J.H. Marco Jansen

In vivo Cytotoxicity of Type I CD20 Antibodies Critically Depends on Fc Receptor ITAM Signaling

Antibody-Fc receptor (FcR) interactions play an important role in the mechanism of action of most therapeutic antibodies against cancer. Effector cell activation through FcR triggering may induce tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC). Reciprocally, FcR cross-linking of antibody may lead to the induction of apoptotic signaling in tumor cells. The relative importance of these bisecting pathways to in vivo antibody activity is unknown. To unravel these roles, we developed a novel mouse model with normal FcR expression but in which FcR signaling was inactivated by mutation of the associated gamma-chain. Transgenic mice showed similar immune complex binding compared with wild-type mice. In contrast, ADCC of cells expressing frequently used cancer targets, such as CD20, epidermal growth factor receptor, Her2, and gp75, was abrogated. Using the therapeutic CD20 antibodies ofatumumab and rituximab, we show that FcR cross-linking of antibody-antigen immune complexes in the absence of gamma-chain signaling is insufficient for their therapeutic activity in vivo. ADCC therefore represents an essential mechanism of action for immunotherapy of lymphoid tumors.

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.

Both activating and inhibitory Fc gamma receptors mediate rituximab-induced trogocytosis of CD20 in mice.

Antigenic modulation by trogocytosis during anti-CD20 mAb treatment with rituximab (RTX) leads to loss of CD20 and therefore can compromise therapy. During trogocytosis, effector cells, such as macrophages, remove CD20 from the surface of antibody-coated cells in an Fc receptor-dependent manner. Importantly, Fcγ receptors (FcγRs) are also crucial in the anti-tumor effects of RTX by inducing antibody dependent cell-mediated cytotoxicity (ADCC). Here we studied the role of FcγR during RTX-induced trogocytosis of CD20 in an intraperitoneal tumor model with EL4-CD20 cells. We found marked RTX-induced trogocytosis of CD20 in FcγRI- or FcγRIII-deficient mice, similar to wild type mice, demonstrating a redundancy for activating FcγR in trogocytosis. Interestingly, in FcRγ-chain-deficient mice, trogocytosis was still apparent, indicating that the inhibitory receptor FcγRIIB alone can also mediate trogocytosis. These data were confirmed by in vitro analysis with blocking antibodies. Decreasing the amount of RTX in vivo resulted in less trogocytosis of CD20, supporting clinical studies with lower RTX dose. Importantly, we show that cells which undergo in vivo trogocytosis can still be killed ex vivo by ADCC but not by complement-mediated cytotoxicity (CDC), underscoring the clinical relevance of trogocytosis. Taken together, our study provides more insights into the mechanism and consequences of RTX-induced trogocytosis of CD20.

An anti-EGFR IgA that displays improved pharmacokinetics and myeloid effector cell engagement in vivo

Antibodies of IgA isotype effectively engage myeloid effector cells for cancer immunotherapy. Here, we describe preclinical studies with an Fc engineered IgA2m(1) antibody containing the variable regions of the EGFR antibody cetuximab. Compared with wild-type IgA2m(1), the engineered molecule lacked two N-glycosylation sites (N166 and N337), two free cysteines (C311 and C472), and contained a stabilized heavy and light chain linkage (P221R mutation). This novel molecule displayed improved production rates and biochemical properties compared with wild-type IgA. In vitro, Fab- and Fc-mediated effector functions, such as inhibition of ligand binding, receptor modulation, and engagement of myeloid effector cells for antibody-dependent cell-mediated cytotoxicity, were similar between wild-type and engineered IgA2. The engineered antibody displayed lower levels of terminal galactosylation leading to reduced asialoglycoprotein-receptor binding and to improved pharmacokinetic properties. In a long-term in vivo model against EGFR-positive cancer cells, improved serum half-life translated into higher efficacy of the engineered molecule, which required myeloid cells expressing human FcαRI for its full efficacy. However, Fab-mediated effector functions contributed to the in vivo efficacy because the novel IgA antibody demonstrated therapeutic activity also in non-FcαRI transgenic mice. Together, these results demonstrate that engineering of an IgA antibody can significantly improve its pharmacokinetics and its therapeutic efficacy to inhibit tumor growth in vivo.

Improved in vivo anti-tumor effects of IgA-Her2 antibodies through half-life extension and serum exposure enhancement by FcRn targeting

Antibody therapy is a validated treatment approach for several malignancies. All currently clinically applied therapeutic antibodies (Abs) are of the IgG isotype. However, not all patients respond to this therapy and relapses can occur. IgA represents an alternative isotype for antibody therapy that engages FcαRI expressing myeloid effector cells, such as neutrophils and monocytes. IgA Abs have been shown to effectively kill tumor cells both in vitro and in vivo. However, due to the short half-life of IgA Abs in mice, daily injections are required to reach an effect comparable to IgG Abs. The relatively long half-life of IgG Abs and serum albumin arises from their capability of interacting with the neonatal Fc receptor (FcRn). As IgA Abs lack a binding site for FcRn, we generated IgA Abs with the variable regions of the Her2-specific Ab trastuzumab and attached an albumin-binding domain (ABD) to the heavy or light chain (HCABD/LCABD) to extend their serum half-life. These modified Abs were able to bind albumin from different species in vitro. Furthermore, tumor cell lysis of IgA-Her2-LCABD Abs in vitro was similar to unmodified IgA-Her2 Abs. Pharmacokinetic studies in mice revealed that the serum exposure and half-life of the modified IgA-Her2 Abs was extended. In a xenograft mouse model, the modified IgA1 Abs exhibited a slightly, but significantly, improved anti-tumor response compared to the unmodified Ab. In conclusion, empowering IgA Abs with albumin-binding capacity results in in vitro and in vivo functional Abs with an enhanced exposure and prolonged half-life.

MCL-1 is required throughout B-cell development and its loss sensitizes specific B-cell subsets to inhibition of BCL-2 or BCL-XL

Pro-survival BCL-2 family members protect cells from programmed cell death that can be induced by multiple internal or external cues. Within the haematopoietic lineages, the BCL-2 family members BCL-2, BCL-XL and MCL-1 are known to support cell survival but the individual and overlapping roles of these pro-survival BCL-2 proteins for the persistence of individual leukocyte subsets in vivo has not yet been determined. By combining inducible knockout mouse models with the BH3-mimetic compound ABT-737, which inhibits BCL-2, BCL-XL and BCL-W, we found that dependency on MCL-1, BCL-XL or BCL-2 expression changes during B-cell development. We show that BCL-XL expression promotes survival of immature B cells, expression of BCL-2 is important for survival of mature B cells and long-lived plasma cells (PC), and expression of MCL-1 is important for survival throughout B-cell development. These data were confirmed with novel highly specific BH3-mimetic compounds that target either BCL-2, BCL-XL or MCL-1. In addition, we observed that combined inhibition of these pro-survival proteins acts in concert to delete specific B-cell subsets. Reduced expression of MCL-1 further sensitized immature as well as transitional B cells and splenic PC to loss of BCL-XL expression. More markedly, loss of MCL-1 greatly sensitizes PC populations to BCL-2 inhibition using ABT-737, even though the total wild-type PC pool in the spleen is not significantly affected by this drug and the bone marrow (BM) PC population only slightly. Combined loss or inhibition of MCL-1 and BCL-2 reduced the numbers of established PC >100-fold within days. Our data suggest that combination treatment targeting these pro-survival proteins could be advantageous for treatment of antibody-mediated autoimmune diseases and B-cell malignancies.

FcRγ-Chain ITAM Signaling Is Critically Required for Cross-Presentation of Soluble Antibody–Antigen Complexes by Dendritic Cells

The uptake of Ag–Ab immune complexes (IC) after the ligation of activating FcγR on dendritic cells (DC) leads to 100 times more efficient Ag presentation than the uptake of free Ags. FcγRs were reported to facilitate IC uptake and simultaneously induce cellular activation that drives DC maturation and mediates efficient T cell activation. Activating FcγRs elicit intracellular signaling via the ITAM domain of the associated FcRγ-chain. Studies with FcRγ-chain knockout (FcRγ−/−) mice reported FcRγ-chain ITAM signaling to be responsible for enhancing both IC uptake and DC maturation. However, FcRγ-chain is also required for surface expression of activating FcγRs, hampering the dissection of ITAM-dependent and independent FcγR functions in FcRγ−/− DCs. In this work, we studied the role of FcRγ-chain ITAM signaling using DCs from NOTAM mice that express normal surface levels of activating FcγR, but lack functional ITAM signaling. IC uptake by bone marrow–derived NOTAM DCs was reduced compared with wild-type DCs, but was not completely absent as in FcRγ−/− DCs. In NOTAM DCs, despite the uptake of ICs, both MHC class I and MHC class II Ag presentation was completely abrogated similar to FcRγ−/− DCs. Secretion of cytokines, upregulation of costimulatory molecules, and Ag degradation were abrogated in NOTAM DCs in response to FcγR ligation. Cross-presentation using splenic NOTAM DCs and prolonged incubation with OVA-IC was also abrogated. Interestingly, in this setup, proliferation of CD4+ OT-II cells was induced by NOTAM DCs. We conclude that FcRγ-chain ITAM signaling facilitates IC uptake and is essentially required for cross-presentation, but not for MHC class II Ag presentation.

Anti-tumor activity of human IgG1 anti-gp75 TA99 mAb against B16F10 melanoma in human FcgammaRI transgenic mice.

Patients suffering from advanced melanoma have a very poor prognosis. Despite recent advances in the understanding of oncogenic mechanisms and therapeutic interventions, the median survival of patients with metastatic disease is less than 12 months. Immunotherapy of melanoma has been intensely investigated and holds great promises. Tyrosinase-related protein-1 or gp75 (TYRP-1/gp75) antigen is a melanosomal polypeptide. It is the most abundant glycoprotein synthesized by pigmented melanocytes and melanomas. It is specific for melanocytes and both primary and metastatic melanomas. In mice, administration of the mouse mAb anti-gp75 TA99 prevents outgrowth of B16F10 melanoma metastases. The activity of TA99 is dependent on the presence and activity of the IgG specific, Fc receptors. TA99 cross-reacts with human gp75, and is currently being used for diagnosis of patients. Here, we sequenced mIgG2a TA99 and found that the locus harboring the endogenous light chain of the fusion partner in the TA99 hybridoma cells is not inactivated, resulting in the production of a mixed pool of mAbs that mitigates binding to gp75. Since human IgG1 (hIgG1) is the most frequently used mAb format in clinical studies, we produced a recombinant hIgG1 TA99 molecule. Whereas it is known that hIgG1 can functionally interact with mouse Fc receptors, we found that hIgG1 TA99 did not exhibit in vivo activity against B16F10 melanoma in wild type C57BL/6 mice. However, results obtained in this study demonstrated anti-tumor activity of hIgG1 TA99 in FcγRIIB knockout mice and in human FcγRI transgenic mice. These results emphasize the need for testing hIgG mAb in mice with functional human FcγRs.

Simultaneous Targeting of FcγRs and FcαRI Enhances Tumor Cell Killing

The efficacy of anticancer monoclonal antibodies (mAbs) is limited by the exhaustion of cellular effector mechanisms. The combination of IgG and IgA to two different tumor targets leads to enhanced cytotoxicity, providing a basis for therapeutic mAb improvements. Efficacy of anticancer monoclonal antibodies (mAb) is limited by the exhaustion of effector mechanisms. IgG mAbs mediate cellular effector functions through FcγRs expressed on effector cells. IgA mAbs can also induce efficient tumor killing both in vitro and in vivo. IgA mAbs recruit FcαRI-expressing effector cells and therefore initiate different effector mechanisms in vivo compared with IgG. Here, we studied killing of tumor cells coexpressing EGFR and HER2 by the IgG mAbs cetuximab and trastuzumab and their IgA variants. In the presence of a heterogeneous population of effector cells (leukocytes), the combination of IgG and IgA mAbs to two different tumor targets (EGFR and HER2) led to enhanced cytotoxicity compared with each isotype alone. Combination of two IgGs or two IgAs or IgG and IgA against the same target did not enhance cytotoxicity. Increased cytotoxicity relied on the presence of both the peripheral blood mononuclear cell and the polymorphonuclear (PMN) fraction. Purified natural killer cells were only cytotoxic with IgG, whereas cytotoxicity induced by PMNs was strong with IgA and poor with IgG. Monocytes, which coexpress FcγRs and FcαRI, also displayed increased cytotoxicity by the combination of IgG and IgA in an overnight killing assay. Coinjection of cetuximab and IgA2-HER2 resulted in increased antitumor effects compared with either mAb alone in a xenograft model with A431-luc2-HER2 cells. Thus, the combination of IgG and IgA isotypes optimally mobilizes cellular effectors for cytotoxicity, representing a promising novel strategy to improve mAb therapy. Cancer Immunol Res; 3(12); 1316–24. ©2015 AACR.

Inhibition of the Classical and Lectin Pathway of the Complement System by Recombinant LAIR-2

Activation of complement may cause severe tissue damage in antibody-mediated allograft rejection and other antibody-mediated clinical conditions; therefore, novel potent complement inhibitors are needed. Previously, we described binding of the inhibitory receptor LAIR-1 and its soluble family member LAIR-2 to collagen. Here, we investigated binding of LAIR-1 and LAIR-2 to the complement proteins C1q and MBL, which both have collagen-like domains, and evaluated the effect of this binding on complement function. We demonstrate specific binding of recombinant LAIR proteins to both C1q and MBL. Surface plasmon resonance experiments showed that LAIR-2-Fc protein bound C1q and MBL with the highest affinity compared to LAIR-2-HIS. We, therefore, hypothesized that LAIR-2-Fc is a potent complement inhibitor. Indeed, LAIR-2-Fc inhibited C4 fixation to IgG or mannan, reduced activation of C4 by aggregated IgG in plasma and inhibited iC3b deposition on cells. Finally, LAIR-2-Fc inhibited complement-mediated lysis of cells sensitized with anti-HLA antibodies in an ex vivo model for antibody-mediated transplant rejection. Thus, LAIR-2-Fc is an effective novel complement inhibitor for the treatment and prevention of antibody-mediated allograft rejection and antibody-mediated clinical conditions.