Page 48 - Tyrosine-Based Bioconjugations - Jorick Bruins
P. 48

Chapter 2
AT1002. To further assess the applicability of SPOCQ for site-specific modification of monoclonal antibodies, we modified AT1002, a potent anti-influenza antibody, with a C-terminal G4Y tag (SI S9).28 To this end, AT1002 with a sortase tag residing on the C-terminus of each light chain was employed to obtain a C-terminally fused G4Y (AT1002[LC]G4Y). Of relevance, the obtained AT1002[LC]G4Y possesses a longer C-terminally fused tag (-G4SLPETG4Y) compared to Tras[LC]G4Y, which was anticipated to contribute favorably with regard to accessibility of the tyrosine by mTyr.29 SPOCQ was attempted on AT1002[LC]G4Y under identical conditions compared to Tras[LC]G4Y, after which SDS-PAGE analysis demonstrated similar conjugation selectivity with high conversion: only fluorescence on the light chain was detected when reacted with mTyr and 1 (Figure 4B). Interestingly, fluorescence of the labelled AT1002 seems more intense than that of trastuzumab under identical condition, which was taken a confirmation that labelling to the light chain with a short spacer may be encumbered by less steric accessibility. Additionally, a protein band corresponding to the conjugated product was observed by SDS- PAGE analysis upon Coomassie staining, indicating a significantly higher conversion for AT1002[LC]G4Y. MS analysis confirmed SPOCQ on AT1002 (Figure 4C, D). As with trastuzumab, some non-labelled material was still present, which may indicate that also in this case a fraction of the formed quinone reacts with lysine or histidine residues.
Our newly developed conjugation method was envisioned to be suitable as a site-specific approach to access antibody–drug conjugates (ADCs). These are a class of promising chemotherapeutics used for targeted treatment of tumors by combining the high cytotoxicity of a drug such as monomethyl auristatin E (MMAE) or maytansine with an antibody that has high binding affinity to the tumor cell of choice30 The ability of ADCs to bring highly toxic compounds selectively to the tumor cells allows the treatment of cancers while reducing the effect on healthy tissue as with traditional chemotherapies, which has led to the recent market approval of Adcetris (for the treatment of non-Hodgkin lymphoma and anaplastic large-cell lymphoma) and Kadcyla (for treatment of HER2-positive breast cancer).31, 32 To this end, AT1002[LC]G4Y was subjected to BCN-monomethyl auristatin F conjugate (BCN-MMAF, 2) after oxidation by mTyr under identical conditions as before, with the exception that DMF was used as co-solvent instead of DMSO. Much to our satisfaction, the desired ADC was seamlessly obtained, as indicated by the expected mass increase of 1459 Da (Figure 4E), which corresponds to oxidation followed by cycloaddition (SPOCQ) with 2.
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