Click Chemistry Selectively Activates an Auristatin Protodrug with either Intratumoral or Systemic Tumor-Targeting Agents

Abstract

The Click Activated Protodrugs Against Cancer (CAPAC) platform enables the activation of powerful cancer drugs at tumors. CAPAC utilizes a click chemistry reaction between tetrazine and trans-cyclooctene. The reaction between activator, linked to a tumor-targeting agent, and protodrug leads to the targeted activation of the drug. Here, tumor targeting is achieved by intratumoral injection of a tetrazine-modified hyaluronate (SQL70) or by infusion of a tetrazine-modified HER2-targeting antigen-binding fragment (SQT01). Monomethyl auristatin E (a cytotoxin hindered in its clinical use by severe toxicity) was modified with a trans-cyclooctene to form the protodrug SQP22, which reduced its cytotoxicity in vitro and in vivo. Treatment of SQP22 paired with SQL70 demonstrated antitumor effects in Karpas 299 and RENCA murine tumor models, establishing the requirement of click chemistry for protodrug activation. SQP22 paired with SQT01 induced antitumor effects in the HER2-positive NCI-N87 xenograft model, showing that tumor-targeted activation could be accomplished via systemic dosing. Observed toxicities were limited, with transient myelosuppression and moderate body weight loss detected. This study highlights the capabilities of the CAPAC platform by demonstrating the activity of SQP22 with two differentiated targeting approaches and underscores the power of click chemistry to precisely control the activation of drugs at tumors.

Bioorthogonal Bond Cleavage Chemistry for On-demand Prodrug Activation: Opportunities and Challenges

“…Encouragingly, the data from Phase I clinical trials have well established the safety of SQ3370-001, which was safely administered at 12-fold the conventional Dox dose in patients, with no dose-limiting toxicity reported. 66 Very recently, a monomethyl auristatin E (MMAE)-based TCO-prodrug 26 was designed using the same chemistry (Figure 6), 67 and tumor-targeted activation was realized via pretargeting (with a tetrazine-modified Fab 25) and "catch and release" strategy (with a tetrazine-modified hydrogel 24). In both strategies, significant tumor suppression effects were observed in the cotreatment group, and no or only minimal weight loss was noticed, indicating that no unspecific release of MMAE occurred.…”

Synthesis of C3-Substituted N1-tert-Butyl 1,2,4-Triazinium Salts via the Liebeskind–Srogl Reaction for Fluorogenic Labeling of Live Cells

“…Chemical reactions compatible with biological systems have applications in chemical biology, biomedicine, imaging, diagnosis, − and therapy. − Among the many bioorthogonal reactions now available, the inverse electron demand Diels–Alder reaction of 1,2,4,5-tetrazines with strained dienophiles meets the strict criteria for use in living organisms, − including humans . In recent years, researchers have made considerable efforts to fine-tune this bioorthogonal reaction for use in diverse applications. ,,, In addition to other promising synthetic methods, , cross-coupling reactions involving tetrazines provide many opportunities for modifying these heterodienes.…”

Enabling Universal Access to Rapid and Stable Tetrazine Bioorthogonal Probes through Triazolyl-Tetrazine Formation

“…Particularly for certain in vivo biomedical applications, the kinetic constants should be greater than 10 4 M –1 s –1 to enable conjugation at diagnostic or therapeutic concentrations, ,, while the reactants must remain stable after tens of hours in the circulation. The inverse electron-demand Diels–Alder (IEDDA) reaction between tetrazine and trans -cyclooctene (TCO) has been recognized as the most rapid bioorthogonal reaction, , demonstrating successful applications in animal models and clinical transformative potential. ,, Various probes or theranostic agents based on this chemistry have been developed for diverse cutting-edge applications, including super-resolution imaging of biomolecules in live cells, site-specific protein multimodification or function regulation, on-demand prodrug release, and pretargeted nuclear imaging and radionuclide therapy. ,,− However, the inverse correlation between the stability and reactivity of these reactants greatly limits their bioorthogonal performance in living systems and clinic translation. , …”