Single Mutation on Trastuzumab Modulates the Stability of Antibody-Drug Conjugates Built Using Acetal-Based Linkers and Thiol-Maleimide Chemistry.

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 Human and Mouse Islet Peptidome: Effects of Obesity and Type 2 Diabetes, and Assessment of Intraislet Production of Glucagon-like Peptide-1.

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.

Discordance between GLP-1R gene and protein expression in mouse pancreatic islet cells.

The insulinotropic actions of glucagon-like peptide 1 receptor (GLP-1R) in ß-cells have made it a useful target to manage type 2 diabetes. Metabolic stress reduces ß-cell sensitivity to GLP-1, yet the underlying mechanisms are unknown. We hypothesized that Glp1r expression is heterogeneous among ß-cells and that metabolic stress decreases the number of GLP-1R-positive ß-cells. Here, analyses of publicly available single-cell RNA-Seq sequencing (scRNASeq) data from mouse and human ß-cells indicated that significant populations of ß-cells do not express the Glp1r gene, 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 α-, ß-, and δ-cells. Experiments with Glp1r reporter mice and a validated GLP-1R antibody indicated that >90% of the ß-cells are GLP-1R positive, contradicting the findings with the scRNASeq data. α-cells did not express Glp1r mRNA and δ-cells expressed Glp1r mRNA but not protein. We also examined the expression patterns of GLP-1R in mouse models of metabolic stress. Multiparous female mice had significantly decreased ß-cell Glp1r expression, 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 ß-cells, absent in α-cells, and expressed at the mRNA, but not protein, level in δ-cells.

Interconversion of Unexpected Thiol States Affects the Stability, Structure, and Dynamics of Antibody Engineered for Site-Specific Conjugation.

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.

Acute health effects from exposure to indoor ultrafine particles-A randomized controlled crossover study among young mild asthmatics.

Particulate matter is linked to adverse health effects, however, little is known about health effects of particles emitted from typical indoor sources. We examined acute health effects of short-term exposure to emissions from cooking and candles among asthmatics. In a randomized controlled double-blinded crossover study, 36 young non-smoking asthmatics attended three exposure sessions lasting 5 h: (a) air mixed with emissions from cooking (fine particle mass concentration): (PM2.5 : 96.1 µg/m3 ), (b) air mixed with emissions from candles (PM2.5 : 89.8 µg/m3 ), and c) clean filtered air (PM2.5 : 5.8 µg/m3 ). Health effects (spirometry, fractional exhaled Nitric Oxide [FeNO], nasal volume and self-reported symptoms) were evaluated before exposure start, then 5 and 24 h after. During exposures volatile organic compounds (VOCs), particle size distributions, number concentrations and optical properties were measured. Generally, no statistically significant changes were observed in spirometry, FeNO, or nasal volume comparing cooking and candle exposures to clean air. In males, nasal volume and FeNO decreased after exposure to cooking and candles, respectively. Participants reported additional and more pronounced symptoms during exposure to cooking and candles compared to clean air. The results indicate that emissions from cooking and candles exert mild inflammation in asthmatic males and decrease comfort among asthmatic males and females.

A Platform for Site-Specific DNA-Antibody Bioconjugation by Using Benzoylacrylic-Labelled Oligonucleotides.

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.

Dichloro Butenediamides as Irreversible Site-Selective Protein Conjugation Reagent.

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.

Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry.

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.

Development and characterisation of a novel glucagon like peptide-1 receptor antibody.

AIMS/HYPOTHESIS: Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. METHODS: A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice. RESULTS: Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs. CONCLUSIONS/INTERPRETATION: Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood.

Use of CRISPR/Cas9-engineered INS-1 pancreatic ß cells to define the pharmacology of dual GIPR/GLP-1R agonists.

Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic ß-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant ß-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical.

High Affinity Recognition of a Selected Amino Acid Epitope within a Protein by Cucurbit[8]uril Complexation.

Supramolecular interactions between the host cucurbit[8]uril (CB[8]) and amino acids have been widely interrogated, but recognition of specific motifs within a protein domain have never been reported. A phage display approach was herein used to select motifs with the highest binding affinity for the heteroternary complex with methyl viologen and CB[8] (MV⋅CB[8]) within a vast pool of cyclic peptide sequences. From the selected motifs, an epitope consisting of three amino acid was extrapolated and incorporated into a solvent-exposed loop of a protein domain; the protein exhibited micromolar binding affinity for the MV⋅CB[8] complex, matching that of the cyclic peptide. By achieving selective CB[8]-mediated conjugation of a small molecule to a recombinant protein scaffold we pave the way to biomedical applications of this simple ternary system.

Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a Ki of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP.

The effect of air quality on sleep and cognitive performance in school children aged 10-12 years: a double-blinded, placebo-controlled, crossover trial.

OBJECTIVES: To investigate the effect of CO2 during sleep on next-morning cognitive performance in young schoolchildren, the authors performed a double-blind fully balanced crossover placebo-controlled study. MATERIAL AND METHODS: The authors included 36 children aged 10-12 years in the climate chamber. The children slept at 21°C in 6 groups each at 3 different conditions separated by 7 days in a random order. Conditions were as follows: high ventilation with CO2 at 700 ppm, high ventilation with added pure CO2 at 2000-3000 ppm, and reduced ventilation with CO2 at 2-3000 ppm and bioeffluents. Children were subjected to a digital cognitive test battery (CANTAB) in the evening prior to sleep and on the next morning after breakfast. Sleep quality was monitored with wrist actigraphs. RESULTS: There were no significant exposure effects on cognitive performance. Sleep efficiency was significantly lower at high ventilation with CO2 at 700 ppm which is considered to be a chance effect. No other effects were seen, and no relation between air quality during sleep and next-morning cognitive performance was observed in the children emitting an estimated 10 lCO2/h per child. CONCLUSIONS: No effect of CO2 during sleep was found on next day cognition. The children were awakened in the morning, and spent from 45-70 min in well-ventilated rooms before they were tested. Hence, it cannot be precluded that the children have benefitted from the good indoor air quality conditions before and during the testing period. The slightly better sleep efficiency during high CO2 concentrations might be a chance finding. Hence, replication is needed in actual bedrooms controlling for other external factors before any generalizations can be made. Int J Occup Med Environ Health. 2023;36(2):177-91.

Mechanistic insights into the rational design of masked antibodies.

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.

Controlled masking and targeted release of redox-cycling ortho-quinones via a C-C bond-cleaving 1,6-elimination.

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, ß-lapachone, we created cathepsin-B-cleavable quinone prodrugs. We applied the strategy for intracellular release of ß-lapachone upon antibody-mediated delivery. Conjugation of protected ß-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.

The Effect of Seasonal Priming on Specific Inhalation Challenges With Birch and Grass Allergen Among Persons With Allergic Rhinitis.

Objectives: Allergic diseases are prevalent in the working population, and work-related airborne pollen exposure might be substantial, especially among outdoor workers, resulting in work-exacerbated effects. Seasonal exposure to pollen may induce a priming effect on the allergic bronchial response resulting in exaggerated effects at the end of the natural pollen season. This was previously observed among people with asthma but may also be of importance for persons with allergic rhinitis. In this study, we examined the effect of seasonal priming on bronchial responsiveness among young adults with allergic rhinitis and no or mild asthma. In addition, we explored the association between the baseline characteristics of participants and the severity of bronchoconstriction. Finally, we evaluated the application of a novel non-linear regression model to the log-dose-response curves. Material and methods: In a crossover design, 36 participants underwent specific inhalation challenges (SICs) with either grass or birch allergen outside and at the end of the pollen season. The differences in bronchial response were evaluated by comparing the dose-response profiles and PD20 estimates derived by applying a non-linear regression model. Results: The results showed that 12 of the 19 grass pollen-exposed participants had a lower PD20 at the end of the season compared with the outside season. For birch, this was true for nine out of the 17 participants. However, no statistically significant effects of the seasonal pollen exposure were found on neither the shape nor the magnitude of the modeled dose-response curves for either birch allergen, p = 0.77, or grass allergen, p = 0.45. The model depicted a good fit for the data. Among the baseline characteristics, only the size of the skin prick test for grass allergen was associated with PD20. Conclusion: This study does not support a priming effect of pollen exposure on the bronchial response from the natural seasonal exposure levels of grass or birch allergens among young adults with allergic rhinitis.

Gut Hormone GIP Induces Inflammation and Insulin Resistance in the Hypothalamus.

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-ß 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 3ß 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.

An RCT of acute health effects in COPD-patients after passive vape exposure from e-cigarettes.

Background: E-cigarette use has been shown to have short-term acute effects among active users but less is known of the acute passive effects, particularly among individuals with existing respiratory diseases. Objective: To investigate local and systemic effects of short-term passive vape exposure among patients with mild or moderate chronic obstructive pulmonary disease (COPD). Methods: In a double-blinded crossover study 16 non-smoking COPD-patients (mean age 68) were randomly exposed for 4 h to passive vape (median PM2.5: 18 µg/m3 (range: 8-333)) and clean air (PM2.5 < 6 µg/m3) separated by 14 days. Particles were measured using an ultrafine particle counter (P-TRAK) and a scanning mobility particle sizer (SMPS). Health effects including Surfactant Protein-A (SP-A) and albumin in exhaled air, spirometry, FeNO, and plasma proteins were evaluated before, right after, and 24 hours after exposure. Participants reported symptoms throughout exposure sessions. Data were analyzed using mixed models. Results: SP-A in exhaled air was negatively affected by exposure to vape and several plasma proteins increased significantly. Throat irritation was more pronounced during passive vape exposure, while FVC and FEV1 decreased, however, not significantly. Conclusions: SP-A in exhaled air and some plasma proteins were affected by passive vape in patients with COPD indicating inflammation, showing that passive vape exposure is potentially harmful.

Pharmacological antagonism of the incretin system protects against diet-induced obesity.

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.