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Abstract 5292: CD28 humanized mouse model for efficacy and safety assessment of CD28-targeting therapies

Abstract Agonist antibodies targeting CD28 have proven to be effective against cancer, but also faced challenges due to severe adverse events triggered by its activation. Human (hCD28) and mouse CD28 (mCD28) have different signaling responses, with CD28 ligands and superagonists inducing pro-inflammatory cytokines upon stimulation in absence of TCR ligation in humans, but not in mice. Expression of CD28i amplifier isoform, which is thought to enhanced the production of cytokines in humans, could partially explain this difference, as it is not expressed in mice. In addition, evidence suggests that the different signaling between hCD28 and mCD28 relies on one amino acid change in the intracellular domain (ICD)[1]. Herein, we describe a CD28 humanized mouse model for assessment of CD28-targeting agents. To improve translatability, we decided to keep the expression of both canonical and CD28i human isoforms to avoid undermining the biological effects of the testing agents. Although keeping the human ICD could favor the evaluation of cytokine production and therefore the safety of the test agents, we decided to keep the mouse ICD to enable a proper interaction of CD28 with its signaling partners, allowing a physiological stimulation of CD28 in efficacy studies. The hCD28 model expresses a chimeric protein, with the whole human extracellular domain, and mouse intracellular domain. Human CD28 is expressed at physiological levels on T cells, and these cells can be activated and proliferate in response to hCD28 ligands and agonist antibodies. Tumor growth inhibition and complete tumor regressions are achieved in a MC38-TAA colorectal cancer model following prophylactic treatment with CD28-TAA bispecific antibody in combination with anti-PD-1. Extension of this study and re-challenged of the tumor-free surviving animals shows that re-challenged hCD28 mice have enhanced survival compared to naïve hCD28 mice inoculated with MC38-TAA, suggesting tumor immunological memory. Furthermore, TGN1412 administration in CD28 mice induces cytokine release, and a slightly body weight loss, but no major change in body temperature. Although the model does not fully reproduce the ability of human CD28 to produce cytokines upon activation due to the mouse ICD, the expression of the amplifier isoform CD28i might contribute to the response to TGN1412. Altogether, data suggest that hCD28 model enables assessment of efficacy and safety of CD28-targeting agents. The hCD28 model was intercrossed with CD3 humanized models to enable assessment of combination therapies and bispecific antibodies targeting both CD3 and CD28. [1] Porciello N, Grazioli P, Campese AF, et al. A non-conserved amino acid variant regulates differential signalling between human and mouse CD28. Nat Commun 2018; 9:1-16 Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Yacine Cherifi, Patricia Isnard-Petit, Kader Thiam. CD28 humanized mouse model for efficacy and safety assessment of CD28-targeting therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5292.

The activation of the adaptor protein STING depends on its interactions with the phospholipid PI4P.

Activation of the endoplasmic reticulum (ER)-resident adaptor protein STING, a component of a cytosolic DNA-sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins. Appropriate localization and activation of STING at the Golgi apparatus required ACBD3 and the PI4P-generating kinase PI4KB. In contrast, STING activation was enhanced when the lipid-shuttling protein OSBP, which removes PI4P from the Golgi apparatus, was inhibited by the US Food and Drug Administration-approved antifungal itraconazole. The increase in the abundance of STING-activating phospholipids at the trans-Golgi network resulted in the increased production of IFN-β and other cytokines in THP-1 cells. Furthermore, a mutant STING that could not bind to PI4P failed to traffic from the ER to the Golgi apparatus in response to a STING agonist, whereas forced relocalization of STING to PI4P-enriched areas elicited STING activation in the absence of stimulation with a STING agonist. Thus, PI4P is critical for STING activation, and manipulating PI4P abundance may therapeutically modulate STING-dependent immune responses.

Heterozygous Variants in the Clpbgene Associated with Neutropenia Affect Neutrophil Function

Introduction The CLPB gene encodes a caseinolytic peptidase belonging to the ATPase family and acts as a mitochondrial chaperone. While biallelic mutations in CLPB are associated with the 3-methylglutaconic aciduria and severe neutropenia, (MIM number: 616271 3), some heterozygous variants seem to induce neutropenia exclusively. In the current study, we evaluated for the first time a function of neutrophils among neutropenic carriers of CLPB mutation. Methods Among 282 individuals with neutropenia studied with targeted Next-Generation Sequencing (panel of 725 genes related to hematological disorders), three neutropenic probands with heterozygous CLPB amino acid substitutions were identified. Neutrophils from patients and control healthy donors were isolated from peripheral blood with a Pancoll density gradient. The percentages of freshly isolated neutrophils undergoing apoptosis or necrosis were determined by flow cytometry using Annexin V - FITC and Propidium Iodide (PI) staining. Phagocytic capacity was evaluated using pHrodo Green S. aureus BioParticles Conjugates. A Boyden's chamber model was used to determine the level of the neutrophil's mobility and chemotaxis response to 10% human serum. Formation of Neutrophils Extracellular Traps (NETs) and induction of the oxidative burst were studied upon phorbol 12-myristate 13-acetate (PMA) stimulation (25 nM) with SYTOX™ Green Nucleic Acid and flow cytometry with CellROX Green assay, respectively. The intracellular and circulating concentrations of anti-microbial compounds (Myeloperoxidase MPO, Lactotransferrin LTF, Cathelicidin-LL37, and defensins) were measured using commercially available ELISA assays. The results were evaluated with an unpaired t-test using GraphPad Prism 9. Quantitative data presented as medians (25%-75%). Results Genetic analysis identified three heterozygous variants of CLPB gene p.Arg327Trp, p.Arg628Cys, p .Arg629Cys in three different families. Two of these mutations were novel p.Arg327Trp and p .Arg629Cys. Interestingly, the p.Arg327Trp variant was located outside the ATPase domain. In total 7 family members who suffered from neutropenia and were carriers of the CLPB variants were available for neutrophil functional assays (Figure 1). Neutrophils collected from CLPB-deficient individuals have a higher rate of apoptosis as compared to healthy neutrophils: 57(52-66)% vs 23(20-27)%, P<0.0001, and necrosis: 0.56(0.31-1.07)% vs 0.13(0.10-0.21)%, P=0.02. CLPB-deficient neutrophils display reduced mobility in the absence of stimulation: 298(191-413) vs 1011(936-1201) migrating neutrophils, P<0.0001, and after 10% human serum treatment: 2090(1746-2458) vs 3736(3071-4828) migrating neutrophils, P=0.0014. Similarly, reduced phagocytosis of S. aureus bioparticles was observed in CLPB-deficient neutrophils: 1890(1679-2063) vs 3352(2472-4440) Fluorescence Units, respectively, P=0.0029. Upon PMA stimulation, neutrophils from CLPB patients produced less reactive oxygen species compared to controls: 311(175-542)FMI vs 775(766-1150)FMI, P=0.08, and their ability to produce NETs is almost entirely abolished: 0.5(0.21-0.79)ng/µl vs 1.3(1.08-3.05)ng/µl, P=0.0075. Moreover, CLPB-deficient neutrophils have reduced intracellular concentration of myeloperoxidase: 35(31-39)ng/mg vs 55(41-63)ng/mg, P=0.0043 and cathelicidin LL-37: 5.5(5-6.3)ng/µg vs 19.22(14.21-23.28)ng/µg, P=0.0003. The concertation of the LL-37 is also reduced in patients' plasma 1.34(0.84-1.78)ng/ml vs 7.17(5.38-8.03)ng/ml, P<0.0001. Both intracellular and circulating concentrations of LTF and defensins did not differ between study groups. Conclusion Neutrophils from neutropenic carriers of CLPB mutations are functionally impaired, especially concerning their ability for NETs formation and cathelicidin LL-37 production.

Modeling cardiac injury using human cardiac slices to test therapeutic approaches for cardiac regeneration and repair

Abstract Background Due to limited regenerative capacity of the adult human heart, myocardial infarction causes irreversible damage by inducing the death of cardiomyocytes and the formation of a fibrotic scar. These remodeling mechanisms cause loss of contractility and eventually heart failure ensues in a significant proportion of patients. Purpose There is a need for a reliable model that can replicate the adult cardiac pathophysiology in order to test treatments against cardiac remodeling. Here, we developed a cardiac tissue culture model that can simulate in vitro the molecular changes occurring after cardiac injury using human cultured cardiac slices. Methods We obtained cardiac slices by sectioning ventricular myocardium from the surgical waste material of adult patients undergoing valve surgery or Morrow myectomy. Cardiac slices were cultured at a liquid-air interface and were viable up to 7 days of culture, even in the absence of stimulation. During this time cardiac slices underwent mechanical and chemical treatments to induce ischemia and/or local injury. Results We found that following these treatments we were able to simulate cardiac remodeling in terms of cardiomyocyte death, activation of cardiac fibroblasts and induction of inflammation and oxidative stress. Following small molecule screening, we discovered that addition of compounds with anti-inflammatory properties (e.g. dexamethasone) or targeting the TGF-β pathway (e.g. SB-431542) into our culture medium helped in preserving the microscopic structure of the slices. Additionally, we found that treatment with these compounds protected cardiac slices against remodeling following ischemic injury. Conclusions In conclusion, our model can emulate cardiac pathophysiology in culture, providing us a reliable platform to test therapies for cardiac regeneration and repair in an adult, multicellular, 3-D environment.

Surfactant Enhanced Oil Recovery Improves Oil Recovery in a Depleted Eagle Ford Unconventional Well: A Case Study

Summary A simple huff “n” puff (HnP) injection and flowback using a nonionic surfactant solution to drive enhanced oil recovery (EOR) in a depleted Eagle Ford “black oil” unconventional well has been executed and analyzed. The pilot injection was performed in December 2020, with pressures below the estimated fracture gradient. More than 12,300 bbl of surfactant solution were injected into the 6,000-ft lateral. In January 2021, the well was put back on production with oil and water flow rate data being gathered and samples collected. Within 3 months of the well being put back onto production after surfactant stimulation, the well produced at oil rates over five times what it had produced before stimulation. The current oil rates (through October 2022; 22 months after stimulation) are still twice the prestimulation rates. Using a long-term hyperbolic fit to historical data as the “most likely” production scenario in the absence of stimulation as a “baseline,” incremental recovery was estimated using the actual oil production data to date. Economic analysis with prevailing West Texas Intermediate (i.e., WTI) prices at the time of production and the known costs of the pilot result in project payout time less than 1 year and project internal rate of return in excess of 80%, with only incremental production to date. These results prove the potential for technoeconomic viability of HnP EOR techniques using surfactants for wettability alteration in depleted unconventional oil wells. The well was chosen from a portfolio of unconventional Eagle Ford black oil window wells that were completed in the 2012–2014 time frame. The goal of the test was to demonstrate successful application of laboratory work to the field and economic viability of surfactant-driven water imbibition as a means of incremental EOR. The field design was based on laboratory work completed on oil and brine samples from the well of interest, with rock sampled from a nearby well at the same depth. The technical and economic objectives of the field test were to (1) inject surfactant solution to contact sufficient matrix surface area that measurable and economically attractive amounts of oil could be mobilized, (2) measure the amount of surfactant produced in the flowback stream to determine the amount of surfactant retained in the reservoir, and (3) prove the concept of using wettability alteration in conjunction with residual well energy in a depleted well to achieve economically attractive incremental recovery. Surfactant selection was completed in the laboratory using oil and brine gathered from potential target wells, and rock from nearby wells completed in the same strata. Several surfactant formulations were tested, and a final nonionic formulation was chosen on the basis of favorable wettability alteration and improved spontaneous imbibition recovery. The design for the pilot relied on rules of thumb derived from unconventional completion parameters. Rates, pressures, and injectant composition were carefully controlled for the single-day “bullhead” injection. Soak time between injection and post-stimulation restart of production was inferred from laboratory-scale imbibition trials. Post-stimulation samples were gathered, while daily oil and water rates were monitored since production restart. Flowback samples were analyzed for total dissolved solids (TDS), ions, and surfactant concentration.

Prevention of Type 1 Diabetes via a Lipid Nanoparticle-Based JAK inhibitor Treatment that Involves Anergy Formation

Abstract Type 1 diabetes is an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. The inhibition of inflammatory cytokines signaling via JAK inhibitors like Tofacitinib (Tofa) has shown promise in the prevention of this disease. To improve on drug bioavailability and reduce deleterious systemic exposure, we identified a lipid nanoparticle (LNp) formulation with favorable Tofa encapsulation efficiency and negligible cellular toxicity. Live animal imaging indicated these particles have the unique property of accumulating in lymphoid tissues, particularly in pancreatic and mesenteric lymph nodes when administrated via oral gavage. Spectral flow analysis revealed that multiple immune cells, like dendritic cells, T cells, and B cells, uptake the particles in these tissues. Short-term administration (5 gavages once every other day) of Tofa-LNp early (3-week-old) or late (10-week-old) in disease prone NOD mice promoted a significant delay of disease onset. Importantly, the therapeutic effect was associated with a profound increase in anergic CD4 T cells in the draining sites of LNp. Anergic cells have been known to form following stimulation in absence of co-stimulation, however, additional in vitro stimulation assays suggest a distinctive path of anergy induction promoted by blocking cytokine signaling. Overall, our results suggest an unexpected and novel immunomodulatory effect of JAK inhibition that warrants further investigation for the development of an effective treatment of type 1 diabetes.

Modulation of calcium signaling “on demand” to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. Over the past two decades, major advances have been made in our understanding of the disease with the identification of germline or somatic mutations in ion channels and pumps. These mutations enhance calcium signaling, the main trigger of aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, thus mimicking some of the known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the a7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3a named a7-5HT3. Its activation by a selective agonist named PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular sodium and calcium concentrations, cell proliferation, steroid production electrophysiological properties, and gene expression. Treatment of a7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10–9 to 10–5 M) induced a significant increase in intracellular calcium concentrations. Interestingly, whereas cells were hyperpolarized in absence of stimulation (around –60 mV), PSEM-817 induced a strong depolarization, with cells rising to a membrane potential of around –10 mV. The stimulation of calcium signaling by PSEM-817 leads to an increase of CYP11B2 (encoding for aldosterone synthase) mRNA expression and aldosterone biosynthesis but did not affect cell proliferation measured at different time points (8 h, 24 h, 48 h, 72 h). Interestingly, calcium kinetic analyses revealed significant differences in response to the modulation of potassium or sodium entry into cells. Gene expression analyses of gene expression by RNAseq highlighted the activation of specific signaling pathways in response to treatment with Angiotensin II, potassium, and PSEM-817. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Behavioral context improves optogenetic stimulation of transplanted dopaminergic cells in unilateral 6-OHDA rats

Stem cell therapy has long been a popular method of treatment for Parkinson’s disease currently being researched in both preclinical and clinical settings. While early clinical results are based upon fetal tissue transplants rather than stem cell transplants, the lack of successful integration in some patients and gradual loss of effect in others suggests a more robust protocol is needed. We propose a two-front approach, one where transplants are directly stimulated in coordination with host activity elicited by behavioral tasks, which we refer to as behavioral context. After a pilot with unilateral 6-OHDA rats transplanted with dopaminergic cells differentiated from mesenchymal stem cells that were optogenetically stimulated during a swim task, we discovered that early stimulation predicted lasting reduction of motor deficits, even in the absence of later stimulation. This led to a follow-up with n = 21 rats split into three groups: one stimulated while performing a swim task (Stim-Swim; St-Sw), one not stimulated while swimming (NoStim-Swim; NSt-Sw), and one stimulated while stationary in a bowl (Stim-NoSwim; St-NSw). After initial stimulation (or lack thereof), all rats were retested two and seven days later with the swim task in the absence of stimulation. The St-Sw group gradually achieved and maintained symmetrical limb use, whereas the NSt-Sw group showed persistent asymmetry and the St-NSw group showed mixed results. This supports the notion that stem cell therapy should integrate targeted stimulation of the transplant with behavioral stimulation of the host tissue to encourage proper functional integration of the graft.

Forward genetic screen of homeostatic antibody levels in the Collaborative Cross identifies MBD1 as a novel regulator of B cell homeostasis.

Variation in immune homeostasis, the state in which the immune system is maintained in the absence of stimulation, is highly variable across populations. This variation is attributed to both genetic and environmental factors. However, the identity and function of specific regulators have been difficult to identify in humans. We evaluated homeostatic antibody levels in the serum of the Collaborative Cross (CC) mouse genetic reference population. We found heritable variation in all antibody isotypes and subtypes measured. We identified 4 quantitative trait loci (QTL) associated with 3 IgG subtypes: IgG1, IgG2b, and IgG2c. While 3 of these QTL map to genome regions of known immunological significance (major histocompatibility and immunoglobulin heavy chain locus), Qih1 (associated with variation in IgG1) mapped to a novel locus on Chromosome 18. We further associated this locus with B cell proportions in the spleen and identify Methyl-CpG binding domain protein 1 under this locus as a novel regulator of homeostatic IgG1 levels in the serum and marginal zone B cells (MZB) in the spleen, consistent with a role in MZB differentiation to antibody secreting cells.

1406. IMPACT (Infection Management Plus Addiction Care Together): A Combined Contingency Management Pilot for Substance Use Disorders & Antibiotic Adherence in the Acute Care Setting

Abstract Background Amidst interrelated problems of increasing infections related to drug use and overdose deaths, contingency management (CM) is an underutilized substance use disorder treatment that leverages incentives for objective behavior change. CM implementation outside of drug treatment settings is limited, despite its regard as gold-standard treatment for stimulant use disorder and potential use to support infection treatment completion. Objective: to describe feasibility and preliminary effectiveness of a novel CM program incentivizing reduced drug use and antibiotic adherence in the acute care setting. Methods We conducted a pilot of twice weekly CM in an urban public hospital and its attached skilled nursing facility with escalating opportunities to earn incentives from a fishbowl based on 1) antibiotic adherence and/or 2) absence of stimulants or opioids on urine drug testing. Eligible participants were people with stimulant and/or opioid use disorders hospitalized for at least 2 weeks of infection treatment. We measured feasibility via visits attempted/completed and cost of gift cards dispensed. We evaluated effectiveness via antibiotic completion, discharge type, and participant perception of intervention effectiveness collected via structured survey. Results Between March-April 2022, n=9 participants were referred to IMPACT, and n=7 were enrolled. Most participants (5/7) required antibiotics for osteomyelitis, and most had stimulant use disorder (methamphetamine, 5/7) and opioid use disorder (fentanyl, 5/7). In 4 of 7 instances, multiple visits were necessary to complete a CM visit, and participants earned between $10-$350 during intervention. Only 1 participant self-directed discharge without antibiotic completion. Most participants reported CM “extremely effective” in supporting antibiotic completion, though were more likely to describe CM as moderately effective (range 5-8/10) in addressing drug use reduction. Conclusion CM for patients with stimulant and/or opioid use disorders needing prolonged antibiotics in acute care settings may be effective in supporting antibiotic completion, and additional exploration is needed to understand both program feasibility and its role in supporting reduced drug use for participants after discharge. Disclosures All Authors: No reported disclosures.

Missense mutations in inositol 1,4,5-trisphosphate receptor type 3 result in leaky Ca2+ channels and activation of store-operated Ca2+ entry.

Mutations in all subtypes of the inositol 1,4,5-trisphosphate receptor Ca2+ release channel are associated with human diseases. In this report, we investigated the functionality of three neuropathy-associated missense mutations in IP3R3 (V615M, T1424M, and R2524C). The mutants only exhibited function when highly over-expressed compared to endogenous hIP3R3. All variants resulted in elevated basal cytosolic Ca2+ levels, decreased endoplasmic reticulum Ca2+ store content, and constitutive store-operated Ca2+ entry in the absence of any stimuli, consistent with a leaky IP3R channel pore. These variants differed in channel function; when stably over-expressed the R2524C mutant was essentially dead, V615M was poorly functional, and T1424M exhibited activity greater than that of the corresponding wild-type following threshold stimulation. These results demonstrate that a common feature of these mutations is decreased IP3R3 function. In addition, these mutations exhibit a novel phenotype manifested as a constitutively open channel, which inappropriately gates SOCE in the absence of stimulation.

7952 Controlled Ovarian Stimulation with LNG-Ius in Situ in Women with Atypical Endometrial Lesions, Undergoing Fertility-Sparing Treatment

<h3>Study Objective</h3> to demonstrate safety of ovarian stimulation with 52mg Levonorgestrel Intrauterine System (LNG-IUS) <i>in situ</i> in patients conservatively treated for atypical endometrial lesions <h3>Design</h3> prospective observational study. <h3>Setting</h3> <b>G</b>ynecology department of University Federico II, Naples, Italy. <h3>Patients or Participants</h3> young women with atypical endometrial hyperplasia (AEH),or FIGO IA G1-G2 endometrial cancer(EC-G1,EC-G2),who underwent fertility-sparing treatment and ovarian stimulation with LNG-IUS <i>in situ.</i> <h3>Interventions</h3> combined fertility-sparing treatment was performed:EC-G1 and EC-G2 were treated by three steps hysteroscopic technique, AEH by superficial endometrial resection. LNG-IUS was inserted after surgery. Hysteroscopic endometrial biopsies were performed at 3 and 6 months. If complete response (CR) was achieved, controlled ovarian stimulation with LNG-IUS <i>in situ</i> was started, oocytes retrieval performed, and mature oocytes cryopreserved. After removal of LNG-IUS, embryo transfer was performed. <h3>Measurements and Main Results</h3> 23 young women with AEH(n=17), EC-G1(n=3), or EC-G2(n=3) were enrolled. CR was achieved in 14/16(87.5%) of AEH, 2/2(100%) of EC-G1 and 2/3(66.7%) of EC-G2 at 3-month follow up and in 11/14(78.6%) of AEH, 2/2(100%) of EC-G1 and 2/2(100%) of EC-G2 at 6-month follow up. 10 patients underwent ovarian stimulation with LNG-IUS <i>in situ</i> (experimental group) and compared with 10 infertile patients undergoing ovarian stimulation in absence of LNG-IUS (control group). The mean number of oocytes retrieved and mature oocytes was 8.10(± 2.77 SD) and 7.00(± 2.11 SD) in experimental group, compared with 6.30(± 3.3 SD) and 4.88(± 2.68 SD) in control group. There were no statistically significant differences in oocytes retrieved(p=0.19) and mature oocytes(p=0.05) between the two groups. Reproductive outcome (pregnancies and live birth rate) was also assessed. Pregnancy rate was 50%, live birth rate 37.5% and miscarriage rate 12.5%; 50% of patients experienced implantation failure. <h3>Conclusion</h3> presence of LNG-IUS during ovarian stimulation has no negative effects on oocyte quality, balancing the potentially dangerous effect of hyperestrogenism on patients with atypical endometrial lesions. The combined approach confirmed as a safe and effective fertility-sparing approach. Promising pregnancy outcomes can be expected.

Selective blockade of rat brain T-type calcium channels provides insights on neurophysiological basis of arousal dependent resting state functional magnetic resonance imaging signals.

A number of studies point to slow (0.1–2 Hz) brain rhythms as the basis for the resting-state functional magnetic resonance imaging (rsfMRI) signal. Slow waves exist in the absence of stimulation, propagate across the cortex, and are strongly modulated by vigilance similar to large portions of the rsfMRI signal. However, it is not clear if slow rhythms serve as the basis of all neural activity reflected in rsfMRI signals, or just the vigilance-dependent components. The rsfMRI data exhibit quasi-periodic patterns (QPPs) that appear to increase in strength with decreasing vigilance and propagate across the brain similar to slow rhythms. These QPPs can complicate the estimation of functional connectivity (FC) via rsfMRI, either by existing as unmodeled signal or by inducing additional wide-spread correlation between voxel-time courses of functionally connected brain regions. In this study, we examined the relationship between cortical slow rhythms and the rsfMRI signal, using a well-established pharmacological model of slow wave suppression. Suppression of cortical slow rhythms led to significant reduction in the amplitude of QPPs but increased rsfMRI measures of intrinsic FC in rats. The results suggest that cortical slow rhythms serve as the basis of only the vigilance-dependent components (e.g., QPPs) of rsfMRI signals. Further attenuation of these non-specific signals enhances delineation of brain functional networks.

Abstract ND06: ABBV-CLS-484: An active site PTPN2/N1 inhibitor that augments the immune response and sensitizes tumors to immune-mediated killing

Abstract Background: Pharmacologic inhibition of PTPN2 and PTPN1 (PTPN2/N1) represents a novel therapeutic approach in immuno-oncology that augments innate and adaptive immune responses in addition to enhancing tumor cell sensitivity to immune-mediated killing. PTPN2/N1 emerged as top hits in an in vivo CRISPR screen to identify tumor-intrinsic targets that enhance sensitivity and overcome resistance to anti-PD-1 treatment. PTPN2/N1 are phosphatases that act as negative regulators in numerous pathways including immune activation. While phosphatases have long been of interest, they are challenging drug targets, and the active site had been considered undruggable. Results: Here we report the discovery of the highly selective, active site PTPN2/N1 small molecule inhibitor, ABBV-CLS-484. Highly optimized ligand-protein interactions have led to the design of sub-nanomolar PTPN2/N1 inhibitors, confirmed through x-ray crystallography. PTPN2/N1 inhibitors increase the activation and function of cytotoxic T cells as well as increase the pro-inflammatory properties of CD103+ dendritic cells and macrophages in vitro. However, they do not cause non-specific activation in the absence of stimulation; rather, they augment signaling in cells that are already activated. PTPN2/N1 inhibition also has effects directly on tumor cells, where it amplifies sensitivity to immune-mediated killing by enhancing the interferon response. ABBV-CLS-484 promotes anti-tumor immunity as monotherapy and in combination with anti-PD-1 leading to dramatic tumor regression, even in models resistant to anti-PD-1 treatment such as 4T1, or those with minimal inflammation such as EMT6. Single-cell RNAseq analyses of tumor-infiltrating immune cells confirmed activation of T cells and demonstrated switching of myeloid-derived suppressor cells towards a proinflammatory phenotype, thereby revealing a distinct mechanism of action of ABBV-CLS-484 compared with PD-1 blockade. Our results show that PTPN2/N1 inhibitors have complementary effects on the immune system and tumor microenvironment that act to promote effective tumor killing. Based on these robust preclinical data, phase I clinical trials of ABBV-CLS-484 alone and in combination with an anti-PD-1 agent have been initiated to establish the safety, tolerability, and efficacy in diverse solid tumor indications. Conclusions: We have discovered a first-in-class PTPN2/N1 inhibitor, which represents a promising novel immunotherapy that both enhances the immune response and increases tumor sensitivity to immune-mediated killing. ABBV-CLS-484 is currently being evaluated in phase I clinical trials in patients with advanced solid tumors, as a monotherapy or in combination with a PD-1 targeting agent (NCT04777994). Citation Format: Christina K. Baumgartner, Marcia N. Paddock, Jennifer M. Frost, Keith M. Hamel, Kathleen A. McGuire, Kyle Halliwill, Zhaoming Xiong, Liang Mu, Kelly Klinge, Prasanthi Geda, Jaqueline Aguado, Marinka Bulic, Elliot P. Farney, Kathleen B. Yates, Robert T. Manguso, Clay Beauregard, Philip R. Kym. ABBV-CLS-484: An active site PTPN2/N1 inhibitor that augments the immune response and sensitizes tumors to immune-mediated killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND06.

DEVELOPMENT OF AN ADRENOCORTICAL CELL MODEL OF CALCIUM SIGNALING MODULATION TO DECIPHER THE MOLECULAR MECHANISMS RESPONSIBLE FOR PRIMARY ALDOSTERONISM

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. The identification of germline or somatic mutations in different genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H and CLCN2) and ATPases (ATP1A1 and ATP2B3) defines PA as a channelopathy. These mutations promote activation of calcium signaling, the main trigger for aldosterone biosynthesis.The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, mimicking some of known mutations identified in PA.We have developed an adrenocortical H295R_S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3α and named α7-5HT3. Mutations have been introduced in the ligand binding domain to allow only synthetic drugs to activate this channel receptor. Activation of α7-5HT3 by a specific drug, PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production, steroidogenic expression and electrophysiological properties.Treatment of α7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10-9 to 10-5 M) induced a significant increase in intracellular calcium concentrations, similarly to potassium (12 mM) or angiotensin II (10-8 M). This stimulation of calcium signaling did not affect cell proliferation, but was responsible for an increase in CYP11B2 expression and aldosterone production after 24 h of treatment. However, while increased intracellular calcium concentrations were observed starting from 10-8 M of PSEM-817, CYP11B2 expression and aldosterone production were only affected starting from 10-7 M, suggesting a dose dependent effect. Finally, whereas cells were hyperpolarized in absence of stimulation (around -60 mV), PSEM-817 induced a strong depolarization, cells rising to a membrane potential around -10 mV.This cell line, in which we can modulate the intracellular calcium concentration "on demand", is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Development of an adrenocortical cell model of calcium signaling modulation to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. The identification of germline or somatic mutations in different genes coding for ion channels and defines PA as a channelopathy. These mutations promote activation of calcium signaling, the main trigger for aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, mimicking some of known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3α and named α7-5HT3. Mutations have been introduced in the ligand-binding domain to allow only synthetic drugs to activate this channel receptor. Activation of α7-5HT3 by a specific drug, PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production, steroidogenic expression and electrophysiological properties. Treatment of α7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10-9 to 10-5 M) induced a significant increase in intracellular calcium concentrations, similarly to potassium (12 mM) or angiotensin II (10-8 M). This stimulation of calcium signaling did not affect cell proliferation, but was responsible for an increase in CYP11B2 expression and aldosterone production after 24 h of treatment. However, while increased intracellular calcium concentrations were observed starting from 10-8 M of PSEM-817, CYP11B2 expression and aldosterone production were only affected starting from 10-7 M, suggesting a dose-dependent effect. Finally, whereas cells were hyperpolarized in absence of stimulation (around −60 mV), PSEM-817 induced a strong depolarization, cells rising to a membrane potential around −10 mV. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

ZnO Nanoparticles Obtained by Green Synthesis as an Alternative to Improve the Germination Characteristics of L. esculentum.

Tomato is an important crop due to its nutritional contributions and organoleptic properties, which make it an appetizing vegetable around the world. In its sowing, the use of seed is the most accessible propagation mechanism for farmers. However, the induction to germination and emergence is often limited in the absence of stimulants that promote the development and growth of the seedling, added to the interference of infectious agents that notoriously reduce the vitality and viability of the seed. Given this, it was proposed as a research objective to determine the effect of zinc oxide nanoparticles (ZnO NPs) mediated by a green route on the germinative characteristics of Lycopersicon esculentum Mill. 1768 “tomato”. The experimental phase consisted of the synthesis of ZnO NPs and its subsequent characterization. After its synthesis, its inoculation was conducted during the germination of seeds of L. esculentum, considering six sample groups for the treatment with zinc nanoparticles (T1: Control; T2: 21.31 ppm; T3: 33.58 ppm; T4: 49.15 ppm; T5: 63.59 and T6: 99.076 ppm). The results indicate that concentrations close to 100 ppm of ZnO NPs are ideal in the treatment of L. esculentum seeds, due to the promotion of enzymatic and metabolic activity to achieve cell elongation; likewise, the biosynthesized nanoparticles showed no phytotoxicity, due to the fact that, in all the treatments, there were processes of germination and emergence. This was linked to the generation of a Zn0-phenolate complex through a chelating effect, which generates compatibility with the seed and, compared to classic inorganic synthesis, usually shows phytotoxicity. In this sense, green synthesis is presented as a great alternative in this type of application.

Platelets are highly efficient and efficacious carriers for tumor-targeted nano-drug delivery

The present work aims to prove the concept of tumor-targeted drug delivery mediated by platelets. Doxorubicin (DOX) attached to nanodiamonds (ND-DOX) was investigated as the model payload drug of platelets. In vitro experiments first showed that ND-DOX could be loaded in mouse platelets in a dose-dependent manner with a markedly higher efficiency and capacity than free DOX. ND-DOX-loaded platelets (Plt@ND-DOX) maintained viability and ND-DOX could be stably held in the platelets for at least 4 hr. Next, mouse Lewis lung cancer cells were found to activate Plt@ND-DOX and thereby stimulate cargo unloading of Plt@ND-DOX. The unloaded ND-DOX was taken up by co-cultured cancer cells which consequently exhibited loss of viability, proliferation suppression and apoptosis. In vivo, Plt@ND-DOX displayed significantly prolonged blood circulation time over ND-DOX and DOX in mice, and Lewis tumor grafts demonstrated infiltration, activation and cargo unloading of Plt@ND-DOX in the tumor tissue. Consequently, Plt@ND-DOX effectively reversed the growth of Lewis tumor grafts which exhibited significant inhibition of cell proliferation and apoptosis. Importantly, Plt@ND-DOX displayed a markedly higher therapeutic potency than free DOX but without the severe systemic toxicity associated with DOX. Our findings are concrete proof of platelets as efficient and efficacious carriers for tumor-targeted nano-drug delivery with the following features: 1) large loading capacity and high loading efficiency, 2) good tolerance of cargo drug, 3) stable cargo retention and no cargo unloading in the absence of stimulation, 4) prolonged blood circulation time, and 5) excellent tumor distribution and tumor-activated drug unloading leading to high therapeutic potency and few adverse effects. Platelets hold great potential as efficient and efficacious carriers for tumor-targeted nano-drug delivery.

Effective Anti–SARS-CoV-2 Immune Response in Patients With Clonal Mast Cell Disorders

BackgroundMast cells are key players in innate immunity and the TH2 adaptive immune response. The latter counterbalances the TH1 response, which is critical for antiviral immunity. Clonal mast cell activation disorders (cMCADs, such as mastocytosis and clonal mast cell activation syndrome) are characterized by abnormal mast cell accumulation and/or activation. No data on the antiviral immune response in patients with MCADs have been published.ObjectiveTo study a comprehensive range of outcomes in patients with cMCAD with PCR- or serologically confirmed coronavirus disease 2019 and to characterize the specific anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune response in this setting.MethodsClinical follow-up and outcome data were collected prospectively over a 12-month period by members of the French Centre de Référence des Mastocytoses rare disease network. Anti–SARS-CoV-2–specific T-cell activity was measured with an ELISA, and humoral responses were evaluated by assaying circulating levels of specific IgG, IgA, and neutralizing antibodies.ResultsOverall, 32 patients with cMCAD were evaluated. None required noninvasive or mechanical ventilation. Two patients were admitted to hospital for oxygen and steroid therapy. The SARS-CoV-2–specific immune response was characterized in 21 of the 32 patients. Most had high counts of circulating SARS-CoV-2–specific, IFN-γ–producing T cells and high titers of neutralizing antispike IgGs. The patients frequently showed spontaneous T-cell IFN-γ production in the absence of stimulation; this production was correlated with basal circulating tryptase levels (a marker of the mast cell burden).ConclusionsPatients with cMCADs might not be at risk of severe coronavirus disease 2019, perhaps due to their spontaneous production of IFN-γ.

The Effect of Noisy Galvanic Vestibular Stimulation on Learning of Functional Mobility and Manual Control Nulling Sensorimotor Tasks.

Galvanic vestibular stimulation (GVS) is a non-invasive method of electrically stimulating the vestibular system. We investigated whether the application of GVS can alter the learning of new functional mobility and manual control tasks and whether learning can be retained following GVS application. In a between-subjects experiment design, 36 healthy subjects performed repeated trials, capturing the learning of either (a) a functional mobility task, navigating an obstacle course on a compliant surface with degraded visual cues or (b) a manual control task, using a joystick to null self-roll tilt against a pseudo-random disturbance while seated in the dark. In the “learning” phase of trials, bilateral, bipolar GVS was applied continuously. The GVS waveform also differed between subjects in each task group: (1) white noisy galvanic vestibular stimulation (nGVS) at 0.3 mA (2) high-level random GVS at 0.7 mA (selected from pilot testing as destabilizing, but not painful), or (3) with the absence of stimulation (i.e., sham). Following the “learning” trials, all subjects were blindly transitioned to sham GVS, upon which they immediately completed another series of trials to assess any aftereffects. In the functional mobility task, we found nGVS significantly improved task learning (p = 0.03, mean learning metric 171% more than the sham group). Further, improvements in learning the functional mobility task with nGVS were retained, even once the GVS application was stopped. The benefits in learning with nGVS were not observed in the manual control task. High level GVS tended to inhibit learning in both tasks, but not significantly so. Even once the high-level stimulation was stopped, the impaired performance remained. Improvements in learning with nGVS may be due to increased information throughput resulting from stochastic resonance. The benefit of nGVS for functional mobility, but not manual control nulling, may be due to the multisensory (e.g., visual and proprioceptive), strategic, motor coordination, or spatial awareness aspects of the former task. Learning improvements with nGVS have the potential to benefit individuals who perform functional mobility tasks, such as astronauts, firefighters, high performance athletes, and soldiers.