Despite this variety of mechanisms of action, most mAbs are not administered as a monotherapy, but usually are combined with other modalities, particularly chemotherapy. the levels of phosphorylated p38 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by all 3 HexAbs and the notable differences in the signaling events triggered by the HexAbs from those incurred by crosslinking veltuzumab or rituximab with a secondary antibody. Thus, the greatly enhanced direct toxicity of these HexAbs correlates with their ability to alter the basal expression of various intracellular proteins involved in regulating cell growth, survival, and apoptosis, with the net outcome leading to cell death. Introduction To address the clinical concerns of undesirable immmunogenicity and suboptimal pharmacokinetics, cancer therapy with monoclonal antibodies (mAbs) has evolved from murine to chimeric, humanized, and now fully human constructs. Parallel to these improvements A939572 have been continuing efforts to develop more effective forms of mAbs, which to date, include different isotypes, smaller single-chain proteins with monomeric or multimeric binding moieties derived from variable domains, specific mutations in the Fc to modulate immune effector functions or circulating half-lives, and bispecific antibodies (bsAbs) of numerous designs that vary in valency, structure, and constituents, among others.1 In the absence of a covalently attached drug, toxin, or radionuclide, the toxicity of a mAb after ligation of its cognate antigen on target cells can be either direct or indirect. Direct toxicity is usually caused primarily by apoptosis, resulting from perturbation of intracellular signal transduction pathways, whereas indirect toxicity requires the involvement of effector cells and complement, which lead to antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and/or monocyte/macrophage phagocytosis. A939572 Despite this variety of mechanisms of action, most mAbs are not administered as a monotherapy, but usually are combined with other modalities, particularly chemotherapy. Because signaling pathway redundancies can result in lack of response to a single mAb, diverse strategies to use 2 mAbs, each against a different epitope of the same antigen or different antigens on the same target cell, have been proposed, and combinations such as anti-CD20 and anti-CD22, 2 anti-CD20 and antiChuman leukocyte antigen DR, 3 anti-CD20 and anti-TRAIL-R1,4 anti-insulinClike growth factor 1 receptor (IGF-1R) and antiCepidermal growth factor receptor (EGFR),5 antiCIGF-1R and antiCvascular endothelial growth factor,6 or trastuzumab and pertuzumab that target different extracellular regions of human epidermal growth factor receptor 27 have been evaluated preclinically, showing enhanced or synergistic antitumor activity both in vitro and in vivo. The first clinical evidence of an apparent advantage of combining 2 mAbs against different cell surface antigens of a cancer cell involved the administration of rituximab, the chimeric anti-CD20 mAb, and epratuzumab, the humanized anti-CD22 mAb, in patients with non-Hodgkin lymphoma (NHL), where the combination was found to enhance antilymphoma efficacy without a commensurate increase in toxicities, based on 3 impartial clinical trials.8 A bsAb targeting both EGFR and IGF-R has been studied, 9 yet the combination of the 2 2 parental mAbs has not been reported to be additive or synergistic. Given the short list of mAbs currently approved in cancer therapy, the available combinations are not large. Nevertheless, where such combinations show improved efficacy, it is of concern, from an economic perspective, whether the costs of combining 2 expensive antibody therapies can be borne by the healthcare system, in addition to the inconvenience and time of conducting individual infusions. Therefore, developing bsAbs, whereby 2 antigen targets can be bound by a single agent, has A939572 been a goal for some time, resulting in a multitude of approaches.10 Earlier methods used for the production of bsAbs made use of either chemical cross-linking of IgG or Fab11,12 or quadromas13 obtained by fusing 2 hybridomas. Subsequent strategies focused on generating recombinant bsAbs composed of tandem scFvs or diabodies,14 CDF and one format of such Fc-lacking constructs, referred to as BiTe, is currently being tested clinically.15 Because, for many therapeutic applications, the presence of an Fc and its effector functions is beneficial, if not essential, for improved in vivo properties, Fc-containing bsAbs, as exemplified by a variety of novel designs, also have been described.16C20 Indeed, a renewed interest in the construction of IgG-like bsAbs has emerged21 to recruit effector cells, which is an important mechanism for most of the currently available anticancer mAbs. We have advanced a new approach of constructing multivalent antibodies using the Dock-and-Lock (DNL) method,22 which enables site-specific self-assembly of 2 modular components only with each other, resulting, after combining, in a covalent structure of defined composition with retained bioactivity.23 With the available DNL method and noting that this administration.