Site‐specific Bioconjugation and Convergent Click Chemistry Enhances Antibody–Chromophore Conjugate Binding Efficiency

Abstract

Photosensitizer (PS)–antibody conjugates (photoimmunoconjugates, PICs) enable cancer cell‐targeted photodynamic therapy (PDT). Nonspecific chemical bioconjugation is widely used to synthesize PICs but gives rise to several shortcomings. The conjugates are heterogeneous, and the process is not easily reproducible. Moreover, modifications at or near the binding sites alter both binding affinity and specificity. To overcome these limitations, we introduce convergent assembly of PICs via a chemo‐enzymatic site‐specific approach. First, an antibody is conjugated to a clickable handle via site‐specific modification of glutamine (Gln) residues catalyzed by transglutaminase (TGase, EC 2.3.2.13). Second, the modified antibody intermediate is conjugated to a compatible chromophore via click chemistry. Utilizing cetuximab, we compared this site‐specific conjugation protocol to the nonspecific chemical acylation of amines using N‐hydroxysuccinimide (NHS) chemistry. Both the heavy and light chains were modified via the chemical route, whereas, only a glutamine 295 in the heavy chain was modified via chemo‐enzymatic conjugation. Furthermore, a 2.3‐fold increase in the number of bound antibodies per cell was observed for the site‐specific compared with nonspecific method, suggesting that multiple stochastic sites of modification perturb the antibody–antigen binding. Altogether, site‐specific bioconjugation leads to homogenous, reproducible and well‐defined PICs, conferring higher binding efficiency and probability of clinical success.

Multiscale imaging of therapeutic anti-PD-L1 antibody localization using molecularly defined imaging agents

“…However, hepatic accumulation was increased twofold for the mIgG1 PD-L1-srt-his antibody compared to the other antibodies, most likely caused by the presence of the Histag [ 46 , 47 ]. Although previous studies showed that random DTPA conjugation via lysine side chains can influence affinity [ 24 ], in vivo pharmacokinetics [ 25 , 48 ] and therapeutic index [ 49 ], we did not observe differences in blood clearance or tumor uptake for site-specifically versus randomly labeled mIgG1 aPD-L1 with DTPA or IH20 . Finally, no statistical differences in biodistribution were observed for IH20 - and IH18 -conjugated chimeric mIgG1 aPD-L1, except for the spleen, where uptake of mIgG1-IH18 was significantly higher, potentially caused by the apparent higher blood concentration 24 h p.i.…”

“…In most of these studies, imaging moieties were coupled to the targeting antibody using non-selective protein modification methods, without spatial control. However, such random conjugation can interfere with the antigen-binding region and consequently alter the binding affinity [ 24 ] and in vivo pharmacokinetics [ 25 , 26 ]. In contrast, site-specific labeling does not interfere with the antigen-binding site and yields a more controlled, homogeneous and therefore reproducible product.…”

Site-selective modification strategies in antibody–drug conjugates

“…Non-glycosylated approaches to antibody modification are not limited to toxin attachment. Other reports have detailed the attachment of azide polymers, 271 photosensitiser motifs 272 or virus nanoparticles. 273 Recent research offers some reservations concerning the selectivity of mTG for the conserved PWEEQYNST sequence (containing Q295) in the Fc region.…”

Photoclick Chemistry: A Bright Idea

“…By exploiting the advantages of the photoSPAAC reaction, the labeling occurred in a one-pot reaction with only a few minutes of light exposure (Figure ). In similar applications, SPAAC photoclick reactions were employed as light-responsive photoaffinity probes for detecting carbohydrate-binding proteins and antibodies. − …”