Novel antibody-drug conjugate with UV-controlled cleavage mechanism for cytotoxin release
Graphical abstract
Introduction
In recent years, antibody-drug conjugates (ADCs) have been developed worldwide with the potential to revolutionize current cancer treatment strategies [1], [2], [3]. Structurally, they are formed through conjugation of an antibody to a highly active cytotoxin via a linker. Currently, nine ADCs have been approved, and more than 80 are at different phases of clinical trials [4], [5].
Essentially, as a prodrug, ADCs are ideally highly stable in the circulatory system and release cytotoxins in the tumor microenvironment. Therefore, the linker, similar to the trigger of the ADC, is always the key in ADC design [6], [7], [8]. To date, three generations of ADCs have been developed: first-generation ADCs that contain acid-labile linkers, such as hydrazone and carbonate bonds, represented by the drugs Mylotarg [9] and Besponsa [10]; and second- and third-generation ADCs that predominantly contain enzyme-cleavable linkers, such as Adcetris [11]. Although the continuous development of ADCs has effectively improved their therapeutic index to a certain extent, off-target toxicity caused by the instability of linkers remains one of the main issues to be resolved. For example, mainstream enzyme-cleavable ADCs mainly utilize cathepsin, which is present in all mammalian tissue cells for drug release [12], but the degradation behaviors of off-target ADC linker structures can lead to toxic events, thereby limiting their further clinical applications (Fig. 1a). A study has shown that more than 23 of the 55 conventional ADCs fail because of poor therapeutic indexes [5]. Therefore, the development of linker technology with a novel drug release mechanism is urgently needed.
In recent years, photocontrol technology has been widely studied as a noninvasive approach, which allows control of active substance release with high spatiotemporal precision [13], [14], [15], [16], [17]. The principle behind this technology is that a photoremovable protecting group (PPG) is linked to an active drug via a covalent bond to mask its activity. However, the bond can be cleaved by external irradiation to release the active component from the parent drug. However, certain intrinsic shortcomings of the current photocontrol technology still exist, such as lack of targeting effects and its proneness to cause unavoidable toxic adverse effects, which are due to nonspecific systemic distribution of the drug. These factors limit their applications for highly toxic cytotoxic drugs. To resolve this issue, a combination photocontrolled drug release system with an effective targeted ADC appears to be an attractive research topic.
Currently, there are only a few reports on photocontrolled ADCs. Nani et al. introduced the structure of cyanine to the ADC linker and designed two photocontrolled ADCs [18], [19] (Fig. 2). Upon irradiation with near-infrared (NIR) light (650–900 nm), the ADCs effectively released the small-molecule cytotoxin, CA-4, and duocarmycin in the irradiated tumor areas in a site-specific manner. Photocontrolled ADCs based on NIR exhibit good tissue penetration, but their structures are complex. In addition, they tend to self-aggregate and be photounstable [20], [21], which to a certain extent limits their applications in biological research and their further development as drugs. Overall, this work has provided us with a reference point to expand our ADC research.
Herein, we report, for the first time, a type of ADC that utilizes ultraviolet (UV) light to achieve photocontrolled drug release. In this design, we carefully introduced a UV light-controlled o-nitrobenzyl group to replace the p-aminobenzyl (PAB) in linkers. In addition, this would help to maximize maintaining the optimum pharmacological properties of ADCs (Fig. 1b). Currently, the o-nitrobenzyl system and its derivatives are among the most commonly used photosensitive protective groups (PPGs) in biological research [22], [23], [24], [25], and some studies have shown that these PPGs have almost no cytotoxicity after photolysis [26], [27]. Our novel type of ADC can be delivered to tumor sites by utilizing the targeting function of the antibody. Upon brief UV irradiation, the o-nitrobenzyl group formed a highly active diradical, followed by dehydrogenation of the carbon atom in the γ-position [25], [26], [27], after which the cytotoxic drug was cleaved and released (Fig. 1B). 1b), finally achieving precise temporal and spatial control. In this study, we synthesized an ADC using the highly active cytotoxic drug monomethyl auristatin E (MMAE) (EC50 = 0.18 nM) and the well-studied antibody mil40, which is a biosimilar of trastuzumab and is in the clinical research stage (CTR20180362) [28], [29], [30]. Our preliminary work revealed that our ADC possessed some promising features, such as good stability and targeting effect. In addition, it rapidly released drugs and effectively destroyed drug-resistant breast and gastric cancer cells only after irradiation with UV light. Our work presented herein preliminarily validated the feasibility of photocontrolled ADCs for achieving drug release upon UV light irradiation. The novel design strategy of UV light-controlled ADCs may provide new perspectives for future research on ADCs and promote the development of photocontrol systems.
Section snippets
Chemistry
As shown in Scheme 1, the linker-MMAE portion, i.e., compounds 1a and 1b, was first synthesized, and 4-Hydroxy-3-methoxyacetophenone and ethyl 4-bromobutyrate were used as starting materials. The substitution reaction was performed under the catalysis of potassium carbonate at 50 °C to obtain compound 3, followed by a nitration reaction in acetic anhydride and nitric acid to obtain compound 4. Compound 5 was obtained by a two-step reaction: compound 4 was hydrolyzed in a hot acidic solution of
Chemistry
Reagents and solvents were purchased from Innochem, Energy Chemical, Aladdin, Adamas-beta, TCI, Ark Pharm, Acros and Alfa Aesar and were used without additional purification. Mil40 is a biosimilar of trastuzumab, which was provided by Zhejiang Haizheng Pharmaceutical Co., Ltd. Reactions were followed by thin-layer chromatography (Dexin Biotechnology Co., Ltd, Yantai, China). Visualization was accomplished with 254 nm UV light. Nuclear magnetic resonance (NMR) spectra were obtained on a
Discussion
Although several admirable works considering photocontrolled ADCs using near infrared light (650–900 nm) have been reported, we developed novel UV-light (360 nm) control ADCs for the following points. First, compared with the complex structure of near infrared light-controlled ADCs, UV light-controlled ADCs have a more reasonable and simple structure and retain the structure of the marketed ADCs. Only a minor structural modification has been made by replacing PAB with the commonly used PPG
Conclusions
This article reports, for the first time, the use of UV light to control drug release by ADCs. The feasibility of such a design was also preliminarily validated. Throughout the study, our ADCs exhibited good stability and photocontrollability while maintaining a targeting effect similar to that of a naked antibody. Brief UV light irradiation resulted in rapid release of small-molecule cytotoxins and effective killing of Herceptin-resistant N87-HerDR and BT474-HerDR cells. Compared with those of
Funding
This research was funded by the National Science and Technology Major Project for Major New Drugs Innovation and Development under grant no. 2018ZX09711003-009 and Chinese National Natural Science Foundation under grant no. 81872736.
CRediT authorship contribution statement
Jiaguo Li: Methodology, Formal analysis, Writing - original draft. Dian Xiao: Conceptualization, Investigation. Fei Xie: Formal analysis. Wei Li: Investigation. Lei Zhao: Software. Wei Sun: Validation, Validation, Writing - review & editing. Xiaohong Yang: Validation, Writing - review & editing, Supervision. Xinbo Zhou: Validation, Resources, Data curation, Writing - review & editing, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors gratefully acknowledge research support from Dan Jiang laboratory for the structural confirmation.
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These authors contribute equally to this work.