Engineered therapeutic antibodies with enhanced effector functions: Clinical application of the Potelligent® Technology

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.

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Enhancing FcγR-mediated antibody effector function during persistent viral infection

Science Immunology ◽

10.1126/sciimmunol.aao3125 ◽

2018 ◽

Vol 3 (27) ◽

pp. eaao3125 ◽

Cited By ~ 3

Author(s):

Andreas Wieland ◽

Alice O. Kamphorst ◽

Rajesh M. Valanparambil ◽

Jin-Hwan Han ◽

Xiaojin Xu ◽

...

Keyword(s):

Viral Infections ◽

Lymphocytic Choriomeningitis ◽

Target Cells ◽

Therapeutic Antibodies ◽

Persistently Infected ◽

Effector Functions ◽

Persistent Viral Infection ◽

Wide Range ◽

Persistent Viral Infections ◽

Lcmv Infection

Persistent viral infections can interfere with FcγR-mediated antibody effector functions by excessive immune complex (IC) formation, resulting in resistance to therapeutic FcγR-dependent antibodies. We and others have previously demonstrated that mice persistently infected with lymphocytic choriomeningitis virus (LCMV) are resistant to a wide range of depleting antibodies due to excessive IC formation. Here, we dissect the mechanisms by which two depleting antibodies overcome the obstacle of endogenous ICs and achieve efficient target cell depletion in persistently infected mice. Efficient antibody-mediated depletion during persistent LCMV infection required increased levels of antibody bound to target cells or use of afucosylated antibodies with increased affinity for FcγRs. Antibodies targeting the highly expressed CD90 antigen or overexpressed human CD20 efficiently depleted their target cells in naïve and persistently infected mice, whereas antibodies directed against less abundant antigens failed to deplete their target cells during persistent LCMV infection. In addition, we demonstrate the superior activity of afucosylated antibodies in the presence of endogenous ICs. We generated afucosylated antibodies directed against CD4 and CD8α, which, in contrast to their parental fucosylated versions, efficiently depleted their respective target cells in persistently infected mice. Efficient antibody-mediated depletion can thus be achieved if therapeutic antibodies can outcompete endogenous ICs for access to FcγRs either by targeting highly expressed antigens or by increased affinity for FcγRs. Our findings have implications for the optimization of therapeutic antibodies and provide strategies to allow efficient FcγR engagement in the presence of competing endogenous ICs in persistent viral infections, autoimmune diseases, and cancer.

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Engineered therapeutic antibodies with improved effector functions

Cancer Science ◽

10.1111/j.1349-7006.2009.01222.x ◽

2009 ◽

Vol 100 (9) ◽

pp. 1566-1572 ◽

Cited By ~ 113

Author(s):

Tsuguo Kubota ◽

Rinpei Niwa ◽

Mitsuo Satoh ◽

Shiro Akinaga ◽

Kenya Shitara ◽

...

Keyword(s):

Therapeutic Antibodies ◽

Effector Functions

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Fc-engineered antibodies with immune effector functions completely abolished

PLoS ONE ◽

10.1371/journal.pone.0260954 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0260954

Author(s):

Ian Wilkinson ◽

Stephen Anderson ◽

Jeremy Fry ◽

Louis Alex Julien ◽

David Neville ◽

...

Keyword(s):

Fusion Proteins ◽

Inflammatory Cytokine ◽

Inflammatory Responses ◽

Therapeutic Antibodies ◽

Fcγ Receptors ◽

Specific Amino Acid ◽

Immune Effector ◽

Effector Functions ◽

Cytokine Responses ◽

Novel Variants

Elimination of the binding of immunoglobulin Fc to Fc gamma receptors (FcγR) is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been described in the literature but none of them completely eliminates binding to all of the Fcγ receptors. Here we describe a set of novel variants having specific amino acid substitutions in the Fc region at L234 and L235 combined with the substitution G236R. They show no detectable binding to Fcγ receptors or to C1q, are inactive in functional cell-based assays and do not elicit inflammatory cytokine responses. Meanwhile, binding to FcRn, manufacturability, stability and potential for immunogenicity are unaffected. These variants have the potential to improve the safety and efficacy of therapeutic antibodies and Fc fusion proteins.

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Enhancing CDC and ADCC of CD19 Antibodies by Combining Fc Protein-Engineering with Fc Glyco-Engineering

Antibodies ◽

10.3390/antib9040063 ◽

2020 ◽

Vol 9 (4) ◽

pp. 63

Author(s):

Sophia Roßkopf ◽

Klara Marie Eichholz ◽

Dorothee Winterberg ◽

Katarina Julia Diemer ◽

Sebastian Lutz ◽

...

Keyword(s):

Amino Acid ◽

Protein Engineering ◽

Amino Acid Sequence ◽

Therapeutic Antibodies ◽

Adcc Activity ◽

Fcγ Receptor ◽

Effector Functions ◽

Complement Dependent Cytotoxicity ◽

Dependent Cell ◽

Cluster Of Differentiation

Background: Native cluster of differentiation (CD) 19 targeting antibodies are poorly effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are crucial effector functions of therapeutic antibodies in cancer immunotherapy. Both functions can be enhanced by engineering the antibody’s Fc region by altering the amino acid sequence (Fc protein-engineering) or the Fc-linked glycan (Fc glyco-engineering). We hypothesized that combining Fc glyco-engineering with Fc protein-engineering will rescue ADCC and CDC in CD19 antibodies. Results: Four versions of a CD19 antibody based on tafasitamab’s V-regions were generated: a native IgG1, an Fc protein-engineered version with amino acid exchanges S267E/H268F/S324T/G236A/I332E (EFTAE modification) to enhance CDC, and afucosylated, Fc glyco-engineered versions of both to promote ADCC. Irrespective of fucosylation, antibodies carrying the EFTAE modification had enhanced C1q binding and were superior in inducing CDC. In contrast, afucosylated versions exerted an enhanced affinity to Fcγ receptor IIIA and had increased ADCC activity. Of note, the double-engineered antibody harboring the EFTAE modification and lacking fucose triggered both CDC and ADCC more efficiently. Conclusions: Fc glyco-engineering and protein-engineering could be combined to enhance ADCC and CDC in CD19 antibodies and may allow the generation of antibodies with higher therapeutic efficacy by promoting two key functions simultaneously.

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