(5): 955-964

[22] SpizzoG, WentP, DirnhoferS, et al.. Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol Oncol, 2006, 103(2): 483-488

[23] BrunnerA, PrelogM, VerdorferI, et al.. EpCAM is predominantly expressed in high grade and advanced stage urothelial carcinoma of the bladder. J Clin Pathol, 2008, 61(3): 307-310

[24] FongD, MoserP, KasalA, et al.. Loss of membranous expression of the intracellular domain of EpCAM is a frequent event and predicts poor survival in patients with pancreatic cancer. Histopathology, 2014, 64(5): 683-692

[25] LeeSJ, ChungKY, KwonJE, et al.. Expression of EpCAM in adenoid cystic carcinoma. Pathology, 2018, 50(7): 737-741

[26] GaghanaLO, MiskadU, CangaraMH, et al.. The Relationship Between Expression of EpCAM Cancer Stem Cell Marker with Histopathological Grading, Lymphovascular Invasion, and Metastases in Colorectal Adenocarcinoma. Asian Pac J Cancer Prev, 2023, 24(3): 929-934

[27] YangY, FeiF, SongY, et al.. Polymorphisms of EpCAM gene and prognosis for non-small-cell lung cancer in Han Chinese. Cancer Sci, 2014, 105(1): 89-96

[28] LiaoY, WuM, JiaY, et al.. EpCAM as a Novel Biomarker for Survivals in Prostate Cancer Patients. Front Cell Dev Biol, 2022, 10: 843604

[29] SpizzoG, FongD, WurmM, et al.. EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis. J Clin Pathol, 2011, 64(5): 415-420

[30] van der FelsCAM, RosatiS, de JongIJ. EpCAM Expression in Lymph Node Metastases of Urothelial Cell Carcinoma of the Bladder: A Pilot Study. Int J Mol Sci, 2017, 18(8): 1802

[31] LiuX, LiJ, CadilhaBL, et al.. Epithelial-type systemic breast carcinoma cells with a restricted mesenchymal transition are a major source of metastasis. Sci Adv, 2019, 5(6): eaav4275

[32] BelloneS, SiegelER, CoccoE, et al.. Overexpression of Epithelial Cell Adhesion Molecule in Primary, Metastatic, and Recurrent/Chemotherapy-Resistant Epithelial Ovarian Cancer Implications for Epithelial Cell Adhesion Molecule-Specific Immunotherapy. Int J Gynecol Cancer, 2009, 19(5): 860-866

[33] FuJ, ShangY, QianZ, et al.. Chimeric antigen receptor-T (CAR-T) cells targeting epithelial cell adhesion molecule (EpCAM) can inhibit tumor growth in ovarian cancer mouse model. J Vet Med Sci, 2021, 83(2): 241-247

[34] van den BrandD, van LithSAM, de JongJM, et al.. EpCAM-Binding DARPins for Targeted Photodynamic Therapy of Ovarian Cancer. Cancers, 2020, 12(7): 1762

[35] LeeS, AhnHJ. Anti-EpCAM-conjugated adeno-associated virus serotype 2 for systemic delivery of EGFR shRNA: Its retargeting and antitumor effects on OVCAR3 ovarian cancer in vivo.. Acta Biomater, 2019, 91: 258-269

[36] ZhengJ, ZhaoS, YuX, et al.. Simultaneous targeting of CD44 and EpCAM with a bispecific aptamer effectively inhibits intraperitoneal ovarian cancer growth. Theranostics, 2017, 7(5): 1373-1388

[37] WangL, QiaoY, ZongH, et al.. IgG-like Bispecific Antibody CD3xEpCAM Generated by Split Intein Against Colorectal Cancer. Front Pharmacol, 2022, 13: 803059

[38] HuehlsAM, CoupetTA, SentmanCL. Bispecific T-cell engagers for cancer immunotherapy. Immunol Cell Biol, 2015, 93(3): 290-296

[39] SeimetzD. Novel Monoclonal Antibodies for Cancer Treatment: The Trifunctional Antibody Catumaxomab (Removab (R)). J Cancer, 2011, 2: 309-316

[40] FramptonJE. Catumaxomab in Malignant Ascites. Drugs, 2012, 72(10): 1399-1410

[41] HeissMM, MurawaP, KoralewskiP, et al.. The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelial cancer: results of a prospective randomized phase II/III trial. Int J Cancer, 2010, 127(9): 2209-2221

[42] EnglishDP, BelloneS, SchwabCL, et al.. Solitomab, an Epithelial Cell Adhesion Molecule/CD3 Bispecific Antibody (BiTE), Is Highly Active Against Primary Chemotherapy-Resistant Ovarian Cancer Cell Lines In Vitro and Fresh Tumor Cells Ex Vivo.. Cancer, 2015, 121(3): 403-412

[43] SongL, XueJ, ZhangJ, et al.. Mechanistic prediction of first-in-human dose for bispecific CD3/EpCAM T-cell engager antibody M701, using an integrated PK/PD modeling method. Eur J Pharm Sci, 2021, 158: 105584

[44] ZhangCM, YuLY, LvJF, et al.. Effects of immuno-related gene polymorphisms on a bispecific antibody targeting colorectal cancer cell. Per Med, 2018, 15(3): 167-179

[45] GiornelliGH. Management of relapsed ovarian cancer: a review. Springerplus, 2016, 5(1): 1197

[46] KeyvaniV, FarshchianM, EsmaeiliSA, et al.. Ovarian cancer stem cells and targeted therapy. J Ovarian Res, 2019, 12(1): 120

[47] NingF, ColeCB, AnnunziataCM. Driving Immune Responses in the Ovarian Tumor Microenvironment. Front Oncol, 2021, 10: 604084

[48] MelicharB, TouskovaM, TosnerJ, et al.. The phenotype of ascitic fluid lymphocytes in patients with ovarian carcinoma and other primaries. Onkologie, 2001, 24(2): 156-160

[49] FagottoF, AslemarzA. EpCAM cellular functions in adhesion and migration, and potential impact on invasion: A critical review. Biochim Biophys Acta Rev Cancer, 2020, 1874(2): 188436

[50] LandskronJ, HellandO, TorgersenKM, et al.. Activated regulatory and memory T-cells accumulate in malignant ascites from ovarian carcinoma patients. Cancer Immunol Immunother, 2015, 64(3): 337-347

[51] SatoE, OlsonSH, AhnJ, et al.. Intraepithelial CD8(+) tumor-infiltrating lymphocytes and a high CD8(+)/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. PNAS, 2005, 102(51): 18538-18543

[52] ZhangL, Conejo-GarciaJR, KatsarosD, et al.. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med, 2003, 348(3): 203-213

[53] LinkeR, KleinA, SeimetzD. Catumaxomab Clinical development and future directions. Mabs, 2010, 2(2): 129-136

[54] HongR, ZhouY, TianX, et al.. Selective inhibition of IDO1, D-1-methyl-tryptophan (D-1MT), effectively increased EpCAM/CD3-bispecific BiTE antibody MT110 efficacy against IDO1(hi)breast cancer via enhancing immune cells activity. Int Immunopharmacol, 2018, 54: 118-124

[55] FerrariF, BelloneS, BlackJ, et al.. Solitomab, an EpCAM/CD3 bispecific antibody construct (BiTE (R)), is highly active against primary uterine and ovarian carcinosarcoma cell lines in vitro.. J Exp Clin Cancer Res, 2015, 34: 123