Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG isotypes. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.

The promise of bispecific antibodies (bsAbs) to yield more effective therapeutics is well recognized; however, the generation of bsAbs in a practical and cost-effective manner has been a formidable challenge. Here we present a technology for the efficient generation of bsAbs with normal IgG structures that is amenable to both antibody drug discovery and development. The process involves separate expression of two parental antibodies, each containing single matched point mutations in the CH3 domains. The parental antibodies are mixed and subjected to controlled reducing conditions in vitro that separate the antibodies into HL half-molecules and allow reassembly and reoxidation to form highly pure bsAbs. The technology is compatible with standard large-scale antibody manufacturing and ensures bsAbs with Fcmediated effector functions and in vivo stability typical of IgG1 antibodies. Proof-of-concept studies with HER2×CD3 (T-cell recruitment) and HER2×HER2 (dual epitope targeting) bsAbs demonstrate superior in vivo activity compared with parental antibody pairs.immunotherapy | pharmacokinetics | anti-tumor

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell–expressed antigen.

Tiam1 is a ubiquitously expressed activator of the small GTPase Rac. Previously, we found that Tiam1 knockout (KO) mice are resistant to DMBA-induced skin tumorigenicity, which correlated with increased apoptosis in keratinocytes of the skin epidermis. Here, we have studied the mechanisms by which Tiam1 protects against apoptosis. We found that Tiam1-KO keratinocytes show increased apoptosis in response to apoptotic stimuli, including growth factor deprivation and heat-shock treatment. Expression of catalytically active Tiam1, but not inactive Tiam1, rescues the apoptosis susceptibility of Tiam1-KO keratinocytes, indicating that this defect is caused by impaired Tiam1-mediated Rac activation. Apoptosis induced by growth factor starvation correlates with impaired ERK phosphorylation in Tiam1-KO keratinocytes. Moreover, Tiam1-KO keratinocytes contain lower levels of intracellular reactive oxygen species (ROS) when compared with wild-type cells. The ROS content of keratinocytes is dependent on both Tiam1 and the activity of NADPH oxidase (Nox), and is required for ERKmediated survival signaling. Indeed, Tiam1 deficiency or the inhibition of intracellular ROS production blocks ERK phosphorylation and sensitizes wild-type keratinocytes to apoptotic stimuli. Our results indicate that the Rac activator Tiam1 controls the intracellular redox balance by Nox-mediated ROS production, which regulates ERK phosphorylation and the susceptibility of keratinocytes to apoptotic signaling.

Complement is recognized as a key player in a wide range of normal as well as disease-related immune, developmental and homeostatic processes. Knowledge of complement components, structures, interactions, and cross-talk with other biological systems continues to grow and this leads to novel treatments for cancer, infectious, autoimmune- or age-related diseases as well as for preventing transplantation rejection. Antibodies are superbly suited to be developed into therapeutics with appropriate complement stimulatory or inhibitory activity. Here we review the design, development and future of antibody-based drugs that enhance or dampen the complement system.

CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo . Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.

Tumor progression requires the dispersion of epithelial cells from neoplastic clusters and cell invasion of adjacent stromal connective tissue. Aiming at demonstrating the precise relationships between cell dispersion and cell invasion, related respectively to expression of E-cadherin/catenin complex and matrix metalloproteinases (MMPs), we developed an original in vitro model of cell dispersion analysis. Our study reports the validation of this model that allowed us to analyze and quantify the cell cohesion level by means of timelapse videomicroscopy and computer analysis based on the observation of spatial and temporal cell distribution. Our model was able to distinguish 2 groups among different human bronchial and mammary epithelial cells previously characterized for the expression of E-cadherin/catenin complex and MMPs and their invasive capacity in the Boyden chamber assay. The first group (16HBE14o ؊ , MCF-7, T47D) that expressed membranous E-cadherin and ␤-catenin, and was negative for MMP-2 expression and non-invasive, displayed a highly cohesive pattern corresponding to a cluster spatial distribution Key words: metastatic process; cell dispersion; E-cadherin/catenin complex; cell invasion; matrix metalloproteinases; cellular sociology; spatial distributionMetastatic progression is a multistep process involving basement membrane disruption, tumor cell dispersion from the primary tumor cluster, stromal connective tissue invasion by tumor cells, neoangiogenesis, intravasation and extravasation and finally colonization of a target organ. The cell dispersion step largely depends on the reorganization of intercellular adhesion molecules, especially the epithelial adherens junction E-cadherin/catenin complex. This complex is composed of E-cadherin that mediates homotypic calcium-dependent cell-cell adhesion, ␣-catenin that is linked to the actin cytoskeleton and ␤-catenin that links E-cadherin to ␣-catenin. 1-3 This adhesive complex maintains the integrity of epithelia and is thus considered a powerful tumor suppressor. 4 Indeed, many studies have described a correlation between tumor cell aggressiveness and the dysfunction of the complex, either by mutational, transcriptional or post-translational alterations of one of these molecules, in vitro in tumor cell lines [3][4][5][6][7][8] and in vivo in different types of cancers. 9 -11 The stromal invasion step has been shown to involve the expression of proteolytic enzymes, particularly matrix metalloproteinases (MMPs) that are able to degrade almost all the extracellular matrix components. The MMP family consists of several subclasses that include the collagenases, the gelatinases, the stromelysins, the MT-MMPs (membrane type-matrix metalloproteinases) and an heterogeneous group including matrilysin. 12-14 The MMP-2 gelatinase and its activator MT1-MMP have been shown to be especially involved in tumor invasion in vitro 15-17 and in vivo. 13,18 -20 MMPs are usually considered to play a major role in vivo in late stages of tumor progression and in metastasis...

The presence of a functional E-cadherin/catenin cell-cell adhesion complex is a prerequisite for normal development and maintenance of epithelial structures in the mammalian body. This implies that the acquisition of molecular abnormalities that disturb the expression or function of this complex is related to the development and progression of most, if not all, epithelial cell-derived tumors, i.e. carcinomas. E-cadherin downregulation is indeed correlated with malignancy parameters such as tumor progression, loss of differentiation, invasion and metastasis, and hence poor prognosis. Moreover, E-cadherin has been shown to be a potent invasion suppressor as well as a tumor suppressor. Disturbed expression profiles of the E-cadherin/catenin complex have been demonstrated in histological sections of many human tumor types. In different kinds of carcinomas, biallelic downregulation of the E-cadherin gene, resulting in tumor-restricted decrease or even complete loss of E-cadherin expression, appears to be caused by a variety of inactivation mechanisms. Gene deletion due to loss of heterozygosity of the CDH1 locus on 16q22.1 frequently occurs in many carcinoma types. However, somatic inactivating mutations resulting in aberrant E-cadherin expression by loss of both wild-type alleles is rare and restricted to only a few cancer types. A majority of carcinomas thus seems to show deregulated E-cadherin expression by other mechanisms. The present evidence proposes transcriptional repression as a powerful and recurrent molecular mechanism for silencing E-cadherin expression. The predominant mechanisms emerging in most carcinomas are hypermethylation of the E-cadherin promoter and expression of transrepressor molecules such as SIP1, Snail, and Slug that bind sequence elements in the proximal E-cadherin promoter. Interestingly, complex differential expression of other cadherins seems to be associated with loss of E-cadherin and to reinforce effects of this loss on tumor progression. Multiple agents can upregulate and stabilize the E-cadherin/catenin complex. Especially for those tumors with transcriptional and thus reversible downregulation of E-cadherin expression, these drug agents offer important therapeutic opportunities.