Enhancing The Efficacy and Specificity Of Antibody-Based T Cell Retargeting Strategies Against Hematological Malignancies

New promising candidates for cancer immunotherapy are bispecific antibodies (bsAbs) which have already shown a convincing anti-tumor effect in first clinical trials. BsAbs are composed of two single-chain fragments variable (scFvs), derived from the variable heavy and light chain of a monoclonal Ab (mAb), which bind to the activating CD3-complex on T cells and a tumor-associated antigen (TAA) on cancer cells. Consequently, the cross-linkage of effector and target cells by bsAbs results in an efficient T cell-mediated cancer cell killing. However, several serious adverse effects were observed in patients after application of Ab constructs. Most critical is an overall activation of T cells which bears the risk of a systemic release of pro-inflammatory cytokines.

With respect to the idea that the CD3 binding of an Ab construct is critical for T cell activation, the CD3 domain has to be optimized in order to prevent or even reduce unspecific T cell responses. For this reason, we cloned several anti-CD3 scFvs derived from different anti-CD3 mAb clones and compared their functionality and safety. The anti-CD3 scFv with lowest risk of side effects was introduced in different anti-TAA bsAbs. Functional and safety studies in vitro revealed that development of each bsAb requires extensive and time-consuming optimization steps to gain high efficacy but low risk of side effects. To improve the process of Ab engineering, we recently introduced a novel modular Ab platform that can be rapidly and cost-effectively adapted for redirection of T cells to any TAA (Arndt and Feldmann et al. accepted for publication in Leukemia 2013). In this novel modular system the dual specificity of a conventional bsAb is distributed to two separate Ab modules, (i) the effector module and (ii) the target module. Only the complex of both Ab modules mediates the cross-linkage of effector and target cells resulting in T cell activation and redirected cancer cell lysis similar to conventional T cell engaging bsAbs. The universal effector module is a well optimized bsAb balancing efficacy and safety. It binds CD3 on T cells and the E5B9 tag of the target module. The individual target module comprises an anti-TAA scFv and the peptide epitope E5B9. For treatment of lymphoid or myeloid malignancies a series of different target modules were designed. In vitro and in vivo data clearly underline that the combination of the established effector module with different target modules efficiently activated T cells against hematological malignancies. Both CD4+ and CD8+ T cells from healthy donors were efficiently activated to kill TAA-positive tumor cell lines at low effector to target cell ratios and Ab concentrations in a tumor-specific manner. Most importantly, we could demonstrate that patient-derived T cells were able to kill autologous malignant cells upon Ab-mediated cross-linkage. Another unique feature of the modular system is that multispecific or multifunctional target modules could be easily included to further improve the therapeutic effect. In order to increase anti-tumor specificity and reduce the risk of tumor escape variants, bispecific target modules that recognize different TAAs at the same time, were constructed. Cytotoxicity assays investigating dual targeting of double-positive tumor cells via the modular system demonstrated that target cell lysis can be considerably improved in comparison to single targeting modules. Moreover, proliferation and cytokine release of redirected T cells could be enhanced by supplying costimulatory immunoligands (e.g. 4-1BBL and Ox40L) via target modules.

In summary, we developed a multifunctional and highly flexible modular system based on an anti-CD3 domain with lowest risk of side effects. In contrast to conventional anti-TAA-anti-CD3 bsAbs, the development of a novel modular system for different clinical indications is much easier and less time-consuming. Once the effector module containing the critical CD3 domain is optimized, the modular system can be flexibly applied to target any TAA simply by replacing the target module. Application of bispecific target modules or providing costimulatory signals via the modular system might prolong immune responses and further increase anti-tumor specificity and activity. Finally, the novel modular platform represents a valuable and promising tool for cancer immunotherapy.