Subsequently, the method was shown to allow for the rapid and selective introduction of a single fluorophore, a cytokine, or an scFv around the antibody. growth of the genetic code to incorporate noncanonical amino acids, chemically orthogonal Chebulinic acid deals with can be launched at predefined sites in a given protein sequence,8,9 which allows for chemoselective oxime ligation10 or click chemistry.11 In addition, a wide arsenal of methods is available for site-specific protein modification with varying degrees of versatility.12 Besides chemical strategies, enzymatic conjugations have emerged recently, as they enable highly controlled modification of antibodies through specific peptide tags.13 Examples of enzymatic processes include sortase ligation,14,15 phosphopantetheinyl transferase,16 and transglutaminase.17 Recently, we demonstrated that selective labeling of monoclonal antibodies can Chebulinic acid be ensured in a single step based on the introduction of a C-terminal tetra-glycyltyrosine tag (G4Y, Figure ?Physique11A).18?20 Specifically, oxidation of the phenol moiety of the tyrosine in the G4Y tag to an ortho-quinone by mushroom tyrosinase (mTyr) and in situ DielsCAlder reaction with bicyclo[6.1.0]nonyne (BCN) leads to selective labeling in high yields and under moderate conditions, with reaction rates exceeding those of the strain-promoted alkyneCazide cycloaddition by at least a factor of 500. This strain-promoted oxidation-controlled ortho-quinone cycloaddition (SPOCQ) was subsequently shown to also proceed with cyclopropenes, albeit significantly slower,21 or could be further accelerated by employing cyclopropanated trans-cyclooctene.22 Open in a separate window Physique 1 (A) Schematic depiction of chemoenzymatic antibody engineering using tyrosinase. (B) Current chemoenzymatic modification of a G4Y- or sortase-tag expressed protein ligation (LPETG)-tagged hole-LC of the antibody by means of tyrosinase or sortase, respectively. Given the symmetrical nature of antibody, the site-specific methods described above lead to dual labeling of the protein. However, in some cases, forming a 2:1 antibody format (i.e., two antigen binding sites and one conjugated molecule of interest) may be more desirable, for example, in the generation of an antibodyCdrug conjugate with an extremely potent cytotoxin such as a pyrrolobenzodiazepine dimer23 or by radiolabeling with -emitters such as thorium-227.24 While these formats have been reported, generation of these conjugates relies on methods with poor control of site and stoichiometry like random lysine conjugation followed by isolation of the mono-functionalized conjugate,24 or required the rearrangement of several disulfide bridges between the light and heavy chains (HCs), resulting in a significant loss of ATF1 binding activity and stability.23 Similarly, various antibody conjugates with a 2:1 format to enhance the therapeutic window, e.g., with IL-225 and -CD3,26 have been reported. The latter conjugates were obtained by the fusion of an immunocytokine or T-cell engager to a single chain of an asymmetric antibody format, known as knob-in-hole (KiH) antibodies.27,28 By changing only a few key amino acids in the CH3 domain that are pivotal to the pairing of the heavy chains, i.e., replacing a small side chain with a large side chain on one heavy chain (knob) and vice versa for the other heavy chain (hole), heterodimerization can be promoted.29,30 While the KiH technology has been applied to create immunocytokines based on genetic encoding of a C-terminal fusion protein on one of the heavy chains,25 a modular approach derived from a single engineered antibody scaffold would allow for easy access to a wide range of mono-functional conjugates. Here, we show that this knob-in-hole technology can be readily applied for the generation of 2:1 antibody conjugates by selective strain-promoted cycloaddition of a single heavy chain (HC) based on Chebulinic acid ortho-quinone chemistry or tetrazine ligation (Physique ?Physique11B). We found that this strategy is suitable for the formation of antibodyCsmall molecule, antibodyCprotein, and antibodyCantibody conjugates. 2.?Results and Discussion 2.1. Two-Stage Antibody Conjugation Based on Tetrazine Ligation A knob-in-hole variant of trastuzumab was generated by the introduction of a single mutation in the knob heavy chain (T366W; knob-HC) and three mutations in the hole heavy chain (T366S, L368A, and Y407V; hole-HC).31 For conjugation, the hole-HC contained a C-terminal sortase LPETG-tag (Supporting Information (SI)).14,31 After transient expression and subsequent purification by protein A affinity chromatography, analysis by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), high-performance liquid chromatography (HPLC), and liquid.