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

Abstract

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.

Efficacy and Tolerability of a GD2-Directed Trifunctional Bispecific Antibody in a Preclinical Model: Subcutaneous Administration Is Superior to Intravenous Delivery

“…Subcutaneous and intravenous treatment showed the same antitumor efficacy with a median survival of >100 days when Surek was injected twice (day 2 and 7 after B78-D14 challenge). As already published, a trAb with an irrelevant tumor specificity (anti-HER-2 Â anti-CD3), which did not bind to B78-D14 cells, was completely ineffective (14). It is established that the potential of trAbs depends on the number of treatment cycles but increasing the number of intravenous injections is often limited by possible side effects.…”

“…10). Surek was generated by quadroma technology and purified by affinity and ion exchange chromatography (14).…”

“…This cell-surface molecule is an attractive target for cancer immunotherapy in humans because it is strongly expressed on small-cell lung cancer and on malignancies of neuroectodermal origin such as neuroblastoma, glioma, and melanoma, while it shows a highly restricted pattern of expression in normal tissue (11,12). Using the B16F0-derived murine melanoma cell line B78-D14, which is engineered to express GD2 (13), and trAb Surek (anti-GD2 Â anti-murine CD3) as a surrogate construct for Ektomab, we previously demonstrated that this approach is highly effective in terms of eliminating melanoma cells (6,14,15).…”

Targeting Human Cancer Stem Cells with Monoclonal Antibodies

“…The Triomab Surek, which possesses the identical anti-GD2 binding arm as TRBs07, but binds to mouse CD3 instead of human CD3, has recently been shown to induce tumor eradication and protection against a second tumor challenge in a mouse melanoma model. Eradication of mouse melanoma was accompanied by the induction of tumor-specific T cells with defined melanoma-associated antigen specificity, induction of a humoral anti-tumor immune response, and generation of a longterm memory response against a second tumor challenge, indicating that Triomabs can generate potent tumor immunity against melanoma cells [137,138].…”

“…Importantly, no additional activation of immune cells is necessary for effective tumor elimination by catumaxomab, which therefore represents a self-supporting system [134,135]. In contrast to mAbs of the BiTE class, Triomabs exert a vaccination effect and induce long-lasting antitumor immunity, most likely due to their Fcmediated interaction with Fcγ-receptors expressed on dendritic cells and macrophages [136][137][138].…”