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Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.

CD37 is a tetraspanin plasma membrane protein abundantly expressed on B-cells and represents a promising therapeutic target for the treatment of B-cell malignancies. Although complement-dependent cytotoxicity (CDC) has proven to be a powerful Fc-mediated effector function for killing hematological cancer cells, CD37 antibody-based therapeutics currently in clinical development are poor inducers of CDC. Here we present DuoHexaBody-CD37, a novel humanized IgG1 bispecific antibody targeting two different CD37 epitopes, with an E430G hexamerization-enhancing mutation, for the potential treatment of B-cell malignancies. The natural process of antibody hexamer formation through intermolecular Fc-Fc interactions between IgG molecules after cell surface antigen binding can be improved by introducing a single point mutation such as E430G in the IgG Fc domain, thereby facilitating more efficient C1q binding and complement activation (Diebolder et al., Science 2014; de Jong et al., PLoS Biol 2016). The hexamerization-enhancing mutation E430G was introduced into two humanized CD37 monoclonal antibodies (mAbs) that bind non-overlapping CD37 epitopes. Different antibody formats and combinations, including the single antibodies, combinations of the mAbs and bispecific mAbs were tested for their capacity to induce CDC and antibody-dependent cellular cytotoxicity (ADCC). The bispecific hexamerization-enhanced antibody variant DuoHexaBody-CD37, showed superior CDC activity compared to the single hexamerization-enhanced mAbs and the combination thereof, both in vitro over a range of different B-cell lines, and ex vivo in tumor cell samples obtained from patients with chronic lymphocytic leukemia (CLL). In a CDC assay using tumor cells obtained from a relapsed/refractory CLL patient who received prior treatment with rituximab, ibrutinib and idelalisib, DuoHexaBody-CD37 induced almost complete lysis (84% lysis at a concentration 100 µg/mL), thereby outperforming the single HexaBody molecules (15% and 23% lysis) and the combination (57%) (Figure 1). In addition to its potent CDC activity, DuoHexaBody-CD37 was also capable of inducing potent ADCC of Daudi cells (EC50 = 12.3 ± 9.5 ng/mL), as assessed using peripheral blood mononuclear cells from 8 healthy human donors in a standard chromium release assay. In assays using whole blood from 6 healthy human donors, DuoHexaBody-CD37 showed efficient B-cell binding and potent and specific depletion of the B-cell population (98% ± 1.3% depletion at 10 µg/mL, EC50 = 0.85 ± 0.284 µg/mL). Furthermore, DuoHexaBody-CD37 induced significant inhibition of tumor growth in vivo in Daudi-luc Burkitt's lymphoma and JVM-3 CLL mouse xenograft models, at doses as low as 0.1 and 1 mg/kg (p In summary, we present a novel therapeutic antibody that, for the first time, combines proprietary DuoBody® and HexaBody® platforms. DuoHexaBody-CD37 induced highly potent CDC and efficient ADCC in preclinical models, suggesting that DuoHexaBody-CD37 may serve as a potential therapeutic mAb for the treatment of human B-cell malignancies. Disclosures Oostindie: Genmab: Employment, Equity Ownership. Van Der Horst:Genmab: Research Funding. Overdijk:Genmab: Employment, Equity Ownership. Strumane:Genmab: Employment, Equity Ownership. Verploegen:Genmab: Employment, Equity Ownership. Lindorfer:Genmab: Research Funding. Cook:Genmab: Research Funding. Chamuleau:Gilead: Research Funding; BMS: Research Funding; celgene: Research Funding; Genmab: Research Funding. Mutis:Gilead: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Novartis: Research Funding; OnkImmune: Research Funding. Schuurman:Genmab: Employment, Other: Warrants. Sasser:Genmab: Employment, Equity Ownership. Taylor:Genmab: Research Funding. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Beurskens:Genmab: Employment, Equity Ownership. Breij:Genmab: Employment, Equity Ownership.

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.

DuoBody®-CD3xCD20 (GEN3013) is a bispecific antibody (bsAb), recognizing the T-cell antigen CD3 and the B-cell antigen CD20, that triggers potent T-cell-mediated lysis of CD20-expressing cells. DuoBody-CD3xCD20 is a full-length bispecific IgG1 generated by controlled Fab-arm exchange (cFAE) [1, 2] and contains an effector function-silenced Fc region. In vitro, DuoBody-CD3xCD20 induced potent activation, proliferation and cytotoxic activity of both CD4+ and CD8+ T cells in the presence of CD20-expressing cells, as measured by flow cytometry and bromodeoxyuridine (BrdU) incorporation assays. DuoBody-CD3xCD20 induced T-cell-mediated cytotoxicity towards a diverse panel of cell lines derived from various B-cell malignancies and endogenous B cells, with EC50 values in the low picomolar range (EC50: 0.2-5.0 pM). The CD20-specific antibody 7D8 [3-5] forms the basis for the CD20-specific Fab arm of DuoBody-CD3xCD20. To study the contribution of this specific Fab arm to the observed potency of DuoBody-CD3xCD20, we compared the target binding characteristics and the capacity to induce T-cell-mediated cytotoxicity of a CD3 bsAb based on 7D8, with CD3 bsAbs using B-cell targeting arms derived from alternative CD20 antibodies or from antibodies against other well-known B-cell membrane molecules CD22, CD24, CD37, CD70, CD79b, CD138 and HLA-DR. In addition, target expression levels of the B-cell targets were assessed in a panel of B-cell lines. Using a classic chromium release assay, the 7D8-based CD3 bsAb displayed cytotoxic activity superior to all other B-cell-targeting CD3 bsAbs tested, including alternative CD20-targeting CD3 bsAbs. This unique cytotoxic activity could not be explained by expression levels of the target antigen, nor by the binding affinity or epitope of the B-cell specific Fab arm. This illustrates the complexity of factors that determine the potency of CD3 bsAbs. The anti-tumor activity of DuoBody-CD3xCD20 was confirmed in vivo in humanized mouse models using three different B-cell lymphoma xenograft models, in prophylactic and therapeutic settings. Non-clinical safety studies with DuoBody-CD3xCD20 in cynomolgus monkeys demonstrated profound and long-lasting B-cell depletion (at least 70 days, at dose levels > 0.1 mg/kg) from both peripheral blood and lymphoid organs. B-cell depletion was reversible, with time to B-cell recovery correlating with the treatment dose. Notably, at the same dose level, B-cell depletion was comparable between subcutaneous and intravenous administration. Pharmacokinetic (PK) analysis demonstrated comparable bioavailability for the two administration routes, although peak plasma levels were lower and delayed after subcutaneous administration. Moreover, lower plasma cytokine levels were observed after subcutaneous administration. Based on these data, Genmab has initiated a First-in-Human clinical trial to evaluate the safety and preliminary efficacy of DuoBody-CD3xCD20 by subcutaneous administration in patients with B-cell malignancies. The study is currently enrolling (EudraCT No: 2017-001748-36). References Labrijn, A.F., et al., Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proc Natl Acad Sci U S A, 2013. 110(13): p. 5145-50. Labrijn, A.F., et al., Controlled Fab-arm exchange for the generation of stable bispecific IgG1. Nat Protoc, 2014. 9(10): p. 2450-63. Teeling, J.L., et al., Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood, 2004. 104(6): p. 1793-800. Teeling, J.L., et al., The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique Epitopes on CD20. Journal of Immunology, 2006. 177(1): p. 362-71. van Meerten, T., et al., HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20 low expressing tumor cells that resist rituximab-mediated lysis. Haematologica, 2010. 95(12): p. 2063-71. Disclosures Hiemstra: Genmab: Employment, Other: Warrants. Engelberts:Genmab: Employment, Other: Warrants. de Jong:Genmab: Employment, Other: Warrants. Schuurhuis:Genmab: Employment, Other: Warrants. Salcedo:Genmab: Employment, Other: Warrants. Verploegen:Genmab: Employment, Equity Ownership. van der Zee:Genmab: Employment, Other: Warrants. Gerritsen:Genmab: Employment, Other: Warrants. Losic:Genmab: Employment, Other: Warrants. Horbach:Genmab: Employment, Other: Warrants. Oliveri:Genmab: Employment, Other: Warrants. Lammerts van Bueren:Genmab: Employment, Other: Warrants. Autzen Usher:Genmab: Employment, Other: Warrants. Schuurman:Genmab: Employment, Other: Warrants. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Breij:Genmab: Employment, Equity Ownership.

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 DARA's 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 Burkitt's lymphoma cell lines. Phagocytosis contributed to DARA's 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.

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.

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.

Hematopoiesis is a highly regulated process resulting in the formation of all blood lineages. Aberrant regulation of phosphatidylinositol-3-kinase (PI3K) signaling has been observed in hematopoietic malignancies, suggesting that regulated PI3K signaling is critical for regulation of blood cell production. An ex vivo differentiation system was used to investigate the role of PI3K and its downstream effector, protein kinase B (PKB/c-akt) in myelopolesis. PI3K activity was essential for hematopoietic progenitor survival. High PKB activity was found to promote neutrophil and monocyte development, while, conversely, reduction of PKB activity was required to induce optimal eosinophil differentiation. In addition, transplantation of beta 2-microglobulin(-/-) NOD/SCID mice with CD34(+) cells ectopically expressing constitutively active PKB resulted in enhanced neutrophil and monocyte development, whereas ectopic expression of dominant-negative PKB induced eosinophil development in vivo. Inhibitory phosphorylation of C/EBP alpha on Thr222/226 was abrogated upon PKB activation in hematopoietic progenitors. Ectopic expression of a nonphosphorylatable C/EBP alpha mutant inhibited eosinophil differentiation ex vivo, whereas neurtrophil development was induced, demonstrating the importance of PKB-mediated C/EBPa. phosphorylation in regulation of granulopoiesis. These results identify an important novel role for PKB in regulation of cell fate choices during hematopoietic lineage commitment.

Activation of granulocyte effector functions, such as induction of the respiratory burst and migration, are regulated by a variety of relatively ill-defined signaling pathways. Recently, we identified a novel Ca2+/calmodulin-dependent kinase I-like kinase, CKLiK, which exhibits restricted mRNA expression to human granulocytes. Using a novel antibody generated against the C-terminus of CKLiK, CKLiK was detected in CD34+-derived neutrophils and eosinophils, as well as in mature peripheral blood granulocytes. Activation of human granulocytes by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF), but not the phorbol ester PMA (phorbol 12-myristate-13-acetate), resulted in induction of CKLiK activity, in parallel with a rise of intracellular Ca2+ [Ca2+]i. To study the functionality of CKLiK in human granulocytes, a cell-permeable CKLiK peptide inhibitor (CKLiK297-321) was generated which was able to inhibit kinase activity in a dose-dependent manner. The effect of this peptide was studied on specific granulocyte effector functions such as phagocytosis, respiratory burst, migration, and adhesion. Phagocytosis of Aspergillus fumigatus particles was reduced in the presence of CKLiK297-321 and fMLP-induced reactive oxygen species (ROS) production was potently inhibited by CKLiK297-321 in a dose-dependent manner. Furthermore, fMLP-induced neutrophil migration on albumin-coated surfaces was perturbed, as well as β2-integrin-mediated adhesion. These findings suggest a critical role for CKLiK in modulating chemoattractant-induced functional responses in human granulocytes.

Background: Human polymorphic nuclear granulocytes (PMNs) such as neutrophils and eosinophils play a critical role in mediating inflammatory responses to microbial and parasitic infections. Exposure of these leukocytes to cytokines leads to an amplification of granulocyte effector functions by a mechanism termed "priming." Although many studies have investigated the effects of granulocyte priming, little is known concerning the molecular mechanisms that lead to this phenomenon. Objective: The purpose of this study was to identify potential markers for granulocyte priming and thus also to gain further insight into the pathogenesis of inflammatory responses. Methods: We used a modified differential display technique, random arbitrary primed–PCR to identify genes regulated during the priming of human polymorphic nuclear granulocytes by GM-CSF in vitro. Genes identified were validated by Northern blot analysis of in vitro and in vivo primed leukocytes. Results: Several genes were identified and their expression characterized in vitro. One of these genes, 5-lipoxygenase–activating protein, was also found to be up-regulated in leukocytes isolated after allergen challenge of allergic asthmatic patients. Conclusion: The use of differential display technology is a rapid and effective means of identifying genes whose expression is regulated by priming in vitro and in vivo. Further analysis will lead to a better understanding of the priming phenotype and may provide further insight into the pathologic mechanisms of inflammatory processes. (J Allergy Clin Immunol 2000;105:760-8.)

Introduction We recently discovered that complement is activated by IgG hexamers that assemble on the surface of cells following antigen binding. The formation of hexamers is dependent on intermolecular interactions in the Fc interface between neighboring IgG molecules. This seminal finding allowed us to identify mutations that enhance the ability of IgG antibodies to bind C1 and induce complement-dependent lysis. Objectives To study the role of Fc-mediated intermolecular contacts in complement-dependent cytotoxicity (CDC) of cancer cells by therapeutic IgG antibodies in biological relevant in vitro and in vivo model systems and to exploit this mechanism to improve CDC activity. M&M Based on an IgG hexamer crystal structure, we generated Fc mutations that were predicted to influence Fc:Fc interactions between IgG antibodies and we screened for improved C’ activation. The effect of these mutations on in vitro C1q binding and CDC-mediated killing of tumor B cell lines and patient-derived chronic lymphatic leukemia (CLL) cells was studied with therapeutic antibodies targeting different B cell antigens. The effect of a CDC-enhancing mutation was further studied in a Burkitt lymphoma xenograft model. Results Here we show that the assembly of IgG hexamers on the cell surface represents an important step for efficient complement activation via the classical pathway by therapeutic antibodies against hematological targets. Critical amino acid residues in the Fc:Fc contacts between IgG molecules were identified and individual mutants with increased complement activation generated. Interestingly, introduction of an enhancing mutation was shown to significantly increase complement activation not only of antibodies capable of inducing CDC, but also of antibodies that were devoid of such activity. We provide a proof-of-concept for CD19, Type I and Type II CD20, CD38 and CD52 antibodies. Importantly, we were able to achieve or enable significant CDC of a panel of primary CLL cells by use of this novel approach. Finally, we show that the enhancing mutation resulted in a significant increase in the inhibition of in vivo tumor growth by a CD20 antibody in a subcutaneous Raji xenograft model in SCID mice. Conclusion The data obtained provide a novel insight into the mechanism of complement-dependent lysis. This mechanism is shown to be relevant for a panel of therapeutic antibodies against hematological targets. Mutations that improved the formation of Fc:Fc-mediated hexamerization resulted in strongly increased CDC of complement-resistant primary CLL cells and improved efficacy in an in vivo model with a Burkitt lymphoma cell line. Disclosures: Beurskens: Genmab: Employment. de Jong:Genmab: Employment. Verploegen:Genmab: Employment. Voorhorst:Genmab: Employment. Strumane:Genmab: Employment. Lindorfer:Genmab: Research Funding. Taylor:Genmab: Research Funding. Schuurman:Genmab: Employment. Parren:Genmab: Employment.

Tissue factor (TF) is aberrantly expressed in a wide variety of solid tumors, and expression has been associated with poor prognosis. In normal physiology, TF is the main initiator of the coagulation cascade, which starts when circulating factor VII(a) (FVII(a)) binds membrane bound TF. The TF:FVIIa complex proteolytically activates FX to generate FXa, eventually resulting in clot formation. In addition, the TF:FVIIa complex activates PAR-2 intracellular signaling, thereby stimulating the production of pro-angiogenic factors, cytokines and adhesion molecules. We developed an antibody-drug conjugate (ADC) composed of a human TF-specific IgG1κ antibody (TF-011), a protease-cleavable valine-citrulline (vc) linker and the microtubule disrupting agent monomethyl auristatin E (MMAE). Unconjugated TF-011 efficiently inhibited TF:FVIIa induced ERK phosphorylation and IL-8 production, but showed only minor inhibition of FXa generation or clot formation. Unconjugated TF-011 efficiently killed TF-positive tumor cells by antibody dependent cell-mediated cytotoxicity (ADCC) in vitro, and showed some anti-tumor efficacy in vivo in a prophylactic setting. Upon target binding, TF-011 was rapidly internalized and co-localization with LAMP-1 was observed already after 1 hour. This suggests efficient lysosomal targeting, a prerequisite for intracellular release of MMAE and subsequent tumor cell killing by an ADC. Indeed, TF-011-vcMMAE efficiently induced tumor cell killing in vitro, which was dependent on and correlated with TF cell surface expression. In addition, TF-011-vcMMAE demonstrated potent anti-tumor efficacy in xenograft models for pancreatic and epidermoid cancer in vivo. Therapeutic treatment at doses as low as 0,3 mg/kg inhibited tumor growth, whereas tumor regression was observed at doses of 1 mg/kg or higher. Importantly, TF-011-vcMMAE also induced tumor cell killing in human biopsy-derived xenograft models, which are thought to represent the genetic and histological heterogeneity of human tumors. Immunohistochemical analysis confirmed that the heterogeneity of TF expression in human tumors was reflected in human biopsy-derived xenografts. TF-011-vcMMAE induced efficient tumor regression in xenograft models for bladder, lung, pancreatic, prostate, ovarian and cervical cancer, with the percentage of TF positive cells ranging from 25-50% to 75-100%. In two tumor models that showed TF expression in less than 25% of tumor cells, TF-011-vcMMAE showed inhibition of tumor growth. In summary, TF-011-vcMMAE is a promising new ADC that showed potent anti-tumor activity in vivo in a wide variety of models, including models that represent the heterogeneous TF expression that is observed in human tumors. The ADC potently kills tumor cells by disrupting microtubules, while preserving the effector functions of the unconjugated antibody. Citation Format: Esther C.W. Breij, David Satijn, Sandra Verploegen, Bart E. de Goeij, Danita H. Schuurhuis, Mischa Houtkamp, Wim K. Bleeker, Paul W. Parren. An antibody-drug conjugate targeting tissue factor with broad anti-tumor efficacy in xenograft models with heterogeneous tissue factor expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1234. doi:10.1158/1538-7445.AM2013-1234

Abstract 4054 Daratumumab (DARA) is a human IgG1 CD38 antibody with broad-spectrum killing activity. DARA induces killing of CD38-expressing tumor cells, including fresh cells from multiple myeloma (MM) patient samples, via diverse mechanisms. These prominently include the Fc-dependent effector mechanisms complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers et al. J. Immunol. 2011). In this study we show that DARA is also able to induce tumor cell killing via antibody-dependent cellular phagocytosis (ADCP) as an additional Fc-dependent effector mechanism. In a first set of experiments, we studied ADCP with human macrophages as effector cells. Calcein-AM-labeled Daudi tumor cells, a Burkitt's lymphoma cell line, were mixed with human macrophages in the absence or presence of DARA. Specific DARA-induced phagocytosis was analyzed in flow cytometry by measuring the percentage of calcein-AM+/CD11b+ double-positive (DP) macrophages. Both classical GM-CSF activated and alternative M-CSF activated macrophages mediated DARA-specific ADCP of the Burkitt's lymphoma cells. To further explore the in vivo contribution of ADCP in the mechanism of action (MoA) of DARA, we studied DARA mediated phagocytosis with murine macrophages. In vitro ADCP with M-CSF-stimulated bone marrow-derived murine macrophages showed a dose-dependent DARA-specific effect on the Burkitt's lymphoma cell lines Ramos and Daudi, resulting in up to 24% and 43% DP macrophages and a 25% and 50% tumor cell reduction, respectively. Furthermore, dose-dependent DARA-specific phagocytosis was observed with patient-derived MM cell lines L363 and UM9, which were transduced with CD38 to obtain levels CD38 expression as they are generally observed in primary MM patient samples. With life-cell imaging we found that ADCP of Daudi and Ramos cells occurred very rapid and efficiently. Interestingly, our recordings document that single macrophages could engage multiple target cells and that they were able to engulf up to six tumor cells sequentially in a 30 min period. This suggests that ADCP might be a very potent MoA of DARA in vivo, which we are currently studying in a mouse xenograft model. In conclusion, in addition to CDC and ADCC, we now show that DARA can also induce killing of CD38 expressing tumor cells via phagocytosis. This very fast and potent MoA might contribute to the treatment efficacy of DARA in hematological tumors, especially at sites where high numbers of macrophages reside, such as the bone marrow. Disclosures: Overdijk: Genmab BV: Employment. Verploegen:Genmab BV: Employment. Groen:Genmab BV: Research Funding. Martens:Genmab BV: Research Funding. Lammerts van Bueren:Genmab BV: Employment. Bleeker:Genmab BV: Employment. Parren:Genmab BV: Employment.