Microenvironment and Immunology Potent Immunomodulatory Effects of the Trifunctional Antibody Catumaxomab

Catumaxomab (CatmAb), a trifunctional bispecific antibody directed against the epithelial cell adhesion molecule (EpCAM) and the T-cell antigen CD3, is approved as intraperitoneal therapy for the treatment of malignant ascites in patients with EpCAM-positive carcinomas. The immunomonitoring results of a phase II/III study using CatmAb revealed a tumoricidal effect associated with reduced VEGF levels, CD69-expressing T cells, and the release of T-helper cell (TH)-1 cytokines. We comprehensively dissected the immunomodulatory effects of the CatmAb on the major subsets of malignant ascites-infiltrating leukocytes and the molecular fingerprint of tumor cell death. Herein we show that in the presence of EpCAM-positive tumor targets, CatmAb markedly enhanced T-cell activation [CD69, CD107A (LAMP1), HLA-DR and PD-1(PDCD1) expression] and stimulated inflammatory CD4TH1 andCD8 TH1 to release IFN-g but failed to trigger TH17 cells. Engagement of CD16-expressing cells caused upregulation of TRAIL (TNFSF10) and costimulatory CD40 and CD80 molecules. CatmAb promoted tumor cell death associated with ATP release and strongly synergized with oxaliplatin for the exposure of the three hallmarks of immunogenic cell death (calreticulin, HMGB1, and ATP). These findings warrant validation as potential biomarkers of efficacy of CatmAb. Cancer Res; 73(15); 4663–73. 2013 AACR. Introduction The trifunctional bispecific monoclonal antibody catumaxomab (CatmAb) has two binding specificities directed at epithelial cell adhesion molecule [EpCAM (through a mouse immunoglobulin G (IgG)-2a] and CD3 (through a rat IgG2b)]. With its Fc portion, the antibody has the potential to engage accessory cells (through FcgRI, FcgRIIa, FcgRIII) such as neutrophils, natural killer (NK) cells, macrophages, monocytes, and dendritic cells (DC; ref. 1). This therapeutic strategy is expected to promote an MHC-unrestricted pattern of killing, targeting EpCAM-positive tumor cells (most tumors of gastrointestinal origin and in some carcinomas of the genitourinary tract, i.e., about 90% of ascites causing carcinoma; refs. 2, 3), sparing EpCAM-negative mesothelial cells of the peritoneal cavity. In vitro studies showed that cytokine release, perforin-dependent killing, antibodydependent cytolysis (ADCC), and activation of accessory cells all acted in concert to eliminate tumor cells (4–6). EpCAMþ tumors have a worse prognosis than their negative counterparts, specifically in triple-negative and node-negative breast cancers (7, 8). In tumor spheroids, CatmAb (alone or together with cisplatin) exerted regressions of tumor volumes associated with massive immune infiltration in an ADCC-dependent manner (9). Pilot and phase I/II studies showed that intraperitoneal administration of CatmAb reduced tumor cell accumulation in ascitic fluids in a sustained manner (10, 11). Next, an open label, multicenter, randomized phase II/III trial in patients with malignant ascites due to epithelial cancer compared the effects of CatmAb with control paracentesis. Puncture-free survival (the primary endpoint of the trial) was significantly higher in the CatmAb compared with the control group (median, 46 vs. 11 days, HR: 0.25; ref. 12). Overall survival in the pooled population showed a positive trend toward the CatmAb group with significant results in the subgroup of gastric cancers (12). Treatment with CatmAb delayed the deterioration of quality of life in patients with malignant ascites (13). Moreover, ascites concentrations of VEGF and CD133þEpCAMþ cancer stem cells significantly dropped following CatmAb treatment, whereas 20% to 30% of CD4þ and CD8þ T cells acquired CD69 expression (14). Therefore, we undertook a comprehensive phenotypic analysis of the immune infiltrates and the tumor cell death fingerprint in ascitic fluids incubated ex vivo with CatmAb. Our findings indicate that CatmAb skews the T-cell cytokine Authors' Affiliations: Institut National de la Sant e et de la Recherche M edicale; Centre d'Investigation Clinique Bioth erapie; D epartement de Chirurgie; Unit e de Th erapie Cellulaire; Institut National de la Sant e et de la Recherche M edicale, Institut de canc erologie Gustave Roussy, Villejuif; and Universit e Paris-Sud, Kremlin Bicêtre, France Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). D. Go er e, C. Flament, N. Chaput, and L. Zitvogel are co-first and co-last authors and have contributed equally to this work. Corresponding Author: Laurence Zitvogel, Institut Gustave Roussy, 114, rueEdouardVaillant, 94805VILLEJUIFCedex, France. Phone: 33-1-42-1150-41; Fax: 33-1-42-11-60-94; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-446