Maaike Nederend

The in vivo mechanism of action of CD20 monoclonal antibodies depends on local tumor burden

Background CD20 monoclonal antibodies are widely used in clinical practice. Antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct cell death have been suggested to be important effector functions for CD20 antibodies. However, their specific contributions to the in vivo mechanism of action of CD20 immunotherapy have not been well defined. Design and Methods Here we studied the in vivo mechanism of action of type I (rituximab and ofatumumab) and type II (HuMab-11B8) CD20 antibodies in a peritoneal, syngeneic, mouse model with EL4-CD20 cells using low and high tumor burden. Results Interestingly, we observed striking differences in the in vivo mechanism of action of CD20 antibodies dependent on tumor load. In conditions of low tumor burden, complement was sufficient for tumor killing both for type I and type II CD20 antibodies. In contrast, in conditions of high tumor burden, activating FcγR (specifically FcγRIII), active complement and complement receptor 3 were all essential for tumor killing. Our data suggest that complement-enhanced antibody-dependent cellular cytotoxicity may critically affect tumor killing by CD20 antibodies in vivo. The type II CD20 antibody 11B8, which is a poor inducer of complement activation, was ineffective against high tumor burden. Conclusions Tumor burden affects the in vivo mechanism of action of CD20 antibodies. Low tumor load can be eliminated by complement alone, whereas elimination of high tumor load requires multiple effector mechanisms.

IgA EGFR antibodies mediate tumour killing in vivo

Currently all approved anti‐cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti‐tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti‐tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10‐EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short‐term syngeneic peritoneal model using EGFR‐transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer.

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.

Staphylococcus aureus Formyl Peptide Receptor–like 1 Inhibitor (FLIPr) and Its Homologue FLIPr-like Are Potent FcγR Antagonists That Inhibit IgG-Mediated Effector Functions

To evade opsonophagocytosis, Staphylococcus aureus secretes various immunomodulatory molecules that interfere with effective opsonization by complement and/or IgG. Immune-evasion molecules targeting the phagocyte receptors for these opsonins have not been described. In this study, we demonstrate that S. aureus escapes from FcγR-mediated immunity by secreting a potent FcγR antagonist, FLIPr, or its homolog FLIPr-like. Both proteins were previously reported to function as formyl peptide receptor inhibitors. Binding of FLIPr was mainly restricted to FcγRII receptors, whereas FLIPr-like bound to different FcγR subclasses, and both competitively blocked IgG-ligand binding. They fully inhibited FcγR-mediated effector functions, including opsonophagocytosis and subsequent intracellular killing of S. aureus by neutrophils and Ab-dependent cellular cytotoxicity of tumor cells by both neutrophils and NK cells. In vivo, treatment of mice with FLIPr-like prevented the development of an immune complex–mediated FcγR-dependent Arthus reaction. This study reveals a novel immune-escape function for S. aureus–secreted proteins that may lead to the development of new therapeutic agents in FcγR-mediated diseases.

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.

Fcγ receptor IIIA genotype is associated with rituximab response in antimyelin-associated glycoprotein neuropathy

Background Treatment with anti-B cell antibody rituximab may ameliorate the disease course in a subgroup of patients with polyneuropathy associated with IgM monoclonal gammopathy. Polymorphisms of leukocyte IgG receptors (FcγR) that influence efficiency of antibody-dependent cell-mediated cytotoxicity determine rituximab efficacy in patients with lymphoma and autoimmune disease. Objective To investigate the association of FcγRIIA and FcγRIIIA polymorphisms with the response to rituximab treatment in a cohort of patients with polyneuropathy associated with IgM monoclonal gammopathy (PNP-IgM) with and without antimyelin-associated glycoprotein antibodies. Methods We determined FcγRIIA-R/H131 and FcγRIIIA-V/F158 genotypes in 27 patients with PNP-IgM using allele-specific PCR and Sanger sequencing. Results The FcγRIIIA-V/V158 genotype was associated with functional improvement (p=0.02) after 1 year. Conclusions FcγRIIIA polymorphisms are potential biomarkers for response to rituximab treatment in polyneuropathy associated with IgM monoclonal gammopathy.

A novel FcγRIIa Q27W gene variant is associated with common variable immune deficiency through defective FcγRIIa downstream signaling

We identified a novel Q27W FcγRIIa variant that was found more frequently in common variable immunodeficiency (CVID) or CVID-like children. We analyzed the possible functional consequence of the Q27W FcγRIIa mutation in human cells. We used peripheral blood mononuclear cells from Q27W FcγRIIa patients and healthy controls, and cultured cells that overexpress the Q27W and common FcγRIIa variants. The Q27W FcγRIIa mutation does not disrupt FcγRIIa surface expression in peripheral blood mononuclear cells. Mononuclear cells express multiple FcγR, precluding careful analysis of Q27W FcγRIIa functional deviation. For functional analysis of FcγRIIa function, we therefore overexpressed the Q27W FcγRIIa and common FcγRIIa variant in IIA1.6 cells that are normally deficient in FcγR. We show that FcγRIIa triggering-induced signaling is obstructed, as measured by both decrease in calcium flux and defective MAPK phosphorylation. In conclusion, we here describe a novel Q27W FcγRIIa variant that causes delayed downstream signaling. This variant may contribute to CVID.

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.