In Vivo Pretargeting Based on Cysteine-Selective Antibody Modification with IEDDA Bioorthogonal Handles for Click Chemistry

Abstract

Pretargeted imaging has emerged as an effective multistep strategy aiming to improve imaging contrast and reduce patient radiation exposure through decoupling of the radioactivity from the targeting vector. The inverse electron-demand Diels− Alder (IEDDA) reaction between a trans-cyclooctene (TCO)conjugated antibody and a labeled tetrazine holds great promise for pretargeted imaging applications due to its bioorthogonality, rapid kinetics under mild conditions, and formation of stable products. Herein, we describe the use of functionalized carbonylacrylic reagents for site-specific incorporation of TCO onto a human epidermal growth factor receptor 2 (HER2) antibody (THIO-MAB) containing an engineered unpaired cysteine residue, generating homogeneous conjugates. Precise labeling of THIO-MAB−TCO with a fluorescent or radiolabeled tetrazine revealed the potential of the TCO-functionalized antibody for imaging the HER2 after pretargeting in a cellular context in a HER2 positive breast cancer cell line. Control studies with MDA−MD-231 cells, which do not express HER2, further confirmed the target specificity of the modified antibody. THIOMAB−TCO was also evaluated in vivo after pretargeting and subsequent administration of an 111 In-labeled tetrazine. Biodistribution studies in breast cancer tumorbearing mice showed a significant activity accumulation on HER2 + tumors, which was 2.6-fold higher than in HER2 − tumors. Additionally, biodistribution studies with THIOMAB without the TCO handle also resulted in a decreased uptake of 111 In−DOTA− Tz on HER2 + tumors. Altogether, these results clearly indicate the occurrence of the click reaction at the tumor site, i.e., pretargeting of SK-BR-3 HER2-expressing cells with THIOMAB−TCO and reaction through the TCO moiety present in the antibody. The combined advantages of site-selectivity and stability of TCO tagged-antibodies could allow application of biorthogonal chemistry strategies for pretargeting imaging with minimal side-reactions and background.

IEDDA: An Attractive Bioorthogonal Reaction for Biomedical Applications

“…MALDI-TOF mass spectrometry and denaturing SDS-PAGE analyses showed that approximately 2.4 TCO groups were attached on the heavy chain of the antibody, while immunoreactivity of the conjugate was almost not influenced by the modifications (94%). Another site-specific labeling method based on reaction between cysteine and carbonylacrylic group was recently reported [81]. 5-OH-TCO was functionalized with a carbonylacrylic group (20) and conjugated to THIOMAB LC-V205C, a modified anti-HER2 antibody with cysteines in the light chain.…”

Clickable Radiocomplexes With Trivalent Radiometals for Cancer Theranostics: In vitro and in vivo Studies

“…This choice was motivated by the higher in vivo stability and by the easy separation of the radiolabelled [ 111 In]In-DOTA-Tz derivative from the unreacted chelator. Given a future in vivo application of such clickable chelator, this advantage might allow to achieve radiocomplexes with higher specific activity reducing the competition risk for the in vivo click reaction with the TCO counterpart (31).…”

Luminol anchors improve the electrochemical-tyrosine-click labelling of proteins

“…Targeting the abundant, surface-exposed and charged Lys residues generally results in the formation of highly heterogeneous mixtures and may significantly impact biological binding. Promising approaches have recently been described to overcome the initial limitations of Lys 10,11 and Cys 12,13 modifications, and much effort is now dedicated to selectively target less exploited amino acids such as methionine, 14 tryptophan, 15,16 histidine 17,18 or tyrosine (Y). 19,20 Y usually occurs at low frequency in proteins (<5%) and is partially buried in the surface, offering unique opportunities for controlled labelling of the most reactive residues.…”