It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional chemistries used to make antibody-drug conjugates (ADCs). Site-specific payload placement allows for control both the drug-to-antibody ratio (DAR) and site, of which play an important role in governing pharmacokinetics (PK), disposition, efficacy ADC. In addition DAR site conjugation, linker composition also plays properties We have previously reported a novel platform comprising...

Clicking into place: Cu-free click chemistry is combined with the aldehyde tag protein modification strategy to produce heterobifunctional fusions. This method relies on linkers that utilize orthogonal triazole and oxime chemistries (see scheme) expand conjugation fGly residue enables site-specific full-length human antibodies.

Aldehyde- and ketone-functionalized biomolecules have found widespread use in biochemical biotechnological fields. They are typically conjugated with hydrazide or aminooxy nucleophiles under acidic conditions to yield hydrazone oxime products that relatively stable, but susceptible hydrolysis over time. We introduce a new reaction, the hydrazino-Pictet-Spengler (HIPS) ligation, which has two distinct advantages ligations. First, HIPS ligation proceeds quickly near neutral pH, allowing for...

Although peptide motifs represent the majority of cleavable linkers used in clinical-stage antibody–drug conjugates (ADCs), sequences are often sensitive to cleavage by extracellular enzymes, such as elastase, which leads systemic release cytotoxic payload. This action reduces therapeutic index causing off-target toxicities that can be dose-limiting. For example, a common side-effect ADCs made using peptide-cleavable is myelosuppression, including neutropenia. Only few reports describe...

Expanded ligation techniques are sorely needed to generate unique linkages for the growing field of functionally enhanced proteins. To address this need, we present a chemical that involves double addition pyrazolone moiety with an aldehyde-labeled protein. This occurs via tandem Knoevenagel condensation-Michael addition. A reacts aldehyde enone, which undergoes subsequent attack by second bis-pyrazolone species. rapid and facile technique is performed under mild conditions in absence...

Oncology treatment has been revolutionized by the introduction of immune checkpoint inhibitor drugs, which enable 20–40% patients to generate anti-tumor responses. Combination approaches with chemotherapeutic drugs may responses in remaining patient cohorts. In this regard, a handful are promising due their ability induce immunogenic cell death target cells. However, these agents systemically delivered and indiscriminately cytotoxic proliferating By contrast, antibody-drug conjugates can...

Sorting of transmembrane cargo proteins to different cellular compartments is mediated by sorting signals that are recognized coat involved in vesicle biogenesis. We have identified a signal the yeast cell fusion protein Fus1p required for its transport from trans-Golgi compartment plasma membrane. Transport surface dependent on Chs5p, component multisubunit exomer complex. show also components Bch1p and Bud7p. Disruption clathrin adaptor complex 1 (AP-1) restores localization absence...

The ability to site-specifically conjugate a protein payload of interest (e.g., fluorophore, small molecule pharmacophore, oligonucleotide, or other protein) has found widespread application in basic research and drug development. For example, antibody-drug conjugates represent class biotherapeutics that couple the targeting specificity an antibody with chemotherapeutic potency drug. While first generation (ADCs) used random conjugation approaches, next-generation ADCs are employing...

Abstract Background: Tissue factor (TF) is a transmembrane glycoprotein that plays an important role in the extrinsic coagulation cascade. TF aberrantly expressed various cancers, and its expression generally associated with poor disease outcomes. XB371 anti-TF antibody-drug conjugate, developed using SMARTag® platform, designed to deliver cytotoxic payload TF-expressing tumors while minimizing adverse events related disruption of function, notably bleeding. composed tandem-cleavage...

Auf einen Klick: Kupferfreie Klick-Chemie und eine Aldehydmarkierungsstrategie werden kombiniert, um heterodifunktionelle Proteinfusionen mit den orthogonalen Triazol- Oxim-Verknüpfungen herbeizuführen (siehe Schema). Durch die Einführung eines Formylglycins ermöglicht Methode ortsspezifische Proteinkonjugation an humane Antikörper. Heterobifunctional protein fusions are gaining interest as next-generation biopharmaceuticals.1–5 Combining proteins with disparate functions can enable...

Hematologically derived tumors make up ∼10% of all newly diagnosed cancer cases in the United States. Of these, non-Hodgkin lymphoma (NHL) designation describes a diverse group cancers that collectively rank among top 10 most commonly worldwide. Although long-term survival trends are improving, there remains significant unmet clinical need for treatments to help patients with relapsed or refractory disease, one cause which is drug efflux through upregulation xenobiotic pumps, such as MDR1....

A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). fully human, recombinant antibody that binds uPAR to form a stable complex blocks uPA-uPAR interactions (2G10) and internalized primarily through endocytosis showed efficacy in mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) 2G10 were designed produced bearing tubulin inhibitor payloads ligated seven different linkers. Aldehyde tag technology...

Abstract Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle low-cost method with fluorophores that appears substantially more robust than similar but chemically distinct procedure. The employs the controlled enzymatic conversion central Cys to reactive formylglycine (fGly) aldehyde within six...

Antibody–drug conjugates (ADCs) are an established modality for the tissue-specific delivery of chemotherapeutics. However, due to hydrophobic nature many cytotoxic payloads, challenges remain in developing chemically stable ADCs with high drug loading. In previous studies, payload structure, unique stimuli-responsive chemistries, and PEGylated cross-linkers have been used decrease ADC hydrophobicity. this work, we investigate effect a new parameter, cross-linker sequence. A support-free...

Trafficking of the chitin synthase Chs2p from endoplasmic reticulum (ER) to bud-neck in late mitosis is tightly regulated by cell cycle via phosphorylation serine residues N-terminus protein. Here, we describe effects on interaction with coat protein complex II (COPII). Identification a cdc5ts mutant, which fails transport Chs2p-3xGFP and instead accumulates intracellular punctae, led us discover that at ER exit sites metaphase arrested wild-type cells. Using an vitro vesicle formation assay...

Trastuzumab and the related ADC, ado-trastuzumab emtansine (T-DM1), both target HER2-overexpressing cells. Together, these drugs have treatment indications in early-stage metastatic settings for HER2+ breast cancer. T-DM1 retains antibody functionalities of trastuzumab adds potency a cytotoxic maytansine payload. Interestingly, clinic, cannot always replace use plus chemotherapy administered together as single agents. We hypothesize that this failure may be due, part, to limited systemic...

The advantages of site-specific over stochastic bioconjugation technologies include homogeneity product, minimal perturbation protein structure/function, and – increasingly the ability to perform structure activity relationship studies at conjugate level. When selecting optimal location for payload placement, many researchers turn in silico modeling identify regions predicted offer solvent-exposed conjugatable sites while conserving function. Here, using aldehyde tag as our technology...

Abstract Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to target antigen-expressing cells and have become an important cancer treatment in recent years. However, the molecular mechanisms by which ADCs are internalized activated within remain unclear. Here we use CRISPR-Cas9 screens identify genes that control toxicity of ADCs. Our results demonstrate critical roles for a range known novel endolysosomal trafficking regulators ADC toxicity. We...