Abstract
The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.
Highlights
In the last decades monoclonal antibodies became powerful and promising drug classes, due to their ability to selectively address cancer-related molecules, infectious cells, virus particles, immune cells and immune-checkpoint-related molecules
For the generation of a suitable masking unit, which targets the Fcg receptors (FcgRs)-binding site of antibodies, we focused on implementation of anti-isotypic single-chain variable fragments
Comparing the induction level reached by the highest concentration of TRZ-Fc4 (100 nM) with the concentration of trastuzumab required for a similar induction (0.014 nM), we found a 7,100-fold reduction in antibody-dependent cellmediated cytotoxicity (ADCC) activation
Summary
Thereby, the different IgG subclasses (IgG1, IgG2, IgG3, IgG4) display unique FcgR and complement component binding profiles [2]. Polymorphisms of FcgRs show an immense influence on the affinity to different subclasses of IgGs, translating in reduced or enhanced efficacy of therapeutic antibodies [4, 5]. Several studies reported a correlation of Fc receptor binding-related internalization of antibodies and antibody-drug conjugates (ADCs) and adverse side effects (e.g. thrombocytopenia) [10, 15,16,17]. A prominent example is the implementation of three single point mutations in the Fc part of an anti-HER2 tubulysin (IgG1) ADC (results from clinical trial phase 1) in order to reduce FcgRrelated side effects [19]. A single point mutation (K322A) is known to limit the interaction of C1q to the IgG1
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