Marije B. Overdijk

Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma

Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARAs mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitts lymphoma cell lines. Phagocytosis contributed to DARAs anti-tumor activity in vivo, in both a subcutaneous and an intravenous leukemic xenograft mouse model. Finally, DARA was shown to induce macrophage-mediated phagocytosis of MM cells isolated from 11 of 12 MM patients that showed variable levels of CD38 expression. In summary, we demonstrate that phagocytosis is a fast, potent and clinically relevant mechanism of action that may contribute to the therapeutic activity of DARA in multiple myeloma and potentially other hematological tumors.

Crosstalk between Human IgG Isotypes and Murine Effector Cells

Development of human therapeutic Abs has led to reduced immunogenicity and optimal interactions with the human immune system in patients. Humanization had as a consequence that efficacy studies performed in mouse models, which represent a crucial step in preclinical development, are more difficult to interpret because of gaps in our knowledge of the activation of murine effector cells by human IgG (hIgG) remain. We therefore developed full sets of human and mouse isotype variants of human Abs targeting epidermal growth factor receptor and CD20 to explore the crosstalk with mouse FcγRs (mFcγRs) and murine effector cells. Analysis of mFcγR binding demonstrated that hIgG1 and hIgG3 bound to all four mFcγRs, with hIgG3 having the highest affinity. hIgG1 nevertheless was more potent than hIgG3 in inducing Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cellular phagocytosis with mouse NK cells, mouse polymorphonuclear leukocytes, and mouse macrophages. hIgG4 bound to all mFcγRs except mFcγRIV and showed comparable interactions with murine effector cells to hIgG3. hIgG4 is thus active in the murine immune system, in contrast with its inert phenotype in the human system. hIgG2 bound to mFcγRIIb and mFcγRIII, and induced potent ADCC with mouse NK cells and mouse polymorphonuclear leukocytes. hIgG2 induced weak ADCC and, remarkably, was unable to induce Ab-dependent cellular phagocytosis with mouse macrophages. Finally, the isotypes were studied in s.c. and i.v. tumor xenograft models, which confirmed hIgG1 to be the most potent human isotype in mouse models. These data enhance our understanding of the crosstalk between hIgGs and murine effector cells, permitting a better interpretation of human Ab efficacy studies in mouse models.

The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcγ Receptor–Mediated Cross-Linking

Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38+ multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking–mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug’s multifaceted mechanisms of action.

Epidermal Growth Factor Receptor (EGFR) Antibody-Induced Antibody-Dependent Cellular Cytotoxicity Plays a Prominent Role in Inhibiting Tumorigenesis, Even of Tumor Cells Insensitive to EGFR Signaling Inhibition

Ab-dependent cellular cytotoxicity (ADCC) is recognized as a prominent cytotoxic mechanism for therapeutic mAbs in vitro. However, the contribution of ADCC to in vivo efficacy, particularly for treatment of solid tumors, is still poorly understood. For zalutumumab, a therapeutic epidermal growth factor receptor (EGFR)-specific mAb currently in clinical development, previous studies have indicated signaling inhibition and ADCC induction as important therapeutic mechanisms of action. To investigate the in vivo role of ADCC, a panel of EGFR-specific mAbs lacking specific functionalities was generated. By comparing zalutumumab with mAb 018, an EGFR-specific mAb that induced ADCC with similar potency, but did not inhibit signaling, we observed that ADCC alone was insufficient for efficacy against established A431 xenografts. Interestingly, however, both zalutumumab and mAb 018 prevented tumor formation upon early treatment in this model. Zalutumumab and mAb 018 also completely prevented outgrowth of lung metastases, in A431 and MDA-MB-231-luc-D3H2LN experimental metastasis models, already when given at nonsaturating doses. Finally, tumor growth of mutant KRAS-expressing A431 tumor cells, which were resistant to EGFR signaling inhibition, was completely prevented by early treatment with zalutumumab and mAb 018, whereas ADCC-crippled N297Q-mutated variants of both mAbs did not show any inhibitory effects. In conclusion, ADCC induction by EGFR-specific mAbs represents an important mechanism of action in preventing tumor outgrowth or metastasis in vivo, even of cancers insensitive to EGFR signaling inhibition.

Role of IgG Fc Receptors in Monoclonal Antibody Therapy of Cancer

Historically, lack of specificity for cancer cells has been a major problem in cancer treatment; however, the development of monoclonal antibodies (mAbs), which combine high specificity with multiple mechanisms of action (MoAs), started a revolution in anti-cancer treatment options which continues to date. As of January 2013, 15 major antibody products were being marketed for cancer treatment in various countries around the globe, 10 of which are unmodified mAbs, which generally have multiple potential MoAs and may act via direct, Fab-domain-related effects or indirect, Fc-domain-related effects. Fc-domain-related effects consist of immune-mediated effector functions, which include complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). ADCC and ADCP depend on the engagement of Fcγ-receptors (FcγR) on immune effector cells by Fc-domains clustered due to antibody–antigen binding. Similarly, CDC depends on the engagement of proteins of the complement system by clustered antibody Fc domains. In this chapter, preclinical and clinical studies with approved anti-cancer mAbs are reviewed, with an emphasis on the role of FcγR-mediated effector functions. The importance of therapeutic antibody–FcγR interactions for human treatment can be deduced from correlations of clinical responses with FcγR polymorphisms, results supported by a wealth of preclinical and in vitro studies.

Abstract 592: Improving therapeutic activity of agonistic DR5 antibodies by inducing target binding-dependent hexamer formation

Death receptor 5 (DR5) is a highly interesting tumor target based on the enhanced sensitivity of cancer cells for DR5-dependent apoptosis. In recent years, multiple therapeutic DR5 antibodies have been evaluated in the clinic for which results however have been disappointing. IgG molecules against membrane-bound targets have shown an ability to form ordered hexameric structures upon antigen binding, a process that is dependent on Fc-Fc interactions between IgG molecules. We identified specific mutations in the human IgG1 Fc domain that enhance such antigen-dependent hexamerization while retaining solution-monomericity and developability characteristics of regular IgG1 molecules (HexaBody technology). We hypothesized that antibody-mediated hexamerization, when applied to DR5-specific antibodies, would enhance DR5 signaling and apoptosis, resulting in strongly improved therapeutic potential. The technology was applied to two non-crossblocking DR5-specific IgG1 antibodies, IgG1-DR5-01 and IgG1-DR5-05, by mutating a glutamic acid residue at position 430 in the Fc domain to glycine (the HexaBody mutants were designated Hx-DR5-01 and Hx-DR5-05). Cytotoxicity of the DR5 antibodies was explored in vitro using the CellTiter-Glo luminescent cell viability assay and the Caspase-Glo 3/7 assay in a broad panel of cancer cell lines, and in vivo in xenograft models. Both Hx-DR5-01 and Hx-DR5-05 induced increased cytotoxicity compared to their wild type (WT) IgG1 counterparts. Moreover, the combination of Hx-DR5-01 and Hx-DR5-05 (referred to as Hx-DR5-01/05) was found to be more potent than either Hx-DR5-01 or Hx-DR5-05 alone, or than the combination of the WT antibodies (IC50 in BxPC3 cells 0.5 and 1.5 μg/ml; maximal cytotoxicity 91% and 25% for Hx-DR5-01/05 and WT IgG1-DR5-01/05 respectively). In contrast to wild type agonistic DR5 antibodies, tumor cell killing by Hx-DR5-01/05 was independent of secondary crosslinking. Potent anti-tumor activity was observed in seven xenograft models for multiple indications, with Hx-DR5-01/05 consistently showing significantly better efficacy than the WT DR5 comparator antibody conatumumab. The cytotoxic activity of DR5 antibodies was significantly enhanced by the introduction of a hexamer-enhancing mutation in the IgG1 Fc domain. Maximal killing activity was obtained by combining two non-crossblocking DR5 antibodies carrying this mutation (Hx-DR5-01 and Hx-DR5-05). The strong cytotoxicity of Hx-DR5-01/05 was completely dependent on target binding but, in contrast to WT antibodies, did not require secondary crosslinking. These promising pre-clinical results support the selection of Hx-DR5-01/05 for clinical development for the treatment of cancer. Citation Format: Marije B. Overdijk, Kristin Strumane, Antonio Ortiz Buijsse, Claudine Vermot-Desroches, Andreas Lingnau, Esther C.W. Breij, Janine Schuurman, Paul W.H.I. Parren. Improving therapeutic activity of agonistic DR5 antibodies by inducing target binding-dependent hexamer formation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 592.