Toward Realization of Bioorthogonal Chemistry in the Clinic

Abstract

In the last decade, the use of bioorthogonal chemistry toward medical applications has increased tremendously. Besides being useful for the production of pharmaceuticals, the efficient, nontoxic reactions open possibilities for the development of therapies that rely on in vivo chemistry between two bioorthogonal components. Here we discuss the latest developments in bioorthogonal chemistry, with a focus on their use in living organisms, the translation from model systems to humans, and the challenges encountered during preclinical development. We aim to provide the reader a broad presentation of the current state of the art and demonstrate the numerous possibilities that bioorthogonal reactions have for clinical use, now and in the near future.

Keywords: Bioconjugation; Bioorthogonal chemistry; Bioorthogonal reaction; Click chemistry; Click-to-release; In vivo chemistry.

Fig. 1

Main classes of bioorthogonal reactions. Bioorthogonal ligation reactions aim to connect two molecular entities, while bioorthogonal release reactions link two entities while expelling a third entity

Fig. 2

Overview of the most promising applications of bioorthogonal ligation and release reactions

Fig. 3

Schematic representation of a pretargeting approach. In the first step, a targeting vector with a bioorthogonal tag is administered and binds to its target. After an interval, during which the targeting vector clears from circulation, a radioactive probe with a bioorthogonal tag is administered, which ligates to the tumor-bound targeting vector

Fig. 4

First TCO-tetrazine pretargeting system as presented by Rossin et al. [48]. A Chemical structures of TCO-conjugated CC49 antibody (top) and radiolabeled tetrazine-DOTA probe (bottom). B Schematic representation of the two steps in an IEDDA-based pretargeting system. C SPECT/computed tomography (CT) image of a mouse administered with CC49-TCO, followed 1 day later by ¹¹¹In-labeled tetrazine. The tumor is indicated by a white arrow [48]. Reprinted and adapted with permission from Rossin, R., et al., “In vivo chemistry for pretargeted tumor imaging in live.” Angew Chem Int Ed Engl, 2010. 49(19): p. 3375–8. Copyright 2010 John Wiley and Sons

Fig. 5

Pretargeting components used in the first clinical trial of bioorthogonal-chemistry-based pretargeting. A Schematic representation of 5B1-TCO and tetrazine-based pretargeting system [53]. B Tetrazine ligation reaction between the tetrazine probe and antibody conjugated TCO (top), and chemical structure of [⁶⁴Cu]Cu-Tz-SarAr (bottom) [50]. Reprinted with permission from Zeglis, B.M., et al., “Optimization of a pretargeted strategy for the PET imaging of colorectal carcinoma via the modulation of radioligand pharmacokinetics.” Mol Pharm, 2015. 12(10): p. 3575–87. Copyright 2015 American Chemical Society. C Positron emission tomography (PET) image of tumor-bearing mouse administered with 5B1-TCO followed by [⁶⁴Cu]Cu-NOTA-PEG₇-Tz 72 h later [53]. Reprinted with permission from Houghton, J.L., et al., “Pretargeted immuno-PET of pancreatic cancer: overcoming circulating antigen and internalized antibody to reduce radiation doses”. J Nucl Med, 2016. 57(3): p. 453–9. Copyright 2016 Society of Nuclear Medicine and Molecular Imaging

Fig. 6

First clinical trial and demonstration of bioorthogonal chemistry in large animals. A Chemical structure of TCO-conjugated bisphosphonate (BP-TCO). B Chemical structure of [⁶⁴Cu]Cu-SarAr-Tz probe. C Pretargeted PET image of a dog with an osteodestructive lesion in left ulna administered with TCO-BP and [⁶⁴Cu]Cu-SarAr-Tz [65]. Reprinted with permission from Maitz, C.A., et al., “Pretargeted PET of osteodestructive lesions in dogs.” Mol Pharm, 2022. 19(9): p. 3153–3162. Copyright 2022 American Chemical Society

Fig. 7

In vivo bioorthogonal ligation of a clearing agent to tagged antibodies for pretargeting. Without clearing agent, antibodies undergo slow clearance from blood. In vivo conjugation of a clearing agent directs circulating antibodies toward rapid excretion (i.e., via the liver) and/or inactivates them. Instead of antibodies, this strategy can likewise be used on other bioorthogonally-tagged drug molecules

Fig. 8

Neutralization and clearance enhancement of a warfarin drug by in vivo conjugation to a clearing agent. Azido-warfarin (WN₃) reacts in vivo with a BCN-PEG₆-OH clearing agent to give an inactivated drug [69]. Reprinted with permission from Ursuegui, S., et al., “An in vivo strategy to counteract post-administration anticoagulant activity of azido-Warfarin.” Nat Commun, 2017. 8: p. 15,242. Copyright 2017 Springer Nature, reproduction under CC BY 4.0

Fig. 9

Schematic representation of prodrug unmasking using a bioorthogonal reaction. In the first step, a targeted prodrug is administered and accumulates in its target tissue. In the second step, an activator molecule is administered that reacts with the prodrug, and subsequently releases the drug in an active form. In addition, prodrug unmasking systems exist in which a targeted activator is administered prior to a non-targeted prodrug

Fig. 10

Bioorthogonally activated prodrug system with optimized ADC and activator. A Schematic representation of IEDDA-based ADC prodrug activation. B Therapeutic efficacy of ADC (tc-ADC) alone or in combination with activator (3), and comparison with ADCs with an enzymatically cleavable linker (vc-ADC) and non-tumor binding antibody (nb-ADC), as evaluated in OVCAR-3 ovarian carcinoma mouse models. C Chemical structures of anti-TAG72 diabody-TCO ADC and tetrazine activator [75]. Reprinted and adapted with permission from Rossin, R., et al., “Chemically triggered drug release from an antibody–drug conjugate leads to potent antitumour activity in mice.” Nat Commun, 2018. 9(1): p. 1484. Copyright 2018 Springer Nature, reproduction under CC BY 4.0

Fig. 11

Hydrogel-based prodrug activation system tested in clinical trials. Top: schematic representation of TCO-modified Doxorubicin prodrug (SQP33) activation by tetrazine-modified hydrogel (SQL70). Bottom: chemical structures of SQL70 biopolymer (left) and SQP33 prodrug (right) [77]. Reprinted with permission from Srinivasan, S., et al., “SQ3370 activates cytotoxic drug via click chemistry at tumor and elicits sustained responses in injected and non-injected lesions.” Adv Ther (Weinh), 2021. 4(3). 13(12): p. 4004–4015. Copyright 2021 John Wiley and Sons

Fig. 12

Schematic representation of an off-target deactivation approach. In the first step, a radioactive antibody is administered. After an interval, a trigger is administered, which leads to release of a radioactive fragment from the antibody after a bioorthogonal release reaction

Fig. 13

Bioorthogonal off-target deactivation for enhanced imaging contrast using ⁸⁹Zr-labeled Trastuzumab. Top: structures of ⁸⁹Zr-labeled Trastuzumab construct comprising a cleavable TCO linker and a tetrazine-based trigger. Bottom: PET images of a tumor-bearing mouse before (scan 1) and after administration of the trigger (scan 2). The tumor and bladder are indicated with red and yellow arrows, respectively. [93]. Reprinted with permission from Vlastara, M., et al., “Click-to-release: cleavable radioimmunoimaging with [(89)Zr]Zr-DFO-trans-cyclooctene-trastuzumab increases tumor-to-blood ratio.” Theranostics, 2023. 13(12): p. 4004–4015. Copyright 2023 Ivyspring

Fig. 14

Off-target deactivation approach with ⁶⁴Cu-labeled trastuzumab. Left: structures of cleavable C₂TCO linker connecting trastuzumab to ⁶⁴Cu-labeled DOTA, and HK-Tz trigger. Right: PET and CT images of mice injected with ⁶⁴Cu-labeled trastuzumab followed by HK-Tz 4 h later, leading to an enhanced tumor-to-background ratio after 2 h. Tumors are denoted by white arrows [94]. Reprinted with permission from Quintana, J.M., et al., Bioconjug Chem, 2024 35(10):1543–1552. Copyright 2024 American Chemical Society