However, Fc-mediated effector mechanisms often seem to be insufficient in properly activating the immune system to act against tumor cells

However, Fc-mediated effector mechanisms often seem to be insufficient in properly activating the immune system to act against tumor cells. patients with acute lymphoblastic leukemia and malignant ascites. In addition, Emicizumab, which is a bispecific antibody targeting clotting factors IXa and X, was recently granted market approval by the FDA in 2017 for the treatment of hemophilia A. However, the generation of these next generation therapeutics is challenging and requires tremendous engineering efforts as two distinct paratopes need to be combined from two different heavy and light chains. This mini review summarizes technologies, which enable the generation of antibodies with dual specificities. Keywords: bispecific antibodies, cognate light chain pairing, common heavy chain, common light chain, heavy chain heterodimerization, knobs into holes, SEED 1. Introduction The therapeutic potential of monoclonal antibodies (mAbs) is enormous Chlorothricin but may be limited due to the simple fact that they are only able to bind one distinct target, while complex diseases in general originate from multiple factors and mediators [1]. In contrast, bispecific antibodies (bsAbs) consist of two physically connected antigen binding moieties (i.e., paratopes), which can simultaneously interact with different epitopes on the same or on different antigens [2,3,4,5]. The most established mode of action of these entities is the redirection of T-cells to tumors [6]. While one paratope Chlorothricin of the so-called T-cell engagers binds a tumor antigen, the other specificity is directed against a part of the T-cell receptor termed CD3. By clustering of CD3 at the tumor site in a target specific manner, the T-cell becomes activated and consequently, the formation of a cytolytic synapse leads to the killing of the tumor cell [7]. The clinical effectiveness of this concept led to the market approval of the antibodies Catumaxomab in 2009 2009 (Fresenius SE & Co. KGaA, withdrawal in 2017 for commercial reasons) and Blinatumomab (Amgen Inc.) in 2014. In addition to T-cell engagement, the common light chain-based bsAb Emicizumab (F. Hoffmann-La Roche AG) was approved for the treatment of hemophilia A by authorities in the US in 2017. This antibody is a mimetic of blood clotting factor VIII, which is not present in hemophilia patients due to a genetic defect [8,9]. By crosslinking factors IX and X, the bsAb restores this part of the coagulation FKBP4 cascade. In addition to these marketed therapeutics, about 30 drugs based on bispecific binding are in clinical development and approximately 60 of these entities are scrutinized in preclinical evaluation [10]. Among these, several therapeutic concepts and modes of action are investigated, such as the dual blocking of signaling pathways. Roches Duligotzumab is a phage-derived human antibody, which is engineered to bind two antigens with one heavy chain and light chain combination [6,11,12]. This so-called Dual Action Fab (DAF) is a two-in-one antibody, which can bind to EGFR and Her3. Deregulated signaling of both receptor tyrosine kinases (RTKs) is involved in the pathogenesis of cancers, such as colorectal and head and neck cancer [13,14]. Resistance mechanisms observed with the standard of care Cetuximab might be circumvented by the application of Duligotzumab [6]. Another molecule that follows this targeting strategy was developed by Merrimack Pharmaceuticals. MM-111 consists of Her2 and Her3 targeting single chain Fv fragments (scFv), where the variable domains of the heavy and light chain are connected by a linker and fused to modified human serum albumin (HSA) for extension of half-life [4,15]. While Her3 signaling is an important resistance mechanism to Her2 inhibitors, dual targeting of both RTKs can enhance Chlorothricin an effective tumor response. Moreover, bsAbs are validated as tumor angiogenesis inhibitors. The CrossMab RG7221 targets the two key angiogenic proteins vascular endothelial growth factor A (VEGF-A) and angiopoetin-2 (Ang-2). The neutralization of two angiogenic factors is expected to lead to superior clinical outcomes compared to monospecific targeting [16]. In the context of immunology, several bsAbs capturing two different cytokines were developed. One example is M1095/ALX-07613 (Merck KGaA; Ablynx), which is a trivalent bispecific Chlorothricin single-domain antibody fragment from camelids (VHH, nanobody) neutralizing both Il17-A and IL17-F for the treatment of inflammatory diseases. Extension of half-life is mediated by an additional VHH domain binding to human serum albumin Chlorothricin [17,18]. Apart from clinical evaluations, novel targeting strategies have emerged using bsAbs. In a proof of concept study, which was recently published by our group, we demonstrated that affinity-optimized paratopes can be used to enhance the selectivity and efficacy of bsAbs.