Page 120 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 7
an antibody targeting a sialylated epitope on CD43,15 a transmembrane cell surface protein expressed on all cell lines of acute myeloid lymphoma (AML).16 For functionalization, we employed the scFv of UCHT1, an a-CD3 antibody,17 and cytokine IL-2,18 known to increase T-cell recruitment and activation, respectively. As hypothesized in chapter 5; by combination of SPOCQ and sortase to conjugate UCHT1 and IL-2, a system would be generated capable of efficiently recruiting and activating the immune system, which as a concept obviously could be extended to other antibodies. Interestingly, SPOCQ on a G4Y-tag on AT1413 was found not to be selective, due to the presence of a cystine knob containing a tyrosine residue in the CDR-H3 loop (chapter 6).
However, by blocking the CDR with an anti-idiotypic antibody, effectively functioning as a temporary protective group for the tyrosine-bearing cystine knob, the G4Y-tag expressed on the light chain could be selectively modified (chapter 6, Figure S1). This was achieved with only a one equivalents of AIA compared to AT1413, resulting in a 1:1 binding of AIA to AT1413 (Figure 3). To the best of our knowledge, the use of antibodies for protection of a protein region during bioconjugation at a distinct site is unprecedented and warrants further investigation. For example, it may pave the way for generation of the AT1413–UCHT1–IL-2 trifunctional antibody, either in a [2]:2:2 format or a [2]:1:1 format (Figure 3).
The generation of solvent-exposed loops for internal tyrosine expression (chapter 6) potentially presents an avenue towards stable SPOCQ conjugates not conjugated at either protein terminus. However, stability turned out to be an issue when a peptide tag containing 12 amino acids was introduced in trastuzumab. By introducing cystine knobs in various solvent-exposed loops on an antibody, a tyrosine residue might be introducible without hampering the antibody stability. For example, Grünewald et. al. reported several stable conjugation sites after insertion of a peptide tag,19 therefore introduction of a “CGYSSC” peptide tag might provide internal tyrosine residues for conjugation without hampering antibody stability.
From a commercial standpoint, using mushroom tyrosinase, an enzyme that can be gathered from the common mushroom, would allow for cheap fabrication of antibody conjugates. Other proteins beside antibodies are also candidates for modification by SPOCQ. By expressing a G4Y- tag on either terminus of enzymes, introduction of a chemical moiety for immobilization of enzymes onto supports such as nanoparticles, microbeads, or surfaces can be achieved.20-23 This approach allows for improved rate of catalysis, recyclability, enzyme stability, and easier product purification for many industrial processes.24-26 Therapeutic proteins can also be modified with SPOCQ for site-specific PEGylation with little effort for an enhanced stability of the protein, and longer in vivo half-lives.27
Tyrosine oxidation is also applicable to proximity based labeling. By introducing the photoreactive moiety diazirine (Figure 4A) via SPOCQ on an N-terminal tyrosine tag, proximity-
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