A framework that overcomes the internalization barrier, broadening the target scope for therapeutic and diagnostic conjugates is established and extended BTR to programmed cell death ligand 1 (PD-L1) and an mRNA-display-derived FAP peptide, suggesting potential broad applicability.
Abstract
Drug conjugates, such as antibody-drug conjugates (ADCs) and small molecule-drug conjugates (SMDCs), are often dependent on efficient receptor-mediated endocytosis for payload release1-3-supported by about 10% of targets4-7. For poorly internalizing targets, drug conjugates dissociate and clear rapidly, limiting efficacy. To overcome the limitation in the internalization-to-release (ITR) pattern, we introduce a binding-to-release (BTR) strategy that decouples drug release from endocytosis by positioning an electrophile for direct cleavage by a proximal nucleophilic residue within the binding pocket. To realize this, we developed phosphorus(V)-phenol exchange (PhoPEx), a sulfur(VI) fluoride exchange-inspired chemistry enabling release of various payloads. This platform demonstrated high specificity from in vitro to clinical specimens, achieving precise detection of fibroblast activation protein (FAP) expression in patient-derived lymph nodes. In therapeutic settings, the FAP-BTR-SMDC achieved 5.9-fold higher monomethyl auristatin E exposure (AUC0-120 h) in tumours than internalization-dependent FAP-ITR-SMDC, matching FAP-ITR-ADC levels while minimizing off-target release. This led to improved ratios: the tumour-to-blood ratio was 14.7- and 3.6-fold higher than that of FAP-ITR-SMDC and FAP-ITR-ADC, respectively, and the tumour-to-liver ratio was 55.1- and 58.7-fold higher, respectively. This biodistribution increased the maximum tolerated dose and led to near-complete tumour regression in various tumour models. We further extended BTR to programmed cell death ligand 1 (PD-L1) and an mRNA-display-derived FAP peptide, suggesting potential broad applicability. This work establishes a framework that overcomes the internalization barrier, broadening the target scope for therapeutic and diagnostic conjugates.
Immunotherapy is experiencing rapid growth by increasingly its use of antibody drug conjugates (ADC). Antibodies are a desired vehicle for immunotherapy because they can deliver a drug to a precise cellular location. Heterogenous conjugates have been shown to affect stability, efficacy and binding to intended target. Successful cellular internalization of an ADC with the controlled release of the cytotoxic drug is required for an effective ADC. The creation of a site-specific, tailor-made ADC with a buildable scaffold is expected to increase an ADC’s functional efficacy and its sensitivity in immunoassays.
The well-established Trastuzumab is conjugated to various linker structures and using NHS chemistry with 4 molar excesses to generate conjugates that range in degree of labeling (DOL) from 3-14. Trastuzumab site specific conjugates have a buildable scaffold that corresponds to the NHS DOLs. The performance of the antibody conjugates is compared by immunoassay results, including immunofluorescence (IF) and cell viability assay.
Antibody drug conjugates that are generated using NHS and site-specific chemistry methods show that lower degrees of labeling for NHS and site specific are comparable in binding results. However, as the DOL increases with high labeling and size of the scaffold grows, there’s a distinction between the effectiveness of NHS and site-specific conjugates with expanding drug scaffolds.
Antibody drug conjugates (ADC) provide the specificity of the monoclonal antibody with the precise delivery of a covalently labeled cytotoxic drug. Therefor generating an ADC that is homogenous will mitigate the undesirable effects of a heterogenous conjugate. Here we show the benefits of creating a buildable drug scaffold that’s accommodating for achieving exact payload conjugated at a specific site on Trastuzumab. This approach will enable all therapeutic antibodies to be conjugated in a uniform manner, accelerating the advancing field of ADC therapeutics.
National Science Foundation EPSCoR Award #2119237
Technological Innovations in Immunology (TECH)
Lisa Arvidson, Katarina Jovic-Dold, J. Halpern et al.· Journal of Immunology· 0 citations
Antibody–drug conjugates (ADCs) have emerged as a powerful class of targeted therapeutics in many clinical areas, such as in oncology. Despite their efficacy, the onset of adverse events has been a major drawback in their clinical use. Among other explanations, the clinical performance of the ADCs has been associated with the chemistry of the linker connecting the antibody and payload. Linkers determine plasma stability, intracellular activation, and payload diffusibility, thereby influencing the therapeutic index, off-tumour toxicity, and by-stander activity. Mechanistic insights increasingly show that linker–payload properties govern catabolite permeability and intratumoral distribution, particularly in antigen-heterogeneous settings. Current developments include enzyme-cleavable and tumour-selective linkers, polarity-modulating masking strategies, alternative self-immolative spacers, and dual-trigger systems designed to enhance selectivity and decouple efficacy from toxicity. In parallel, linker behaviour intersects with broader mechanisms of tumour resistance. This review focuses on understanding these processes, which are essential for designing the next generation of linkers capable of improving stability, safety, and long-term therapeutic effectiveness across diverse tumour contexts.
Sara N. Albino, Margarida M. Domingos, T. Pacheco et al.· Pharmaceutics· 0 citations
Antibody-drug conjugates (ADCs) represent an emerging pharmaceutical category that leverages antibodies to facilitate drug delivery to target cells. A key challenge for ADCs is increasing the drug-antibody ratio (DAR) to improve efficacy without compromising their properties, such as pharmacokinetics. Here, we present a novel polypeptide-based platform that substantially increases the DAR of antibody-payload ADCs while minimally compromising the ADCs’ inherent characteristics. A polypeptide-based antibody-drug conjugate lead (polypeptide ADC 6c), capable of achieving a DAR of up to 61, was synthesized using the versatile click reaction method based on our polypeptide-based platform. 6c could efficiently recognize antigens, be subsequently endocytosed, and achieve subnanomolar activity at the cellular level. Moreover, 6c exhibited excellent pharmacokinetic parameters and potent antitumor efficacy in mice. Collectively, this study provides and characterizes a highly variable polypeptide backbone platform that can be used to efficiently develop innovative ADCs with high payloads for the treatment of various cancers
Ruyao Tang, Fusheng Guo, Jinyi Bai et al.· JACS Au· 0 citations
The introduction of cell- and biological-based (CBb) vectors for drug delivery has paved new pathways in cancer therapy, capitalizing on the distinct properties inherent to various microbial systems. In this review, we take a critical look at a range of promising drug delivery platforms, including polymeric micelles, bacterial outer membrane vesicles (OMVs), liposomes, fungi, yeast, bacteria, red blood cells stem cells, virus and microalgae. Our focus is on evaluating their efficacy, targeting capabilities, and biocompatibility. Key findings suggest that Daptomycin-Doxorubicin (Dap-DOX) micelles enhance cytotoxicity and selectively accumulate in tumors through the enhanced permeability and retention (EPR) effect. Likewise, bacterial-derived magnetosomes appear to offer significant tumor suppression and show reduced cardiac toxicity compared to traditional doxorubicin (DOX) treatments. Additionally, RBC-based systems have demonstrated effective targeting and delivery of DOX to cancer sites, surpassing the performance of free drug formulations. The discussed works mainly focused on the use of DOX, however we also reviewed papers where other chemotherapeutic agents have been used. Among the reviewed platforms, microalgae stand out as particularly promising due to their biocompatibility, poor ability to multiply inside human body, and ability to boost drug loading efficiency, facilitating targeted delivery. Evidence points to microalgae’s potential to enhance the therapeutic index of anticancer drugs, as exemplified by the quick drug release behavior of cell vesicles loaded with doxorubicin in acidic tumor microenvironments. Despite these promising developments, challenges persist, especially in the optimization of microbial engineering and the scalability required for clinical applications. We suggest that microalgae offer a compelling platform for future research and development in cancer drug delivery, highlighting the need for further exploration to unlock their full potential in oncological therapies.
Maria Zavali, A. Georgas, Aggeliki Visvardi Bouga et al.· Frontiers in Molecular Biosc...· 0 citations
Antibody-drug conjugates (ADCs) have transformed cancer treatment by covalently linking the monoclonal antibody with cytotoxic payload, yet their clinical potential remains constrained by intrinsic limitations: heterogeneous drug-to-antibody ratios, linker instability, manufacturing complexity, and drug resistance. These challenges highlight the need for fundamentally different drug formulation and delivery platforms. Antibody-encapsulated drugs (AEDs) leverage the single protein encapsulation technology to enable one antibody to noncovalently encapsulate a predefined number of payload molecules. AEDs allow for a fixed drug-to-antibody ratio, mitigate premature drug release, simplify manufacturing, and expand the range of compatible payloads and protein molecules. Preclinical investigation of trastuzumab-encapsulated actinomycin D, a HER2-targeted AED, has demonstrated potent antitumor activity across cancer models with varying HER2 expression levels, alongside a favorable toxicity profile in animal models. The broader translational feasibility of the single protein encapsulation platform is further supported by ongoing clinical trials of albumin-encapsulated therapeutics. Together, these advances position AED as a promising next-generation targeted cancer therapy that complements and potentially extends beyond conventional ADCs, offering a compelling strategy to overcome existing resistance mechanisms and therapeutic limitations.
Linrong Li, Armando E Giuliano, Qiang Sun et al.· Cell investigation· 0 citations
Prostate-specific membrane antigen (PSMA) is a significantly overexpressed receptor in prostate cancer, making it an ideal target. Hence, targeted delivery of a payload via a small molecule to PSMA would facilitate localized cytotoxicity, thereby eradicating the tumor. Herein, we describe a small-molecule drug conjugate (DUPA-SS-Exa, 10) comprising 2-[3-(1,3-dicarboxy propyl)-ureido]pentanedioic acid (DUPA), a PSMA binding ligand, conjugated to exatecan (8) payload via a glutathione-sensitive disulfide traceless linker. Exatecan (8) has been extensively delivered via antibody–drug conjugates (ADCs) and has shown favourable results. The DUPA-SS-Exa (10) conjugate was computationally evaluated to assess PSMA binding affinity in a dynamic environment, and ΔGbind was found to be −115.85 ± 4.52 kcal mol−1. An in vitro cell cytotoxicity assay performed against the PSMA-positive LNCaP cell line showed an IC50 of 117.01 ± 6.67 nM for conjugate (10). DUPA-SS-Exa (10) demonstrated high specificity, showing negligible cytotoxicity against PSMA-negative PC3 cells and non-cancerous HEK293 cells. Furthermore, DUPA-SS-Exa (10) is stable at acidic pH, in PBS, and in serum. Beyond its stability, the conjugate proved significantly safer than free exatecan (8) in B16F10 melanoma models, while exatecan (8) caused severe toxicity, leading to significant weight loss and mortality in mice models. These results establish DUPA-SS-Exa (10) as a stable, highly specific, and potent therapeutic strategy for PSMA-expressing tumors with a favorable safety profile.
A new machine-learning framework aims to improve the success rate of computational protein design while moving away from results that reproduce sequences found in nature.
