First, CD16 was determined for NK cell engagement. proof of theory, genetically-encoded, cysteine meditope tags were added to the N- and/or C-termini of fluorescent proteins, nanobodies, and affibodies, each expressed in bacteria, purified to homogeneity and efficiently conjugated to different memAbs and meFabs. We further show that multiple T-cell and Her2-targeting bispecific molecules using this strategy potently activate T-cell signaling pathways imaging of tumor xenographs. Collectively, the platform offers the opportunity to build and exchange an array of functional moieties, including protein biologics, among any cysteine memAb or Fab to rapidly create, test and optimize stable, multifunctional biologics. Graphical Abstract: Monoclonal antibodies (mAbs) have long been considered magic bullets that seek and eliminate diseased cells(1). Due to their specificity and favorable pharmacological properties and positive clinical outcomes, mAbs have become a major therapeutic modality. However, their efficacy is frequently limited, often because of development of resistance to the mAbs and/or suppression of the immune system(2). To exploit the favorable properties of mAbs and circumvent mechanisms of resistance, tremendous efforts have been made, both in academia and industry, to add additional functionality to mAbs. These efforts include conjugation of ultra-potent cytotoxins, fusion of cytokines, and generation of bispecific or multispecific mAbs to produce an artificial immunological synapse. Although each of these methods has merits, they all invariably require specialized chemistries and/or multiple rounds of optimization, which are labor rigorous and must be uniquely developed for each system(3, 4). Herein, we describe a highly stable and facile means to attach small molecules and proteins to mAbs by using the meditope technology. This enables the rapid, efficient exchange of nearly any functionality among meditope-enabled mAbs and eliminates considerable re-engineering efforts. The meditope technology entails the conversation of a cyclic, twelve amino acid peptide and a unique binding site within the Fab arm of cetuximab, which we envisioned could be used as a hitch for attaching drugs and biologics to antibodies(5). The initial affinity of the meditope-Fab conversation was too poor for in vivo use. Through considerable structure-activity relationships, we have significantly improved the affinity of the conversation (KD = 860 pM) and exhibited that this binding site could be easily grafted onto additional mAbs (5C11). Recently, we synthesized and integrated a nonnatural amino acidity in the meditope and utilized click chemistry to generate an interlocked mechanised relationship to bolt meditopes bearing differing functionalities onto the mAb(12). Even though the mechanised relationship offers a way for stably attaching small-molecules, the usage of a nonnatural amino acidity precludes the immediate conjugation of protein. Here, we released a disulfide relationship between your meditope peptide Cethromycin and Fab arm to make a stable discussion for antibody functionalization only using natural proteins. We attached a range of protein and small-molecules to multiple mAbs with this technology. We discovered that antigen binding can be indistinguishable from parental mAbs. Considerably, we discovered that the disulfide relationship stabilizes the Fab, raising the melting temperatures by 10 levels. The stabilization from the Fab is within sharp comparison to existing ways of functionalization, which decreases the stability from the Fab typically. Ultimately, the system permits an individual mAb to become functionalized for medication delivery quickly, tumor imaging, biologic delivery, or immune system cell recruitment with no need for more re-engineering. Outcomes and Dialogue Disulfide Introduction As the interlocked mechanised relationship described above needs nonnatural proteins(12), we looked into if the meditope-Fab discussion could template and catalyze the forming of a disulfide relationship using only normally encoded Cethromycin proteins (13, 14). To this final end, we determined Ser6 for the meditope and Ala175 for the weighty chain from the Fab as applicants for disulfide development. In Cethromycin the crystal framework of meditope-enabled trastuzumab with meditope (pdb 5U5F), Ser6 and Ala175 are juxtaposed and really should become amendable to changes (Structure 1). We mutated Ala175 in the trastuzumab mAb to cysteine (known as 175Cys throughout), and produced Nes and Cethromycin purified the modified mAb then.