Gold(III)-Induced Amide Bond Cleavage In Vivo: A Dual Release Strategy via π-Acid Mediated Allyl Substitution.

Selective cleavage of amide bonds holds prominent significance by facilitating precise manipulation of biomolecules, with implications spanning from basic research to therapeutic interventions. However, achieving selective cleavage of amide bonds via mild synthetic chemistry routes poses a critical challenge. Here, we report a novel amide bond-cleavage reaction triggered by Na[AuCl4] in mild aqueous conditions, where a crucial cyclization step leads to the formation of a 5-membered ring intermediate that rapidly hydrolyses to release the free amine in high yields. Notably, the reaction exhibits remarkable site-specificity to cleave peptide bonds at the C-terminus of allyl-glycine. The strategic introduction of a leaving group at the allyl position facilitated a dual-release approach through π-acid catalyzed substitution. This reaction was employed for the targeted release of the cytotoxic drug monomethyl auristatin E in combination with an antibody-drug conjugate in cancer cells. Finally, Au-mediated prodrug activation was shown in a colorectal zebrafish xenograft model, leading to a significant increase in apoptosis and tumor shrinkage. Our findings reveal a novel metal-based cleavable reaction expanding the utility of Au complexes beyond catalysis to encompass bond-cleavage reactions for cancer therapy.

Optimized LC-MS/MS methods for quantifying antibody-drug conjugate payloads in cell culture media containing phenol red.

Aim: Phenol red is commonly used in cell culture media, but can be detrimental to bioanalysis of in vitro samples as it may impact instrument reliability. Many researchers do their final stage of culture in 'phenol red free' media, but in collaborative work this is not always feasible.Materials & methods: A comparison was made between typical extraction methods to reduce phenol red matrix interferences, including organic solvent precipitation and solid phase extraction.Results: The final method was demonstrated to be precise and accurate for the measurement of a target analyte by LC-MS/MS, and was applied to an in vitro ADC deconjugation study.Conclusion: This method allows for for continued bioanalytical support of in vitro models used in drug development.

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A New Age of Drug Delivery: A Comparative Perspective of Ferritin-Drug Conjugates (FDCs) and Antibody-Drug Conjugates (ADCs).

Ferritin-drug conjugates (FDCs) and antibody-drug conjugates (ADCs) respectively represent the innovative and traditional mainstream approaches in drug delivery systems, each offering unique advantages and challenges. This viewpoint delves into the evolving landscape of drug delivery technologies, specifically focusing on FDCs and ADCs. Each method exhibits unique advantages and inherent challenges, shaping their roles in therapeutic applications. The article provides a comparative analysis of two delivery systems, FDCs and ADCs, in terms of targeting accuracy, drug loading capacity, and the nature of the payload itself. This comparison offers valuable insights into the distinct advantages and disadvantages associated with each system, enabling a clearer understanding of their potential applications and limitations in therapeutic contexts. This analysis is crucial for optimizing the use of these delivery systems across varying medical contexts, offering a comprehensive overview of their impact on the field of drug delivery.

Antibody-Calixarene Drug Conjugate: A General Drug Delivery Platform for Tumor-Targeted Therapy.

Antibody-drug conjugates (ADCs), which combine the precise targeting capabilities of antibodies with the powerful cytotoxicity of small-molecule drugs, have evolved into a promising approach for tumor treatment. However, the traditional covalent coupling method requires the design of a specific linker tailored to the properties of the small-molecule drugs, which greatly limits the development of ADCs and the range of drugs that can be used. Herein, a novel type of antibody-calixarene drug conjugates (ACDCs) that function similarly to ADCs by delivering drugs to their targets using antibodies but without the requirement of covalent conjugation of the drugs with antibodies is presented. By replacement of conventional linkers with supramolecular linkers, the ACDCs can load various chemotherapeutic drugs through host-guest interactions. Furthermore, ACDCs are readily reduced upon reaching the hypoxic microenvironment, resulting in rapid release of the drugs. With this precise drug encapsulation and controlled release mechanism, ACDCs deliver drugs to tumor tissues effectively and achieve a significantly enhanced antitumor effect. Considering that the ACDCs can be easily prepared by combining antibody-calixarene conjugates derived from tumor-targeting antibodies with various small-molecule drugs, ACDCs may provide a promising platform technology to accelerate ADC development and thus improve the therapeutic efficacy of chemotherapy.

Natural-source payloads used in the conjugated drugs architecture for cancer therapy: Recent advances and future directions.

Drug conjugates are obtained from tumor-located vectors connected to cytotoxic agents via linkers, which are designed to deliver hyper-toxic payloads directly to targeted cancer cells. These drug conjugates include antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), small molecule-drug conjugates (SMDCs), nucleic acid aptamer-drug conjugates (ApDCs), and virus-like drug conjugate (VDCs), which show great therapeutic value in the clinic. Drug conjugates consist of a targeting carrier, a linker, and a payload. Payloads are key therapy components. Cytotoxic molecules and their derivatives derived from natural products are commonly used in the payload portion of conjugates. The ideal payload should have sufficient toxicity, stability, coupling sites, and the ability to be released under specific conditions to kill tumor cells. Microtubule protein inhibitors, DNA damage agents, and RNA inhibitors are common cytotoxic molecules. Among these conjugates, cytotoxic molecules of natural origin are summarized based on their mechanism of action, conformational relationships, and the discovery of new derivatives. This paper also mentions some cytotoxic molecules that have the potential to be payloads. It also summarizes the latest technologies and novel conjugates developed in recent years to overcome the shortcomings of ADCs, PDCs, SMDCs, ApDCs, and VDCs. In addition, this paper summarizes the clinical trials conducted on conjugates of these cytotoxic molecules over the last five years. It provides a reference for designing and developing safer and more efficient conjugates.

Application of Microfluidic Devices for Automated Two-Step Radiolabeling of Antibodies.

Radioimmunoconjugates (RICs) composed of tumor-targeting monoclonal antibodies and radionuclides have been developed for diagnostic and therapeutic application. A new radiolabeling method using microfluidic devices is expected to facilitate simpler and more rapid synthesis of RICs. In the microfluidic method, microfluidic chips can promote the reaction between reactants by mixing them efficiently, and pumping systems enable automated synthesis. In this study, we synthesized RICs by the pre-labeling method, in which the radiometal is coordinated to the chelator and then the radiolabeled chelator is incorporated into the antibodies, using microfluidic devices for the first time. As a result of examining the reaction parameters including the material of mixing units, reaction temperature, and flow rate, RICs with radiochemical purity (RCP) exceeding 90% were obtained. These high-purity RICs were successfully synthesized without any purification simply by pumping three solutions of a chelating agent, radiometal, and antibody into microfluidic devices. Under the same conditions, the RCP of RICs labeled by conventional methods was below 50%. These findings indicate the utility of microfluidic devices for automatic and rapid synthesis of high-quality RICs.

On Demand Bioorthogonal Switching of an Antibody-Conjugated SPECT Probe to a Cytotoxic Payload: from Imaging to Therapy.

Antibody-drug conjugates (ADCs) for the treatment of cancer aim to achieve selective delivery of a cytotoxic payload to tumor cells while sparing normal tissue. In vivo, multiple tumor-dependent and -independent processes act on ADCs and their released payloads to impact tumor-versus-normal delivery, often resulting in a poor therapeutic window. An ADC with a labeled payload would make synchronous correlations between distribution and tissue-specific pharmacological effects possible, empowering preclinical and clinical efforts to improve tumor-selective delivery; however, few methods to label small molecules without destroying their pharmacological activity exist. Herein, we present a bioorthogonal switch approach that allows a radiolabel attached to an ADC payload to be removed tracelessly at will. We exemplify this approach with a potent DNA-damaging agent, the pyrrolobenzodiazepine (PBD) dimer, delivered as an antibody conjugate targeted to lung tumor cells. The radiometal chelating group, DOTA, was attached via a novel trans-cyclooctene (TCO)-caged self-immolative para-aminobenzyl (PAB) linker to the PBD, stably attenuating payload activity and allowing tracking of biodistribution in tumor-bearing mice via SPECT-CT imaging (live) or gamma counting (post-mortem). Following TCO-PAB-DOTA reaction with tetrazines optimized for extra- and intracellular reactivity, the label was removed to reveal the unmodified PBD dimer capable of inducing potent tumor cell killing in vitro and in mouse xenografts. The switchable antibody radio-drug conjugate (ArDC) we describe integrates, but decouples, the two functions of a theranostic given that it can serve as a diagnostic for payload delivery in the labeled state, but can be switched on demand to a therapeutic agent (an ADC).

Recent Advances in Targeted Cancer Therapy: Are PDCs the Next Generation of ADCs?

Antibody-drug conjugates (ADCs) comprise antibodies, cytotoxic payloads, and linkers, which can integrate the advantages of antibodies and small molecule drugs to achieve targeted cancer treatment. However, ADCs also have some shortcomings, such as non-negligible drug resistance, a low therapeutic index, and payload-related toxicity. Many studies have focused on changing the composition of ADCs, and some have even further extended the concept and types of targeted conjugated drugs by replacing the targeted antibodies in ADCs with peptides, revolutionarily introducing peptide-drug conjugates (PDCs). This Perspective summarizes the current research status of ADCs and PDCs and highlights the structural innovations of ADC components. In particular, PDCs are regarded as the next generation of potential targeted drugs after ADCs, and the current challenges of PDCs are analyzed. Our aim is to offer fresh insights for the efficient design and expedited development of innovative targeted conjugated drugs.

Design and synthesis of novel site-specific antibody-drug conjugates that target TROP2.

The approval of Trodelvy® validates TROP2 as a druggable but challenging target for antibody-drug conjugates (ADCs) to treat metastatic triple-negative breast cancer (mTNBC). Here, based on the TROP2-targeted antibody sacituzumab, we designed and developed several site-specific ADC candidates, which employ MMAE (monomethyl auristatin E) as the toxin, via IgG glycoengineering or affinity-directed traceless conjugation. Systematic evaluation of these site-specific ADCs in homogeneity, hydrophilicity, stability, and antitumor efficiency was conducted. The results indicate that the site-specific ADCs gsADC 3b made from one-step glycoengineering exhibit good aggregation stability and in vivo efficacy, providing a new format of ADCs that target TROP2.

Antibody-Drug Conjugates-Evolution and Perspectives.

Antineoplastic therapy is one of the main research themes of this century. Modern approaches have been implemented to target and heighten the effect of cytostatic drugs on tumors and diminish their general/unspecific toxicity. In this context, antibody-drug conjugates (ADCs) represent a promising and successful strategy. The aim of this review was to assess different aspects regarding ADCs. They were presented from a chemical and a pharmacological perspective and aspects like structure, conjugation and development particularities alongside effects, clinical trials, safety issues and perspectives and challenges for future use of these drugs were discussed. Representative examples include but are not limited to the following main structural components of ADCs: monoclonal antibodies (trastuzumab, brentuximab), linkers (pH-sensitive, reduction-sensitive, peptide-based, phosphate-based, and others), and payloads (doxorubicin, emtansine, ravtansine, calicheamicin). Regarding pharmacotherapy success, the high effectiveness expectation associated with ADC treatment is supported by the large number of ongoing clinical trials. Major aspects such as development strategies are first discussed, advantages and disadvantages, safety and efficacy, offering a retrospective insight on the subject. The second part of the review is prospective, focusing on various plans to overcome the previously identified difficulties.

A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs.

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.

Electrocatalytic Reduction of Disulfide Bonds across Chemical Modalities.

The chemical properties of disulfides are leveraged in a wide array of applications, ranging from protein-drug conjugates for cancer treatment to self-healing materials. However, disulfide reduction strategies remain severely underdeveloped despite being the key to efficiently accessing the desired targets. Specifically, no homogeneous catalyst has been reported for this reaction, and conditions that allow the use of mild and green reductants (e.g., via electrochemical reduction) are not known. Herein, we unveil a vitamin B12-catalyzed, electrochemically driven protocol for efficiently reducing disulfide bonds in various aqueous buffers over a broad pH range. This robust and simple method is suitable for disulfide reductions of substrates ranging from small molecules to large proteins. Finally, one-pot reduction and conjugation of disulfide bonds in a monoclonal antibody were demonstrated to produce antibody conjugates.

Advancing Tumor-Targeted Chemo-Immunotherapy: Development of the CAR-M-derived Exosome-Drug Conjugate.

Traditional antibody-drug conjugates (ADCs) mainly suppress tumor growth through either chemotherapy with cytotoxic payloads or immunotherapy with immuno-modulators. However, a single therapeutic modality may limit their exploration. Herein, we developed a new type of drug conjugate termed CAR-EDC (CAR-M-derived exosome-drug conjugate) by using CAR-exosomes from CAR-M cells as the targeting drug carrier that contains a high level of CXCL10. CAR-exosomes could significantly enhance the immunological activation and migratory capacity of T lymphocytes and promote their differentiation into CD8+ T cells. It also increased the proportion of M1 macrophages. The CAR-EDC, covalently loaded with SN-38, was internalized into Raji cells through endocytosis mediated by the CAR molecules. It exerted excellent antitumor activity in vivo by virtue of not only chemotherapy by SN38 but also immunotherapy by CXCL10-mediated antitumor immunity. Generally, this study provides an exosome-drug conjugate system with enhanced antitumor effects over traditional ADCs through the synergism of chemotherapy and immunotherapy.

Imaging and Monitoring HER2 Expression in Tumors during HER2 Antibody-Drug Conjugate Therapy Utilizing a Radiolabeled Site-Specific Single-Domain Antibody Probe: 68Ga-NODAGA-SNA004-GSC.

The overexpression of HER2 is pivotal in the initiation and progression of breast cancer. Developing HER2-targeted radiotracers is crucial for noninvasive assessment of HER2 expression, patient selection for HER2-targeted therapy, monitoring treatment response, and identifying resistance. Here, we reported a nonsite-specific coupled radiotracer, 68Ga-NOTA-SNA004-His6, and a site-specific coupled radiotracer, 68Ga-NODAGA-SNA004-GSC, based on a novel HER2 nanobody, SNA004. Both radiotracers exhibited high affinity, specific targeting, and rapid clearance in vitro and in vivo. Additionally, these tracers and trastuzumab showed noncompetitive binding to HER2. Compared to 68Ga-NOTA-SNA004-His6, 68Ga-NODAGA-SNA004-GSC demonstrated significantly reduced renal and liver uptake. PET/CT imaging with 68Ga-NODAGA-SNA004-GSC sensitively detected the responsiveness of various tumor models to trastuzumab and its antibody-drug conjugates (ADCs). Overall, the site-specific coupled radiotracer 68Ga-NODAGA-SNA004-GSC offered significant advantages in biodistribution and signal-to-noise ratio, making it a valuable tool for monitoring HER2 expression levels before, during, and after trastuzumab and ADC treatment.

Antibody-drug conjugates for targeted cancer therapy: Recent advances in potential payloads.

Antibody-drug conjugates (ADCs) represent a promising cancer therapy modality which specifically delivers highly toxic payloads to cancer cells through antigen-specific monoclonal antibodies (mAbs). To date, 15 ADCs have been approved and more than 100 ADC candidates have advanced to clinical trials for the treatment of various cancers. Among these ADCs, microtubule-targeting and DNA-damaging agents are at the forefront of payload development. However, several challenges including toxicity and drug resistance limit the potential of this modality. To tackle these issues, multiple innovative payloads such as immunomodulators and proteolysis targeting chimeras (PROTACs) are incorporated into ADCs to enable multimodal cancer therapy. In this review, we describe the mechanism of ADCs, highlight the importance of ADC payloads and summarize recent progresses of conventional and unconventional ADC payloads, trying to provide an insight into payload diversification as a key step in future ADC development.

Site-Specific Antibody Prodrugs via S-Arylation: a Bioconjugation Approach Toward Masked Tyrosine Analogues.

The utility of antibody therapeutics is hampered by potential cross-reactivity with healthy tissue. Over the past decade, significant advances have been made in the design of activatable antibodies, which increase, or create altogether, the therapeutic window of a parent antibody. Of these, antibody prodrugs (pro-antibodies) are masked antibodies that have advanced the most for therapeutic use. They are designed to reveal the active, parent antibody only when encountering proteases upregulated in the microenvironment of the targeted disease tissue, thereby minimizing off-target activity. However, current pro-antibody designs are relegated to fusion proteins that append masking groups restricted to the use of only canonical amino acids, offering excellent control of the site of introduction, but with no authority over where the masking group is installed other than the N-terminus of the antibody. Here, we present a palladium-based bioconjugation approach for the site-specific introduction of a masked tyrosine mimic in the complementary determining region of the FDA approved antibody therapeutic ipilimumab used as a model system. The approach enables the introduction of a protease cleavable group tethered to noncanonical polymers (polyethylene glycol (PEG)) resulting in 47-fold weaker binding to cells expressing CTLA-4, the target antigen of ipilimumab. Upon exposure to tumor-associated proteases, the masking group is cleaved, unveiling a tyrosine-mimic (dubbed hydroxyphenyl cysteine (HPC)) that restores (>90% restoration) binding affinity to its target antigen.

Synthesis and evaluation of antibody-drug conjugates with high drug-to-antibody ratio using dimaleimide-DM1 as a linker- payload.

The notable characteristics of recently emerged Antibody-Drug Conjugates (ADCs) encompass the targeting of Human Epidermal growth factor Receptor 2 (HER2) through monoclonal antibodies (mAbs) and a high ratio of drug to antibody (DAR). The achievements of Kadcyla® (T-DM1) and Enhertu® (T-Dxd) have demonstrated that HER2-targeting antibodies, such as trastuzumab, have shown to be competitive in terms of efficacy and price for development. Furthermore, with the arrival of T-Dxd and Trodelvy®, high-DAR (7-8) ADCs, which differ from the moderate DAR (3-4) ADCs that were formerly regarded as conventional, are being acknowledged for their worth. Following this trend of drug development, we endeavored to develop a high-DAR ADC using a straightforward approach involving the utilization of DM1, a highly potent substance, in combination with the widely recognized trastuzumab. To achieve a high DAR, DM1 was conjugated to reduced cysteine through the simple design and synthesis of various dimaleimide linkers with differing lengths. Using LC and MS analysis, we have demonstrated that our synthesis methodology is uncomplicated and efficacious, yielding trastuzumab-based ADCs that exhibit a remarkable degree of uniformity. These ADCs have been experimentally substantiated to exert an inhibitory effect on cancer cells in vitro, thus affirming their value as noteworthy additions to the realm of ADCs.

An Efficient Method for Vault Nanoparticle Conjugation with Finely Adjustable Amounts of Antibodies and Small Molecules.

Vaults are eukaryotic ribonucleoproteins consisting of 78 copies of the major vault protein (MVP), which assemble into a nanoparticle with an about 60 nm volume-based size, enclosing other proteins and RNAs. Regardless of their physiological role(s), vaults represent ideal, natural hollow nanoparticles, which are produced by the assembly of the sole MVP. Here, we have expressed in Komagataella phaffi and purified an MVP variant carrying a C-terminal Z peptide (vault-Z), which can tightly bind an antibody's Fc portion, in view of targeted delivery. Via surface plasmon resonance analysis, we could determine a 2.5 nM affinity to the monoclonal antibody Trastuzumab (Tz)/vault-Z 1:1 interaction. Then, we characterized the in-solution interaction via co-incubation, ultracentrifugation, and analysis of the pelleted proteins. This showed virtually irreversible binding up to an at least 10:1 Tz/vault-Z ratio. As a proof of concept, we labeled the Fc portion of Tz with a fluorophore and conjugated it with the nanoparticle, along with either Tz or Cetuximab, another monoclonal antibody. Thus, we could demonstrate antibody-dependent, selective uptake by the SKBR3 and MDA-MB 231 breast cancer cell lines. These investigations provide a novel, flexible technological platform that significantly extends vault-Z's applications, in that it can be stably conjugated with finely adjusted amounts of antibodies as well as of other molecules, such as fluorophores, cell-targeting peptides, or drugs, using the Fc portion as a scaffold.

Liquid-phase separations coupled with ion mobility-mass spectrometry for next-generation biopharmaceutical analysis.

INTRODUCTION: The pharmaceutical industry continues to expand its search for innovative biotherapeutics. The comprehensive characterization of such therapeutics requires many analytical techniques to fully evaluate critical quality attributes, making analysis a bottleneck in discovery and development timelines. While thorough characterization is crucial for ensuring the safety and efficacy of biotherapeutics, there is a need to further streamline analytical characterization and expedite the overall timeline from discovery to market. AREAS COVERED: This review focuses on recent developments in liquid-phase separations coupled with ion mobility-mass spectrometry (IM-MS) for the development and characterization of biotherapeutics. We cover uses of IM-MS to improve the characterization of monoclonal antibodies, antibody-drug conjugates, host cell proteins, glycans, and nucleic acids. This discussion is based on an extensive literature search using Web of Science, Google Scholar, and SciFinder. EXPERT OPINION: IM-MS has the potential to enhance the depth and efficiency of biotherapeutic characterization by providing additional insights into conformational changes, post-translational modifications, and impurity profiles. The rapid timescale of IM-MS positions it well to enhance the information content of existing assays through its facile integration with standard liquid-phase separation techniques that are commonly used for biopharmaceutical analysis.

Development of Peptide Paratope Mimics Derived from the Anti-ROR1 Antibody and Long-Acting Peptide-Drug Conjugates for Targeted Cancer Therapy.

Antibody-based targeted therapy in cancer faces a challenge due to uneven antibody distribution in solid tumors, hindering effective drug delivery. We addressed this by developing peptide mimetics with nanomolar-range affinity for Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) using computational methods. These peptides showed both specific targeting and deep penetration in vitro and in vivo. Additionally, we created peptide-drug conjugates (PDCs) by linking targeting peptides to toxin drugs via various linkers and enhancing their in vivo half-life with fatty side chains for albumin binding. The antitumor candidate II-3 displayed exceptional affinity (KD = 1.72 × 10-9 M), internalization efficiency, anticancer potency (IC50 = 0.015 ± 0.002 µM), and pharmacokinetics (t1/2 = 2.6 h), showcasing a rational approach for designing PDCs with favorable tissue distribution and strong tumor penetration.