Peter Ruf

Immunomonitoring results of a Phase II/III study of malignant ascites patients treated with the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3)

Patients with malignant ascites secondary to primary carcinomas benefit from intraperitoneal therapy with the trifunctional antibody catumaxomab (anti-EpCAM × anti-CD3). Here, we report the analysis of peritoneal fluid samples from 258 patients with malignant ascites randomized to catumaxomab or control groups to investigate the molecular effects of catumaxomab treatment. In the catumaxomab group, tumor cell numbers and peritoneal levels of VEGF decreased, whereas the activation status of CD4(+) and CD8(+) T-cell populations increased more than two-fold after treatment. Notably, CD133(+)/EpCAM(+) cancer stem cells vanished from the catumaxomab samples but not from the control samples. In vitro investigations indicated that catumaxomab eliminated tumor cells in a manner associated with release of proinflammatory Th1 cytokines. Together, our findings show that catumaxomab therapy activates peritoneal T cells and eliminates EpCAM(+) tumor cells, establishing a molecular and cellular basis to understand in vivo efficacy within the immunosuppressed malignant ascites tissue microenvironment.

Pharmacokinetics, immunogenicity and bioactivity of the therapeutic antibody catumaxomab intraperitoneally administered to cancer patients

AIMS Catumaxomab is the first EMEA approved trifunctional anti-EpCAM×anti-CD3 antibody for the treatment of cancer patients with malignant ascites. A phase II pharmacokinetic study was conducted to determine local and systemic antibody concentrations and anti-drug antibody (ADA) development. METHODS Thirteen cancer patients with symptomatic malignant ascites were treated with four ascending doses of 10, 20, 50, and 150 µg catumaxomab intraperitoneally (i.p.) infused on days 0, 3, 6 or 7 and 10. The pharmacokinetics of catumaxomab were studied by implementation of supportive data from a non clinical mouse tumour model. Additionally, ADA development was monitored. RESULTS Ten out of 13 patients were evaluable for pharmacokinetic analysis. Catumaxomab became increasingly concentrated in ascites during the course of treatment, attaining effective concentrations in the ng ml−1 range. Catumaxomab remained immunologically active even after several days in the circulation. The observed systemic catumaxomab exposure was low (<1%), with a maximal median plasma concentration (Cmax) of 403 pg ml−1. The mean elimination half-life in the plasma was 2.13 days. All patients developed ADA, but not before the last infusion. High observed inter-individual variability and low systemic exposure may be explained by the inverse correlation between tumour burden, effector cell numbers and systemic antibody bioavailability as demonstrated in a defined mouse tumour model. CONCLUSIONS Based on the high and effective local concentrations, low systemic exposure and acceptable safety profile, we confirmed that the i.p. application scheme of catumaxomab for the treatment of malignant ascites is appropriate.

Cancer therapy with trifunctional antibodies: linking innate and adaptive immunity

Trifunctional antibodies (trAbs) are promising novel anticancer biologics with a particular mode of action capable of linking innate with adaptive immunity. Based on their unique structure, trifunctional IgG-like heterodimeric antibodies, consisting of nonhuman mouse and rat immunoglobulin halves are able to redirect T lymphocytes, as well as accessory cells, to the tumor site. This recruitment of immune cells is accompanied by cellular activation events elicited by anti-CD3, as well as Fcγ-receptor engagement of trAbs supported by a proinflammatory Th1-biased cytokine milieu. All necessary immunological factors required for long-term vaccination-like effects are stimulated along trAb-mediated therapeutic interventions. Thus, the concerted interplay of antibody-dependent cellular cytotoxicity plus the polyclonal T-cell cytotoxicity and Fcγ-receptor-driven induction of long-lasting immune responses after the initial tumor cell elimination represent the major hallmarks of trAb-mediated treatment of malignant diseases.

Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model

BackgroundTrifunctional bispecific antibodies (trAb) are a special class of bispecific molecules recruiting and activating T cells and accessory immune cells simultaneously at the targeted tumor. The new trAb Ektomab that targets the melanoma-associated ganglioside antigen GD2 and the signaling molecule human CD3 (hCD3) on T cells demonstrated potent T-cell activation and tumor cell destruction in vitro. However, the relatively low affinity for the GD2 antigen raised the question of its therapeutic capability. To further evaluate its efficacy in vivo it was necessary to establish a mouse model.MethodsWe generated the surrogate trAb Surek, which possesses the identical anti-GD2 binding arm as Ektomab, but targets mouse CD3 (mCD3) instead of hCD3, and evaluated its chemical and functional quality as a therapeutic antibody homologue. The therapeutic and immunizing potential of Surek was investigated using B78-D14, a B16 melanoma transfected with GD2 and GD3 synthases and showing strong GD2 surface expression. The induction of tumor-associated and autoreactive antibodies was evaluated.ResultsDespite its low affinity of approximately 107 M-1 for GD2, Surek exerted efficient tumor cell destruction in vitro at an EC50 of 70ng/ml [0.47nM]. Furthermore, Surek showed strong therapeutic efficacy in a dose-dependent manner and is superior to the parental GD2 mono-specific antibody, while the use of a control trAb with irrelevant target specificity had no effect. The therapeutic activity of Surek was strictly dependent on CD4+ and CD8+ T cells, and cured mice developed a long-term memory response against a second challenge even with GD2-negative B16 melanoma cells. Moreover, tumor protection was associated with humoral immune responses dominated by IgG2a and IgG3 tumor-reactive antibodies indicating a Th1-biased immune response. Autoreactive antibodies against the GD2 target antigen were not induced.ConclusionOur data suggest that Surek revealed strong tumor elimination and anti-tumor immunization capabilities. The results warrant further clinical development of the human therapeutic equivalent antibody Ektomab.

Potential of the trifunctional bispecific antibody surek depends on dendritic cells: rationale for a new approach of tumor immunotherapy.

Trifunctional bispecific antibodies (trAbs) used in tumor immunotherapy have the unique ability to recruit T cells toward antigens on the tumor cell surface and, moreover, to activate accessory cells through their immunoglobulin Fc region interacting with activating Fcγ receptors. This scenario gives rise to additional costimulatory signals required for T cell-mediated tumor cell destruction and induction of an immunologic memory. Here we show in an in vitro system that most effective trAb-dependent T-cell activation and tumor cell elimination are achieved in the presence of dendritic cells (DCs). On the basis of these findings, we devise a novel approach of cancer immunotherapy that combines the specific advantages of trAbs with those of DC-based vaccination. Simultaneous delivery of trAbs and in vitro differentiated DCs resulted in a markedly improved tumor rejection in a murine melanoma model compared with monotherapy.

Potent CD4 + T cell-associated antitumor memory responses induced by trifunctional bispecific antibodies in combination with immune checkpoint inhibition

Combinatorial approaches of immunotherapy hold great promise for the treatment of malignant disease. Here, we examined the potential of combining an immune checkpoint inhibitor and trifunctional bispecific antibodies (trAbs) in a preclinical melanoma mouse model using surrogate antibodies of Ipilimumab and Catumaxomab, both of which have already been approved for clinical use. The specific binding arms of trAbs redirect T cells to tumor cells and trigger direct cytotoxicity, while the Fc region activates accessory cells eventually giving rise to a long-lasting immunologic memory. We show here that T cells redirected to tumor cells by trAbs strongly upregulate CTLA-4 expression in vitro and in vivo. This suggested that blocking of CTLA-4 in combination with trAb treatment enhances T-cell activation in a tumor-selective manner. However, when mice were challenged with melanoma cells and subsequently treated with antibodies, there was only a moderate beneficial effect of the combinatorial approach in vivo with regard to direct tumor destruction in comparison to trAb therapy alone. By contrast, a significantly improved vaccination effect was obtained by CTLA-4 blocking during trAb-dependent immunization. This resulted in enhanced rejection of melanoma cells given after pre-immunization. The improved immunologic memory induced by the combinatorial approach correlated with an increased humoral antitumor response as measured in the sera and an expansion of CD4+ memory T cells found in the spleens.

Immunotherapy with the trifunctional anti-CD20 × anti-CD3 antibody FBTA05 in a patient with relapsed t(8;14)-positive post-transplant lymphoproliferative disease.

B-cell lymphomas are common malignancies during infancy and childhood with excellent long-term cure rates. However, relapse or refractory disease associated with very poor prognosis (especially for Burkitt lymphoma and Burkitt leukemia) occurs in 6–15% of patients [1,2]. Post-transplant lymphoproliferative disease (PTLD) constitutes a heterogeneous group of lymphoproliferative disorders occurring as a severe complication after solid organ transplantation and hematopoietic stem cell transplantation (HSCT). Acquired by up to 15% of pediatric transplant recipients, most cases of childhood PTLD are of B-cell origin and associated with Epstein-Barr virus (EBV) infection or reactivation [3,4]. Long-lasting immunosuppressive therapy required to avoid graft rejection as well as the lack of EBV-specific immunity at the time of transplantation contributes to the high incidence and unfavorable prognosis of PTLD in the pediatric population and up to 20% of affected children eventually succumb to the disease [3]. While for early localized disease, modulation of immunosuppressive therapy is sufficient, multiagent chemotherapy comprises the primary treatment option for advanced stages of PTLD. Compared to relapse of Burkitt lymphoma, prognosis of relapsed PTLD (even when Burkitt-like, t(8;14) translocation positive) is significantly better, but stage IV PTLD displays a similarly dismal outcome with a five-year event free survival rate of only 10% [5]. For these high-risk patients, very limited treatment options are available, particularly when HSCT is not feasible [1,6]. We here report the case of an eight-year-old boy with isolated testicular relapse of stage IV, t(8;14) translocation positive PTLD, who received the trifunctional antibody FBTA05, anti-CD3 anti-CD20 (TRION Pharma, Planegg, Germany), as a single agent consolidation treatment without HSCT (Figure 1). Our patient was initially diagnosed with extrahepatic biliary atresia and liver transplantation (split liver from his mother) was performed during his first year of life. His immunosuppressive therapy consisted of tacrolimus and mycophenolate mofetil. At the age of eight years, EBV-associated, t(8;14) positive (Burkitt-like) monomorphic stage IV PTLD (abdominal lymph nodes and bone marrow) was diagnosed. Initial therapy included the anti-CD20 antibody rituximab and modification of immunosuppressive therapy (mTOR inhibition) while chemotherapy was omitted due to the patient’s poor general condition. After three months, multilocular tumor progression occurred, despite good primary response to rituximab. Therefore mTOR inhibition was discontinued and moderate-intensive polychemotherapy according to the PET PTLD 2005 protocol (six cycles of mCOMP (cyclophosphamide, prednisolone, vincristine, methotrexate) combined with rituximab) was initiated. Complete morphological, radiological and metabolic remission was achieved by this therapy regimen. Nevertheless, four months after completion of chemotherapy, the patient suffered from local bilateral testicular relapse of the t(8;14) positive PTLD. Second remission could be achieved by intensive multi-agent chemotherapy according to the B-NHL BFM protocol (6 cycles including intrathecal therapy) again combined with rituximab. After bilateral orchidectomy (performed after four courses of chemotherapy), no histological evidence for residual lymphoma could be detected. Due to relapse after stage IV primary disease, t(8;14) translocation and the need for long-lasting immunosuppression, the risk for further relapse was estimated extremely high. In consent with the parents, the patient therefore subsequently received the trifunctional antibody FBTA05 (LymphomunR , TRION Pharma) as