Protease-Activation of Fc-Masked Therapeutic Antibodies to Alleviate Off-Tumor Cytotoxicity

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.

Reversible Chemical Modification of Antibody Effector Function Mitigates Unwanted Systemic Immune Activation

“…This technology is akin to other prodrug strategies for biologics . Several masked antibody technologies have been developed to reactivate antibodies in the tumor microenvironment that require the presence of tumor-associated proteases. , These technologies are typically aimed at modulating antibody paratope binding, though a protease-releasable masking technology for Fc masking has recently been described . Instead of protease activity that can be variable among patients and tumor sites, the conjugation technology described here relies on chemical kinetics that occur at defined rates under physiological conditions.…”

A humanized trivalent Nectin-4-targeting nanobody drug conjugate displays potent antitumor activity in gastric cancer

“…Consequently, our Nectin-4 NDCs may potentially maintain a comparable half-life in cynomolgus monkeys and the human body. We opted for the incorporation of the HSA mechanism to extend the half-life of Nb, similar to the approach involving the Fc domain binding [ 56 , 57 ]. Both mechanisms achieve a prolonged half-life through binding to FcRn, thereby avoiding lysosomal degradation.…”

“…Both mechanisms achieve a prolonged half-life through binding to FcRn, thereby avoiding lysosomal degradation. However, the binding of Nectin-4 NDC to HSA effectively avoids adverse reactions, such as thrombocytopenia associated with the binding of the Fc domain to Fcγ receptors [ 56 , 57 ].…”

Advancing Cancer Therapy: A Review of Recent Progress in Monoclonal Antibodies

“…The majority of clinically effective mAbs that are now on the market target tumor cells with lethal effects by means of many mechanisms. The majority use complementdependent cytotoxicity (CDC) or ADDC to interact with immune system components [32,33]. Many also modify tumor cell signal transduction or work to remove important cellsurface antigens.…”