Cancer therapies remain limited by poor selectivity and off-target toxicity, making biodistribution as critical as the drugs themselves. Antibodies have transformed cancer treatment by enabling the selective delivery of highly potent pharmaceutical ingredients (APIs) to diseased tissues, thereby increasing efficacy while reducing systemic side effects. This targeted approach has revived the clinical use of cytotoxic agents, which were previously considered too toxic without precise biodistribution. Building on this principle, antibody–drug conjugates (ADCs) combine these powerful drugs with antibodies to expand the therapeutic window and achieve controlled delivery to specific tumor sites. However, the low drug-to-antibody ratio and hydrophobic payloads often compromise ADC stability and limit the dosing. Nanoparticles offer an alternative route to achieve high-capacity drug delivery with controlled release, but few platforms combine efficient targeting with stable, site-specific conjugation. Metal–organic frameworks (MOFs) are distinguished by their high porosity, modular chemistry, and high drug-loading capacity. However, their translation has been hindered by the challenge of conjugating proteins to their external surfaces in a stable and oriented manner. Herein, we report the first fragment antibody (Fab)- conjugated MOF nanoparticles, using a phosphate anchoring group on the zirconium-based MOF PCN-128. This site-specific conjugation ensures optimal Fab orientation for selective binding to HER2 receptors and efficient cellular internalization of the drug. We confirmed stable conjugation, specific HER2 recognition, and enhanced cytotoxicity when loaded with the potent payload MMAF. Together, these results establish Fab-functionalized MOFs as a versatile, high-capacity drug delivery platform and open a new path for next-generation targeted cancer therapy.

Actively targeted nanoparticle systems have the potential to improve delivery to tumors over untargeted systems however the design rules to achieve this have not been fully elucidated. A HER2-targeted polymer drug delivery system composed of a 32-arm star polymer (SD) conjugated with the TOP1 inhibitor molecule SN-38, with a trastuzumab antigen binding fragment (HER2-Fab), has been used to target cancer cells overexpressing this receptor. The HER2-Fab was attached to the SD at two different densities (average of 1 or 3 Fabs per star polymer) and compared to the native star polymer without Fab. In vitro experimentation showed that both the targeted star polymers (HER2-SDs) had better binding and uptake in HER2-positive cell lines (SK-BR3 and HEK293) compared to the non-targeted SD. In vivo biodistribution studies showed enhanced accumulation of HER2-targeted SDs in tumors, but not normal tissues, particularly at the later (96 h post-dose) timepoint. The HER2-SDs demonstrated increased localization with tumor cells rather than in stromal regions, greater penetration into the tumor core and a more homogenous distribution in the tumor section than the untargeted SD. The targeted star polymer conjugated to SN-38 was tested for anti-tumor activity in a HER2-positive gastric cancer xenograft in mice and showed significantly greater efficacy compared to untargeted SDs.

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvβ3.

Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.

Natural products that contain ortho-quinones show great potential as anticancer agents but have been largely discarded from clinical development because their redox-cycling behaviour results in general systemic toxicity. Here we report conjugation of ortho-quinones to a carrier, which simultaneously masks their underlying redox activity. C-benzylation at a quinone carbonyl forms a redox-inactive benzyl ketol. Upon a specific enzymatic trigger, an acid-promoted, self-immolative C-C bond-cleaving 1,6-elimination mechanism releases the redox-active hydroquinone inside cells. By using a 5-lipoxygenase modulator, β-lapachone, we created cathepsin-B-cleavable quinone prodrugs. We applied the strategy for intracellular release of β-lapachone upon antibody-mediated delivery. Conjugation of protected β-lapachone to Gem-IgG1 antibodies, which contain the variable region of gemtuzumab, results in homogeneous, systemically non-toxic and conditionally stable CD33+-specific antibody-drug conjugates with in vivo efficacy against a xenograft murine model of acute myeloid leukaemia. This protection strategy could allow the use of previously overlooked natural products as anticancer agents, thus extending the range of drugs available for next-generation targeted therapeutics.

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.

The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor that is predominantly expressed on pancreatic β-cells in mice. Endogenous and synthetic ligands for GLP-1R exhibit distinct intracellular signaling cascades and promote insulin secretion to various degrees. Ligand binding typically induces receptor internalization followed by recycling back to the membrane or degradation within the lysosome. We hypothesized that endogenous and synthetic GLP-1R ligands would have distinct rates of internalization and recycling in β-cells. To test this hypothesis, we combined a recently-validated, highly-specific GLP-1R antibody (Glp1r0017) with flow cytometry and applied ligands (GLP-1, exendin-4 [Ex4], glucagon, and tirzepatide [TZP]) to characterize GLP-1R trafficking in freshly-isolated mouse islet cells. Under basal conditions, ~90% of β-cells were GLP1R+, with very low or absent binding in α- and δ-cells. Increasing ligand concentrations (0.01-100 nM) demonstrated distinct rates of internalization between ligands. Most potent was Ex4, followed by the native ligand GLP-1. TZP, a dual-receptor ligand for GLP-1R and GIPR, was more modest in inducing internalization. While showing different potencies, Ex4, GLP-1, and TZP were able to induce maximal internalization. In contrast, maximal treatment with glucagon only resulted in ~70% of β-cells being GLP-1R+. To test the rate of recycling back to the plasma membrane, we treated islet cells with Ex4 or GLP-1 with a dose that induced ~50% internalization, removed the ligand, and then monitored GLP1R+ β-cells over time. Within the first 15 minutes, GLP1R+ β-cells increased linearly and rapidly. This plateaued from 30-60 minutes, resulting in 60-70% of β-cells being GLP1R+ at the end of the experiment. In summary, our assay allows for investigation of GLP-1R trafficking in live β-cells. Understanding into the factors that regulate GLP-1R internalization and recycling may shed light on the insulinotropic properties of these ligands. Disclosure S. M. Gray: None. K. Sloop: Employee; Self; Eli Lilly and Company. P. Ravn: None. J. Campbell: None. D. A. D’alessio: Advisory Panel; Self; Eli Lilly and Company, Sun Pharmaceutical Industries Ltd., Research Support; Self; Eli Lilly and Company, Merck & Co., Inc. Funding National Institutes of Health (F32DK121420, R01DK123075, R01DK101991); Lilly Research Award Program

To characterize the impact of metabolic disease on the peptidome of human and mouse pancreatic islets, LC-MS was used to analyze extracts of human and mouse islets, purified mouse alpha, beta, and delta cells, supernatants from mouse islet incubations, and plasma from patients with type 2 diabetes. Islets were obtained from healthy and type 2 diabetic human donors, and mice on chow or high fat diet. All major islet hormones were detected in lysed islets as well as numerous peptides from vesicular proteins including granins and processing enzymes. Glucose-dependent insulinotropic peptide (GIP) was not detectable. High fat diet modestly increased islet content of proinsulin-derived peptides in mice. Human diabetic islets contained increased content of proglucagon-derived peptides at the expense of insulin, but no evident prohormone processing defects. Diabetic plasma, however, contained increased ratios of proinsulin and des-31,32-proinsulin to insulin. Active GLP-1 was detectable in human and mouse islets but 100–1000-fold less abundant than glucagon. LC-MS offers advantages over antibody-based approaches for identifying exact peptide sequences, and revealed a shift toward islet insulin production in high fat fed mice, and toward proglucagon production in type 2 diabetes, with no evidence of systematic defective prohormone processing.

Many bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site-specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA-antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic-labelled oligonucleotides prepared with this reagent can be site-specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA-antibody bioconjugates was confirmed by a new LC-MS protocol and the bioconjugate probes were used in fluorescence or super-resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site-specific, well-defined and plasma-stable DNA-antibody bioconjugates for biological applications.

Antibody-drug conjugates have become one of the most actively developed classes of drugs in recent years. Their great potential comes from combining the strengths of large and small molecule therapeutics: the exquisite specificity of antibodies and the highly potent nature of cytotoxic compounds. More recently, the approach of engineering antibody-drug conjugate scaffolds to achieve highly controlled drug to antibody ratios has focused on substituting or inserting cysteines to facilitate site-specific conjugation. Herein, we characterize an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge during manufacture. A combination of mass spectrometry and biophysical techniques have been used to understand how the additional disulfide bridge forms, interconverts, and changes the stability and structural dynamics of the antibody intermediate. This quantitative and structurally resolved model of the local and global changes in structure and dynamics associated with the engineering and subsequent disulfide-bonded variant can assist future engineering strategies.

We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation.

The hypothalamus plays a critical role in controlling energy balance. High-fat diet (HFD) feeding increases the gene expression of proinflammatory mediators and decreases insulin actions in the hypothalamus. Here, we show that a gut-derived hormone, glucose-dependent insulinotropic polypeptide (GIP), whose levels are elevated during diet-induced obesity, promotes and mediates hypothalamic inflammation and insulin resistance during HFD-induced obesity. Unbiased ribonucleic acid sequencing of GIP-stimulated hypothalami revealed that hypothalamic pathways most affected by intracerebroventricular (ICV) GIP stimulation were related to inflammatory-related responses. Subsequent analysis demonstrated that GIP administered either peripherally or centrally, increased proinflammatory-related factors such as Il-6 and Socs3 in the hypothalamus, but not in the cortex of C57BL/6J male mice. Consistently, hypothalamic activation of IκB kinase-β inflammatory signaling was induced by ICV GIP. Further, hypothalamic levels of proinflammatory cytokines and Socs3 were significantly reduced by an antagonistic GIP receptor (GIPR) antibody and by GIPR deficiency. Additionally, centrally administered GIP reduced anorectic actions of insulin in the brain and diminished insulin-induced phosphorylation of Protein kinase B and Glycogen synthase kinase 3β in the hypothalamus. Collectively, these findings reveal a previously unrecognized role for brain GIP signaling in diet-induced inflammation and insulin resistance in the hypothalamus.

Objective: Glucose-dependent insulinotropic polypeptide is an intestinally derived hormone that is essential for normal metabolic regulation. Loss of the GIP receptor (GIPR) through genetic elimination or pharmacological antagonism reduces body weight and adiposity in the context of nutrient excess. Interrupting GIPR signaling also enhances the sensitivity of the receptor for the other incretin peptide, glucagon-like peptide 1 (GLP-1). The role of GLP-1 compensation in loss of GIPR signaling to protect against obesity has not been directly tested. Methods: We blocked the GIPR and GLP-1R with specific antibodies, alone and in combination, in healthy and diet-induced obese (DIO) mice. The primary outcome measure of these interventions was the effect on body weight and composition. Results: Antagonism of either the GIPR or GLP-1R system reduced food intake and weight gain during high-fat feeding and enhanced sensitivity to the alternative incretin signaling system. Combined antagonism of both GIPR and GLP-1R produced additive effects to mitigate DIO . Acute pharmacological studies using GIPR and GLP-1R agonists demonstrated both peptides reduced food intake, which was prevented by co-administration of the respective antagonists. Conclusions: Disruption of either axis of the incretin system protects against diet-induced obesity in mice. However, combined antagonism of both GIPR and GLP-1R produced additional protection against diet-induced obesity, suggesting additional factors beyond compensation by the complementary incretin axis. While antagonizing the GLP-1 system decreases weight gain, GLP-1R agonists are used clinically to target obesity. Hence, the phenotype arising from loss of function of GLP-1R does not implicate GLP-1 as an obesogenic hormone. By extension, caution is warranted in labeling GIP as an obesogenic hormone based on loss-of-function studies. (C) 2019 The Author(s). Published by Elsevier GmbH.

The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor that is insulinotropic in β-cells. Multiple endogenous and synthetic ligands exist for the GLP-1R and each ligand interacts with the receptor differently to produce specific activation of intracellular signaling pathways. Ligand binding typically induces receptor internalization, leading to either i) receptor degradation or ii) recycling to the cell membrane. We hypothesized that each specific GLP-1R ligand would produce a unique trafficking profile in primary β-cells. To test this hypothesis, we developed flow cytometry-based assay that can be utilized in primary dispersed mouse islets. First, we validated a GLP-1R antibody (GLP-1R-APC) for flow cytometry application. The GLP-1R-APC stained β-cells, but not α-cells, aligning with the reported expression pattern in islets. Post-sort analysis confirmed GLP-1R expression only in the GLP-1R-APC+ population. Finally, GLP-1R-APC did not stain any cell populations in GLP1R-/- islets. Next, we used this assay to quantify GLP-1R internalization in wild type β-cells. To induce internalization, we treated dispersed islets with either vehicle or a GLP-1R ligand for 30 minutes prior to antibody staining. After vehicle treatment, 90% of the β-cells stained GLP-1R-APC+. Treatment with either GLP-1 or exendin-4 reduced the number of GLP-1R-APC+ β-cells by 97 and 89%, respectively, suggesting near complete internalization of all GLP-1Rs. In contrast, treatment with the GLP-1R antagonist exendin-9 produced staining similar to PBS controls. Finally, we used image-stream technology, a combination of brightfield microscopy and flow cytometry, to visually confirm GLP-1R internalization. Here, vehicle-treated cells produced GLP-1R staining only on the cell membrane, which was nearly ablated by the treatment with a ligand. In conclusion, we have developed an assay that allows for quantification of GLP-1R trafficking in primary mouse islets. Disclosure S.M. Gray: None. P. Ravn: Employee; Self; AstraZeneca. K. Sloop: None. J. Campbell: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. D. D’Alessio: Advisory Panel; Self; Eli Lilly and Company. Consultant; Self; Intarcia Therapeutics. Research Support; Self; Ansh Labs, Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novo Nordisk A/S.

The glucagon-like peptide 1 receptor (GLP-1R) is insulinotropic in β-cells and the target of multiple classes of diabetes drugs. People with T2D secrete normal amounts of GLP-1, but are less responsive to its actions; the mechanisms for this are unknown. Studies have shown that metabolic stress decreases GLP-1R in islets, however, it is unknown if this occurs in all, or just a subset, of β-cells. We hypothesized that GLP-1R expression is heterogeneous in β-cells, such that not all β-cells express the GLP-1R, and that metabolic stress would decrease the number of GLP-1R+ β-cells. To test this, we first performed single-cell RNA sequencing (scRNAseq) in mouse and human islet cells and found that the majority of β-cells were GLP-1R negative, supporting significant heterogeneity. To validate the scRNAseq results, we generated a GLP-1R reporter mouse by crossing GLP-1R-Cre with mTmG mice. Using FACS to separate and qPCR to characterize positive (GFP+) from negative (tdTomato+) dispersed islet cells, we found GLP-1R+ cells were enriched for Ins2 and Sst, while GLP-1R- cells were enriched for Gcg, suggesting GLP-1R promoter activity is localized to β- and d-cells. In addition, β-cell markers were not present in the tdTomato+ population, suggesting very few GLP-1R negative β-cells. Next, we stained dispersed islets from the reporter mice with a validated GLP-1R antibody and found >90% congruence with GFP+ cells and no staining in tdTomato+ cells. In dispersed islets from wild type mice, ∼90% of β-cells were GLP-1R+; very few of the α- or d-cells were GLP-1R+. Metabolic stress induced through chronic high-fat feeding or using multiparous models did not alter the number of GLP-1R+ β-cells. In conclusion, assessment of GLP-1R expression and GLP-1R protein revealed the majority of β-cells are GLP-1R+, a finding that conflicts with the interpretation of scRNAseq data. These findings suggest caution in interpreting the results of scRNAseq for low abundant transcripts such as the GLP-1R. Disclosure S.M. Gray: None. B.M. Chazotte: None. E.C. Ross: None. B. Svendsen: None. P. Ravn: Employee; Self; AstraZeneca. K. Sloop: None. J. Campbell: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. D. D’Alessio: Advisory Panel; Self; Eli Lilly and Company. Consultant; Self; Intarcia Therapeutics. Research Support; Self; Ansh Labs, Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novo Nordisk A/S.

The insulinotropic actions of glucagon-like peptide 1 receptor (GLP-1R) in beta-cells have made it a useful target to manage type 2 diabetes. Metabolic stress reduces beta-cell sensitivity to GLP-1, yet the underlying mechanisms are unknown. We hypothesized thatGlp1rexpression is heterogeneous among beta-cells and that metabolic stress decreases the number of GLP-1R-positive beta-cells. Here, analyses of publicly available single-cell RNA-Seq sequencing (scRNASeq) data from mouse and human beta-cells indicated that significant populations of beta-cells do not express theGlp1rgene, supporting heterogeneous GLP-1R expression. To check these results, we used complementary approaches employing FACS coupled with quantitative RT-PCR, a validated GLP-1R antibody, and flow cytometry to quantify GLP-1R promoter activity, gene expression, and protein expression in mouse alpha-, beta-, and delta-cells. Experiments withGlp1rreporter mice and a validated GLP-1R antibody indicated that >90% of the beta-cells are GLP-1R positive, contradicting the findings with the scRNASeq data. alpha-cells did not expressGlp1rmRNA and delta-cells expressedGlp1rmRNA but not protein. We also examined the expression patterns of GLP-1R in mouse models of metabolic stress. Multiparous female mice had significantly decreased beta-cellGlp1rexpression, but no reduction in GLP-1R protein levels or GLP-1R-mediated insulin secretion. These findings suggest caution in interpreting the results of scRNASeq for low-abundance transcripts such as the incretin receptors and indicate that GLP-1R is widely expressed in beta-cells, absent in alpha-cells, and expressed at the mRNA, but not protein, level in delta-cells.

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceutical products for oncology, with the cytotoxic pyrrolobenzodiazepine (PBD) family of "warheads" well-established in the clinic. While PBDs offer high potency, they are also characterized by their hydrophobicity, which can make formulation of the ADC challenging. Several approaches have been investigated to improve the physicochemical properties of PBD-containing ADCs, and herein a supramolecular approach was explored using cucurbit[8]uril (CB[8]). The ability of CB[8] to simultaneously encapsulate two guests was exploited to incorporate a 12-mer polyethylene glycol harboring a methyl viologen moiety at one terminus (MV-PEG12), together with a PBD harboring an indole moiety at the C2' position (SG3811). This formulation approach successfully introduced a hydrophilic PEG to mask the hydrophobicity of SG3811, improving the physical stability of the ADC while avoiding any loss of potency related to chemical modification.