Bispecific antibodies (bsAb) are considered as a promising improvement of traditional monospecific antibodies for instance because effector functions are enhanced and the risk of drug resistance is reduced . Among the many different formats, trAb are most advanced with catumaxomab being the first EMA-approved bsAb so far . Here, we present preclinical data of the new trAb Surek targeting the melanoma-associated ganglioside GD2 and mCD3 on T cells. A critical point of investigation was the relatively low affinity of the antibodies’ tumor binding arm (~107 M-1) recognizing a sugar epitope. However, in spite of this fact, Surek showed therapeutic effectiveness in the treatment of the GD2-positive B78-D14 melanoma model. The therapeutic outcome was superior to the parental monospecific antibody treatment, specific and dose-dependent with a cumulative dose of 150μg required to accomplish complete tumor rejection (Figures 3A, B, C). In comparison with the high-affinity EpCAM-specific surrogate antibody BiLu (~109 M-1), which was evaluated in a similar melanoma model, the required therapeutic dosage is about 15–30 times higher [18, 29]. This can be partially ascribed to the different affinities of the tumor binding arms. However, the finding that Surek is capable of redirecting T-cell cytotoxicity at all (EC50 = 70 ng/ml [0.47 nM]) may be explained by multivalent binding of redirected T cells opsonized with a multitude of Surek antibody molecules. This hypothesis implies a much higher affinity of the monovalent CD3 binding arm of Surek which was indeed confirmed by comparative FACS binding studies (Figure 1A). Depletion experiments further confirmed that the in vivo mode of action of Surek is also strictly dependent on T cells. Interestingly, both CD8+ as well as CD4+ T cells are essential for therapeutic effectiveness (Figure 3D) suggesting that CD4+ T helper cells contribute to the tumor destruction either directly or indirectly e.g. by promoting T-cell activation via cross-talk with accessory immune cells.
A considerable concern of anti-GD2 antibodies is the triggering of neurotoxicity . Antibody binding to GD2 expressed as a minor constituent in normal peripheral nerves causes severe pain requiring analgetic medication [32–34]. Importantly, we did not observe any apparent signs of neurotoxicity in mice after administration of Surek. Of note, the GD2 binding arm of the therapeutic equivalent antibody Ektomab (anti-GD2 x anti-hCD3) showed no cross- reactive binding to 32 different normal human tissues including peripheral nerves. The only exception was cerebellum (unpublished data). Thus, the relatively low affinity of Ektomab obviously avoids its monospecific binding to healthy tissue and seems to be advantageous in terms of circumventing dose-limiting neurotoxicity. Moreover, the high specificity of the parental antibody Me361, which provides the GD2-binding arm of Ektomab and Surek, prevents significant cross-reactive binding with prominent ganglioside species like GM1 or GM3 . We found that even with the most similar ganglioside structure GD3, there was only minimal cross-detection at high antibody concentrations of > 100 μg/ml (Figure 1B). Selecting therapeutic mAbs with an appropriate affinity is often a matter of debate that can only be answered in the clinic. For instance, high-affinity EpCAM-specific mAbs were shown to cause severe acute pancreatitis, whereas low-affinity counterparts lacked sufficient effector functions .
TrAb represent an attractive approach in cancer therapy because they link innate and adaptive immunity . For this reason it was important to evaluate whether the trAb Surek and other trAb like BiLu [18, 29] or catumaxomab  are equally competent in inducing long-term vaccination. Indeed, a proportion of 50% of Surek-treated long-term survivors were resistant to a lethal second tumor challenge without any further antibody injection (Figure 4A). Anti-tumor immunization was associated with the formation of tumor-reactive antibodies which, however, only marginally contribute to tumor protection as shown by plasma transfer experiments (Figure 5). In fact, the measured humoral immune response which was dominated by IgG2a and IgG3 isotypes indicates an IL-12-mediated and Th1-associated T-cell immunity [39, 40]. Indeed, activation of dendritic and T cells was observed by Eissler et al. 48h after in vivo application of Surek accompanied by IL-12, IFN-γ and TNF release. Moreover, protective tumor-specific T cells recognizing melanoma-derived peptides like tyrosinase-related protein 2 (trp2) and gp100 were induced after immunization of mice with irradiated B78-D14 tumor cells and intact Surek antibody but not with the bsF(ab′)2 counterpart lacking the Fc region . Thus, Surek effectively eliminates B78-D14 melanoma cells in vivo followed by the induction of long-term anti-tumor immunization. Of note, we did not detect autoreactive antibodies directed against the target structure GD2 indicating that Surek therapy does not break tolerance to self antigens. This is an important finding because ganglioside-directed autoimmune reactions were shown to cause severe neurological disorders in some patients .
Immunotherapeutic approaches are especially attractive for the treatment of immunogenic cancers like melanoma. This is underscored by the recent approval of the monoclonal antibody Ipilimumab , which blocks the negative T-cell regulator CTLA-4 . However, severe autoimmune effects like hepatitis or enterocolitis are frequently observed in the course of Ipilimumab treatment. In contrast to nonspecific immune modulators, trAb represent a targeted immunotherapeutic approach. They offer the advantage of redirecting and activating T cells and antigen-presenting cells simultaneously at the tumor site and thus combining passive and active immunization . Hence, trAb may be especially effective in the treatment of immunogenic melanoma. Our data demonstrate that the trAb Surek, which targets the melanoma-associated antigen GD2, effectively eliminates melanoma cells accompanied by the generation of an immunological memory. The typical characteristics of targeted cell destruction and induced long-term immune responses are observed for Surek. These results warrant further clinical development of its therapeutic equivalent Ektomab.