WHI Parren (2015) Antibody-mediated phagocytosis contributes to the antitumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma, mAbs, 7:2, 311-320,

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.

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.

The CD38 molecule, with its high expression on Multiple Myeloma (MM), is considered a suitable target for antibody therapy of MM. We developed daratumumab (DARA), a human CD38 monoclonal antibody (mAb) with direct and Fc-mediated cell killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M. J Immunol 2011), and antibody-dependent cellular phagocytosis (ADCP) by macrophages (both murine and human). In addition, DARA induces apoptosis upon secondary cross-linking and modulates CD38 enzymatic function. DARA is currently in phase I, II and III clinical evaluation in patients with MM. Besides DARA, several other anti-CD38 mAb are in development; SAR650894 (SAR; clone 38SB19; Sanofi-Aventis) for MM and other CD38+ hematological malignancies, MOR03087 (MOR; Morphosys) for relapsed/refractory MM and Ab79 (Millenium/Takeda) which is in preclinical development. Similar mechanisms of action (MoA) are described for these mAb; nevertheless direct comparison studies would be critical for differentiation among these antibodies. . In this study, the efficacy of these anti-CD38 mAb was directly compared to DARA with respect to binding, apoptosis, CD38 ectoenzyme activity, and the induction of ADCC, ADCP and CDC. Surrogate antibodies of MOR, SAR and Ab79 were generated on the basis of protein sequences, as published in their corresponding patents families, and were attached to the backbone of DARA. Binding to CD38 expressing Daudi tumor cells was assessed by flow cytomery. All CD38 antibodies showed similar EC50 (~0.1 µg/mL) and maximal binding, except MOR which showed a lower apparent affinity (~0.3 µg/mL). Previously, CD38 amino acid residues Q272 and S274 were reported as critical for DARA binding. ELISA analyses using CD38 point mutants revealed MOR, SAR and Ab79 not to be affected by mutation of these residues. All CD38 mAb were equally potent in inducing ADCC of Daudi cells (40-60% lysis, 0.02 µg/mL), in classic Cr51-release ADCC assay using human PBMC effector cells (E:T ratio 100:1). Important differences were observed with respect to induction of CDC. SAR was unable to induce CDC in Daudi cells at concentrations up to 30 µg/mL, while DARA induced more than 80% lysis at concentrations above 1 µg/mL. Ab79 and MOR induced CDC, yet maximum lysis was 50% and 20%, respectively. Evaluation of Annexin V/propidium iodide (AnnV/PI) staining and activation of caspase-3 showed that only SAR induced AnnV/PI+ in Ramos cells (~40%) after 48 h exposure without Fc crosslinking, but did not activate caspase-3. In the presence of Fc crosslinking antibodies, all anti-CD38 mAb induced AnnV/PI+, caspase-3 mediated apoptosis. In enzyme activity assays using purified CD38 protein, SAR inhibited generation of cGDPR (indicative of the combined CD38 cyclase activity generating fluorescent cGDPR and hydrolase activity converting cGDPR into GDPR)more effectively than DARA (~70% vs. ~20% inhibition at 30 µg/mL). Ab79 had a modest effect on CD38 enzyme activity (~10% inhibition). MOR did not affect CD38 enzyme activity at all. The capacity to induce ADCP was only tested for DARA, MOR and Ab79 using mouse macrophages (mφ) as effector cells and Daudi target cells. mφ, isolated and matured from bone marrow cells, and calcein-AM labelled target cells (E:T ratio 1:1) were cultured in the presence of 0.0006-5 µg/mL antibody for 4 h. Non-phagocytosed target cells and mφ were collected and ADCP was evaluated by flow cytometry. All CD38 mAb induced mφ-mediated phagocytosis, as observed by a concentration dependent increase in the number of double positive mφ and killing of target cells. Ab79 was as effective as DARA (EC50 ~0.01 µg/mL) in ADCP induction, whereas MOR was less effective (EC50 0.04 µg/mL). In summary, DARA and surrogate mAb of MOR, SAR and Ab79 showed similar binding to cells and induced similar amounts of ADCC. Differences between these mAb involved the ability to directly induce apoptosis, to inhibit the enzymatic activity of CD38 and to induce ADCP. The most striking difference was observed for the ability to induce CDC, the MoA which is currently believed the most important mechanism of MM cell killing in the clinic. DARA efficiently induced high levels of CDC at low concentrations, whereas the other CD38 mAb were unable or less capable to induce CDC. Disclosures Lammerts van Bueren: Genmab: Employment, warrents Other. Jakobs:Genmab: Employment, warrents Other. Kaldenhoven:Genmab: Employment, warrents Other. Roza:Genmab: Employment, warrents Other. Hiddingh:Genmab: Employment. Meesters:Genmab: Employment, warrents Other. Voorhorst:Genmab: Employment, warrents Other. Gresnigt:Genmab: Employment, warrents Other. Wiegman:Genmab: Employment, warrents Other. Ortiz Buijsse:Genmab: Employment, warrents Other. Andringa:Genmab: Employment, warrents Other. Overdijk:Genmab: Employment, warrents Other. Doshi:Janssen R&D: Employment. Sasser:Janssen R&D: Employment. de Weers:Genmab: Employment, warrents Other. Parren:Genmab: Employment, inventor on patents regarding daratumumab Patents & Royalties, stock and warrents Other.

Paratuberculosis is a chronic granulomatous inflammation of the small intestine of cattle and other ruminants, caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). The disease can be found in ruminant herds worldwide, causing substantial economic losses at farm level due to premature culling and production losses.In previous studies, it has been shown that immune responses to recombinant MAP Hsp70 proteins were predominantly cell mediated. As protective immunity to the intracellular mycobacterial pathogens is thought to be cell-mediated in origin, we have studied the use of a recombinant MAP Hsp70 as a subunit vaccine in cattle experimentally infected with MAP.The results of the current study demonstrate that recombinant MAP Hsp70 can be successfully used as a subunit vaccine against bovine paratuberculosis, significantly reducing shedding of bacteria in feces during the first 2 years following experimental infection.

The online version of this article has a Supplementary Appendix. BackgroundMultiple myeloma is a hematologic malignancy characterized by a clonal expansion of malignant plasma cells in the bone marrow, which is accompanied by the development of osteolytic lesions and/or diffuse osteopenia. The intricate bi-directional interaction with the bone marrow microenvironment plays a critical role in sustaining the growth and survival of myeloma cells during tumor progression. Identification and functional analysis of the (adhesion) molecules involved in this interaction will provide important insights into the pathogenesis of multiple myeloma. Design and MethodsMultiple myeloma cell lines and patients' samples were analyzed for expression of the adhesion molecule N-cadherin by immunoblotting, flow cytometry, immunofluorescence microscopy, immunohistochemistry and expression microarray. In addition, by means of blocking antibodies and inducible RNA interference we studied the functional consequence of N-cadherin expression for the myeloma cells, by analysis of adhesion, migration and growth, and for the bone marrow microenvironment, by analysis of osteogenic differentiation. ResultsThe malignant plasma cells in approximately half of the multiple myeloma patients, belonging to specific genetic subgroups, aberrantly expressed the homophilic adhesion molecule N-cadherin. N-cadherin-mediated cell-substrate or homotypic cell-cell adhesion did not contribute to myeloma cell growth in vitro. However, N-cadherin directly mediated the bone marrow localization/retention of myeloma cells in vivo, and facilitated a close interaction between myeloma cells and N-cadherin-positive osteoblasts. Furthermore, this N-cadherin-mediated interaction contributed to the ability of myeloma cells to inhibit osteoblastogenesis. ConclusionsTaken together, our data show that myeloma cells frequently display aberrant expression of Ncadherin and that N-cadherin mediates the interaction of myeloma cells with the bone marrow microenvironment, in particular the osteoblasts. This N-cadherin-mediated interaction inhibits osteoblast differentiation and may play an important role in the pathogenesis of myeloma bone disease.Key words: N-cadherin, multiple myeloma, osteoblast differentiation.Citation: Groen RWJ, de Rooij MFM, Kocemba KA, Reijmers RM, de Haan-Kramer A, Overdijk MB, Aalders L, Rozemuller H, Martens ACM, Bergsagel PL, Kersten MJ, Pals ST, and Spaargaren M. N-cadherin-mediated Haematologica 2011;96(11):1653-1661. doi:10.3324/haematol.2010 This is an open-access paper. interaction with multiple myeloma cells inhibits osteoblast differentiation. N-cadherin-mediated interaction with multiple myeloma cells inhibits osteoblast differentiation

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Tetraspanin CD37 has recently received renewed interest as a therapeutic target for B-cell malignancies. Although complement-dependent cytotoxicity (CDC) is a powerful Fc-mediated effector function for killing hematological cancer cells, CD37-specific antibodies are generally poor inducers of CDC. To enhance CDC, the E430G mutation was introduced into humanized CD37 monoclonal IgG1 antibodies to drive more efficient IgG hexamer formation through intermolecular Fc-Fc interactions after cell surface antigen binding. DuoHexaBody-CD37, a bispecific CD37 antibody with the E430G hexamerization-enhancing mutation targeting two non-overlapping epitopes on CD37 (biparatopic), demonstrated potent and superior CDC activity compared to other CD37 antibody variants evaluated, in particular ex vivo in patient-derived chronic lymphocytic leukemia cells. The superior CDC potency was attributed to enhanced IgG hexamerization mediated by the E430G mutation in combination with dual epitope targeting. The mechanism of action of DuoHexaBody-CD37 was shown to be multifaceted, as it was additionally capable of inducing efficient antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vitro. Finally, potent antitumor activity in vivo was observed in cell line-and patient-derived xenograft models from different B-cell malignancy subtypes. These encouraging preclinical results suggest that DuoHexaBody-CD37 (GEN3009) may serve as a potential therapeutic antibody for the treatment of human B-cell malignancies.