Page 71 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Monofunctionalization of Knob-in-hole Antibodies
To facilitate analysis of the Fc region of the antibody by LC-MS, it was deglycosylated by endo S2 and digested below the hinge region by IdeS to generate the Fc/2 and F(ab’)2 fragments.22, 32 Following these steps, one of the heavy chains was found to have a molecular weight of 25565 Da, corresponding to the hole-HC containing the sortase-tag (calc.: 25564 Da). The second peak, corresponding to the knob-HC, was found to have a molecular weight of 24220 Da (calc.: 24221 Da).The KiH trastuzumab containing the sortase tag (abbreviated as Tras[KiH-HC]ST) was subjected to sortase-mediated ligation to introduce trans-cyclooctene (Tras[KiH-HC]TCO) or a methyltetrazine moiety (Tras[KiH-HC]MeTz) for conjugation via inverse-electron demand Diels- Alder (IEDDA), also known as tetrazine ligation.33 The products resulting from sortagging were purified using protein A affinity column chromatography, after which HPLC analysis showed full conversion of the starting material to the anticipated product with a single light chain and two distinct heavy chains (Figure 2B, C). In the obtained chromatograms, the unmodified light chain and knob-HC eluted at 6.47–6.50 min and 7.66–7.67 min, respectively, while elution of the hole- HC shifts from 7.51 min (sortase tag, A) to 7.83 min (TCO–group, B) or 7.77 min (MeTz-group, C). LC-MS analysis confirmed formation of the anticipated products, with no observed sortase- mediated hydrolysis (SI).
With the mono-functionalized antibodies in hand, Tras[KiH-HC]TCO was first reacted with a methyltetrazine-labelled fluorophore (MeTz-TAMRA). As expected, SDS-PAGE analysis showed a slight upward shift for the band corresponding to the hole-HC (Figure 3, lane 2), and the formation of a single fluorescently labelled band could be identified. HPLC analysis displayed an efficiency exceeding 90%, and LC-MS confirmed the formation of the desired hole-HC IEDDA product, whilst the knob-HC remained unmodified (SI).
Next, we pursued the preparation of monovalent antibody-protein conjugates based on this strategy. In particular we considered the strategy for application in cancer immunotherapy by combining the targeting power of a monoclonal antibody with cytokine IL-2 or an -CD3 T-cell engager. To this end, a MeTz moiety was attached to the C-terminus of either the cytokine interleukin 2 (IL-2)14 or to the short-chain variable fragment to UCHT1 (-CD3 scFv)34 or using sortase. Subsequently, Tras[KiH-HC]TCO was incubated with an excess MeTz–IL-2 (Figure 3, lane 3) or MeTz–UCHT1 (Figure 3, lane 5), resulting in either MeTz-functionalized protein reacting with TCO-functionalized Tras[KiH-HC]TCO antibody. To be precise, MeTz–IL-2 showed clean labelling of the hole-HC, resulting in the formation of a new band with the expected molecular weight of ~70 kDa, i.e. 50 kDa for the HC and 18 kDa for MeTz–IL-2. HPLC analysis showed full conversion of the TCO-labelled hole-HC, which was confirmed by LC-MS analysis (SI). Similarly, the reaction of Tras[KiH-HC]TCO with MeTz–UCHT1 showed near full conversion to the expected 80 kDa band, with HPLC and LC-MS confirming efficient formation of the expected bifunctional antibody via this method.
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