In this analysis, the parental anti-EGFR and anti-IGF1R antibodies were used for identification of the light and heavy chains (Fig. 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for RO462005 bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies. KEYWORDS:Antibody engineering, bispecific monovalent antibodies, dual targeting, single-chain antibody, tethered single-chain antibodies == Introduction == Monoclonal antibodies are monospecific and bivalent for RO462005 a single antigen. Although the bivalent binding of the antibodies to their antigens provides the immune system with many advantages, such as increasing the binding events and biologic responses, a single antibody with specificity to 2 distinct antigens is preferred for many therapeutic applications.1For example, a monovalent bispecific antibody that binds RO462005 to T cells and to a target expressed on a tumor cell surface can facilitate T-cell redirected target cell killing.2Many existing bispecific antibody (BsAb) formats bind bivalently to each antigen, but their structures deviate substantially from that of a canonical IgG structure.3-5Some of these BsAbs have inferior pharmacokinetic properties, including rapid clearance and shorter half-life relative to their IgG counterparts, thus limiting their therapeutic applications.6-12 Several solutions have been described for the generation of BsAb with an IgG chain-like sequence and structure (IgG-Bs).13For example, IgG-Bs have RO462005 been produced by expressing the IgGs separately and subsequently mixing them,14,15but these methods require both site-specific mutations and reduction and oxidation, which complicate downstream development. Moreover, these approaches require the production of 2 separate cell lines expressing the 2 2 antibody pairs. Other IgG-Bs have been generated by culturing 2 differentEscherichia coli, each transformed with DNA encoding one half-antibody obtained by introducing specific mutations in the CH3 domain of the heavy chain.16This approach has limitations, including mutagenesis in the CH3 domains, inability to use a single cell line for production, and, most notably, the final product does not have the conservedN-297 linked glycans in the CH2 domains that are believed to be important to maintain the structure and function of antibodies.17-21 A computational design strategy has RO462005 been applied to express the IgG-Bs in a single mammalian cell by identifying complementary point mutations at the heavy and light chain interfaces.22When expressed using the same mammalian cell, the heavy and light chains assemble into IgG-Bs based on their complementary mutations. However, this approach is laborious because 15 to 20 point mutations are required, and these mutations are not universally applicable to all IgG isotypes. Moreover, this method is only applicable to kappa but not lambda light chains, and only to the VH3 and VH1 antibody gene families. Lastly, a crossover approach has been developed for the production of IgG-Bs antibodies.23This approach involved the molecular exchange of heavy chain and light chain domains within the antigen-binding fragment (Fab), which deviates from the canonical domain structure of IgG, and included mutations in the CH3 domains to favor heterodimerization. Other designs of IgG-Bs have been described, but with the exception of IgG-Bs that are restricted to the same heavy chain on both binding arms24and the two-in-one IgG,25these other IgG-Bs are either variations or optimizations of the methods described thus far.26-28Although these approaches generate IgG-Bs, the ITM2B deviation from the IgG chain sequence and structure remains a substantial issue, 6-12and novel antibody engineering solutions are thus still needed. Generating IgG-Bs with an IgG-like chain sequence and structure is challenging for a 2 reasons. First, the homodimeric structure of IgG must be maintained. Second, 2 genes, one for the light chain and another for the heavy chain, are required. The 2 2 chains are independently translated and assembled into a homodimeric bivalent IgG due to their complementary domain interfaces. We set out to investigate if a single-chain IgG design without any domain interface mutations (herein termed innovative monoclonal antibody (iMab)) could overcome inefficiencies inherent with the use of 2 independent genes to form a full-length IgG-Bs molecule with monovalent antigen binding and with IgG1-like sequence and structure. The key design element of the iMab is based on the hypothesis that the light and heavy chains of different antigen specificity (i.e., 2 different light and heavy chains), if in close proximity (i.e., co-transcribed, co-translated, co-folded, co-secreted), will force the monovalent bispecific formation of the iMab. The iMab design offers a unique engineering solution to overcome some of the limitations of the described previously IgG-Bs,13-28such as the potential formation of half-antibodies and homodimers, which can be difficult to eliminate and require complex multi-step purification methods. ==.