During an earlier study we found that a SPOCQ-alkyne reaction on a C-terminally placed tyrosine residue in the light chains of a transiently expressed tras[LC]G4Y antibody with bis-BCN (BCN-PEG3-BCN, 1, Scheme 1A) yields intramolecularly crosslinked light chains (Scheme 1B, see Supporting Information, Fig. ). In view of the efficiency of this conversion, we reasoned that using an analogous trifunctional linker, tri-BCN (2), with identical distance between the BCN handles as for bis-BCN 1, it should be feasible to obtain an antibody with one free BCN handle, which could then be subjected to further chemical derivatization. Indeed, the SPOCQ-alkyne reaction of tri-BCN 2 with tras[LC]G4Y resulted in the installation of a free BCN handle onto the antibody (Scheme 1C, see Supporting Information, Fig. ). Assessment of the effect of the number of equivalents on crosslinking efficiency revealed that crosslinking was achieved over the range of 0.5-10 equivalents, although mostly compromised by formation of higher oligomers and functionalization of each light chain with one BCN handle, except when sub-stoichiometric amounts were applied (see Supporting Information, Fig. ). Indeed, exposure of the crosslinked mixtures obtained by the different equivalents of tri-BCN to tetrazine-TAMRA resulted in formation of fluorescent bands at 25 kDa, 50 kDa, and higher oligomers. Clearly, using tri-BCN construct 2 yielded poor control over the outcome of crosslinking, and presented a sub-optimal route to the formation of 2:1 bispecific constructs. To counter this, we redesigned our linker by substituting one BCN moiety for a TCO handle. Since BCN has 150-fold higher reactivity with an ortho-quinone when compared to TCO, we reasoned that the presence of a TCO handle instead of one of the BCN handles, would favor crosslinking of the two light chains, and lead to cleaner production of the mono-functionalized mAb. On top of this, as TCO reacts faster with tetrazine than BCN, we expected that construction of our targeted bispecifics would be expedited. Therefore, we synthesized BCN2-TCO (3) linker (Scheme 1A, see Supporting Information), in which the spacer length between the two BCN handles was identical to the distance in our tri-BCN construct (2). Indeed, a SPOCQalkyne reaction of the tras[LC]G4Y with BCN2-TCO linker 3 resulted in installation of TCO handle on the mAb by intramolecularly crosslinked light chains of tras[LC]G4Y via the two BCN units in linker 3 (Fig. ). As expected, formation of higher oligomers was suppressed substantially, especially when at least 10 equivalents of linker 3 was used. Importantly, a SPAAC reaction between TCO-functionalized mAb and azido-TAMRA did not yield a fluorescent product, confirming our assumption that BCN outcompetes TCO in the SPOCQ reaction on the oxidized tyrosine residues, and that the mAb was now functionalized with one TCO handle. For the generation of MeTz-rbFabCD3 (6a), OKT3 mAb (anti-CD3 mAb) was first treated with immobilized papain resulting in two Fab units that were rebridged with MeTz-PEG3-Br2PD (4) after treatment with TCEP, as was described before (Scheme 1D, see Supporting Information for details, Fig. ). Having prepared tetrazinefunctionalized Fab fragments of both OKT3 mAb and HER2 mAb, i.e., MeTz-rbFabCD3 6a and MeTz-rbFabHER2 6b, we performed the IEDDA reaction on tras[LC]G4X-TCO (X = SPOCQ product between tyrosine and BCN). For this, TCOfunctionalized antibody was incubated with MeTz-rbFabCD3 (6a) or MeTz-rbFabHER2 (6b) (0.9 equiv.) at 4 °C for 2 h in PBS pH 5.5 (Scheme 1E, see Supporting Information Fig. ). Analysis of the reaction mixture using nonreducing SDS-PAGE revealed successful formation of the conjugate, after which the desired product was purified using protein A purification and subsequent size exclusion chromatography (see Supporting Information, Fig. and). The cleanest fractions of each SEC purification were collected for characterization and biological evaluation. For characterization of the formed species, and assessment of the purity of the collected fractions, size exclusion chromatography-native mass spectrometry (SEC-nMS) was performed on both the HER2×CD3 (10a) and HER2×HER2 (10b) conjugates (Fig. ). This revealed that even after protein A and SEC purification, we were not able to fully isolate the targeted compounds as pure fractions. Specifically, the SEC-UV chromatograms revealed the presence of a minor amount of earlier eluting species (around 3.6 min), and a more significant later eluting shoulder on the peak of the main compound at 4.4 min. MS-analysis revealed that this peak corresponds to unreacted tras[LC]G4-TCO (9), which is attributed to our use of sub-stoichiometric amounts of MeTz-rbFab (6a/6b) in the preparation of 2:1 constructs. Gaussian fitting of the partially coeluting species yielded relative intensity of 71% of 2:1 HER2×CD3 TDBA (10a) in the purified fraction while 78% for 2:1 HER2×HER2 construct (10b). Therefore, the SEC-nMS reveals that obtained fractions mostly contained the targeted constructs, with relative intensities of leftover starting material tras[LC]G4-TCO (9) of 24% and 15% in 10a and 10b, respectively.