Pathogen-associated T follicular helper cell plasticity is critical in anti-viral immunity.

T follicular helper (Tfh) cells are critical in providing help for B cells in the germinal center reaction. Tfh cell plasticity, especially with regard to their expression of effector Th cytokines, has been described, but lacks in-depth analysis with genetic approaches. In this study, we systemically compared transcriptomic profiles of Tfh cells derived from various types of immune responses and found gene clusters corresponding to effector Th cells were differentially induced in response to pathogens or immune responses. Of special interest, a subset of Tfh cells producing IFN-γ was generated in an influenza virus infection, partially dependent on the innate cytokine IL-12. Lineage-tracing mouse model revealed unique developmental regulation of IFN-γ+ Tfh cells, while selective ablation of these cells impaired the induction of IgG2c+ germinal center B cells and the control of influenza infection. These results indicate that pathogen-associated Tfh cell plasticity is necessary for host immunity, which has implications in vaccine design.

Systematic characterization of the tumor microenvironment in Chinese patients with hepatocellular carcinoma highlights intratumoral B cells as a potential immunotherapy target.

Hepatocellular carcinoma (HCC) is an immunogenic malignancy, which exhibits low responsiveness to programmed cell death protein­1 (PD­1)/programmed death ligand­1 (PD­L1) antibodies. Therefore, the identification of novel immunotherapeutic targets to treat HCC is imperative. Systematic characterization of the HCC tumor microenvironment (TME) can provide novel insight into additional therapeutic approaches. In the present study, the RNA­sequencing (RNA­seq) data of 360 patients with HCC were integrated from The Cancer Genome Atlas to assess the expression of membrane spanning 4­domains A1 (MS4A1; encoding CD20) in tumors and normal liver tissues. Immunofluorescence and multiplex tissue fluorescence analyses were performed and combined with flow cytometry staining to measure CD20/CD19 expression at the protein level. In addition, published single cell RNA­seq data of CD45+ cells were derived from 16 treatment­naïve patients from Beijing Shijitan Hospital with HCC to illustrate the characteristics of CD19+ B cells. The results indicated that the HCC TME included nuclear receptor subfamily 4 group A member 2+ (NR4A2) B cells. Patients with HCC and high density of intratumoral B cells demonstrated compromised antitumor immunity manifested by low percentages of cytotoxic CD8+ T cells and high density of regulatory T cells. Furthermore, PD­L1 expression was significantly correlated with the B cell signature marker CD20. The present study indicated that tumor­infiltrating B cells may play a negative role in antitumor immunity and serve as a promising target for HCC immunotherapy, alone or in combination with anti­PD­L1/PD­1 antibodies.

Mouse IL-2/CD25 Fusion Protein Induces Regulatory T Cell Expansion and Immune Suppression in Preclinical Models of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is associated with an IL-2-deficient state, with regulatory T cells (Tregs) showing diminished immune regulatory capacity. A low dose of IL-2 has shown encouraging clinical benefits in SLE patients; however, its clinical utility is limited because of the requirement of daily injections and the observation of increase in proinflammatory cytokines and in non-Tregs. We recently showed that a fusion protein of mouse IL-2 and mouse IL-2Rα (CD25), joined by a noncleavable linker, was effective in treating diabetes in NOD mice by selectively inducing Treg expansion. In this report, we show that mouse IL-2 (mIL-2)/CD25 at doses up to 0.5 mg/kg twice a week induced a robust Treg expansion without showing signs of increase in the numbers of NK, CD4+Foxp3-, or CD8+ T cells or significant increase in proinflammatory cytokines. In both NZB × NZW and MRL/lpr mice, mIL-2/CD25 at 0.2-0.4 mg/kg twice a week demonstrated efficacy in inducing Treg expansion, CD25 upregulation, and inhibiting lupus nephritis based on the levels of proteinuria, autoantibody titers, and kidney histology scores. mIL-2/CD25 was effective even when treatment was initiated at the time when NZB × NZW mice already showed signs of advanced disease. Furthermore, we show coadministration of prednisolone, which SLE patients commonly take, did not interfere with the ability of mIL-2/CD25 to expand Tregs. The prednisolone and mIL-2/CD25 combination treatment results in improvements in most of the efficacy readouts relative to either monotherapy alone. Taken together, our results support further evaluation of IL-2/CD25 in the clinic for treating immune-mediated diseases such as SLE.

Azatricyclic Inverse Agonists of RORγt That Demonstrate Efficacy in Models of Rheumatoid Arthritis and Psoriasis.

Structure-activity relationship studies directed toward the replacement of the fused phenyl ring of the lead hexahydrobenzoindole RORγt inverse agonist series represented by 1 with heterocyclic moieties led to the identification of three novel aza analogs 5-7. The hexahydropyrrolo[3,2-f]quinoline series 5 (X = N, Y = Z=CH) showed potency and metabolic stability comparable to series 1 but with improved in vitro membrane permeability and serum free fraction. This structural modification was applied to the hexahydrocyclopentanaphthalene series 3, culminating in the discovery of 8e as a potent and selective RORγt inverse agonist with an excellent in vitro profile, good pharmacokinetic properties, and biologic-like in vivo efficacy in preclinical models of rheumatoid arthritis and psoriasis.

Tricyclic-Carbocyclic RORγt Inverse Agonists-Discovery of BMS-986313.

SAR efforts directed at identifying RORγt inverse agonists structurally different from our clinical compound 1 (BMS-986251) led to tricyclic-carbocyclic analogues represented by 3-7 and culminated in the identification of 3d (BMS-986313), with structural differences distinct from 1. The X-ray co-crystal structure of 3d with the ligand binding domain of RORγt revealed several key interactions, which are different from 1. The in vitro and in vivo PK profiles of 3d are described. In addition, we demonstrate robust efficacy of 3d in two preclinical models of psoriasis-the IMQ-induced skin lesion model and the IL-23-induced acanthosis model. The efficacy seen with 3d in these models is comparable to the results observed with 1.

Substituted diaryl ether compounds as retinoic acid-related orphan Receptor-γt (RORγt) agonists.

The discovery of a series of substituted diarylether compounds as retinoic acid related orphan receptor γt (RORγt) agonists is described. Compound 1 was identified from deck mining as a RORγt agonist. Hit-to-lead optimization led to the identification of lead compound 5, which possesses improved potency (10x). Extensive SAR exploration led to the identification of a potent and selective compound 22, that demonstrated an improved pharmacokinetic profile and a dose-dependent pharmacodynamic response. However, when dosed in a MC38 syngeneic tumor model, no evidence of efficacy was observed. ©2020 Elsevier Science Ltd. All rights reserved.

Bicyclic Ligand-Biased Agonists of S1P1: Exploring Side Chain Modifications to Modulate the PK, PD, and Safety Profiles.

Sphingosine-1-phosphate (S1P) binds to a family of sphingosine-1-phosphate G-protein-coupled receptors (S1P1-5). The interaction of S1P with these S1P receptors has a fundamental role in many physiological processes in the vascular and immune systems. Agonist-induced functional antagonism of S1P1 has been shown to result in lymphopenia. As a result, agonists of this type hold promise as therapeutics for autoimmune disorders. The previously disclosed differentiated S1P1 modulator BMS-986104 (1) exhibited improved preclinical cardiovascular and pulmonary safety profiles as compared to earlier full agonists of S1P1; however, it demonstrated a long pharmacokinetic half-life (T1/2 18 days) in the clinic and limited formation of the desired active phosphate metabolite. Optimization of this series through incorporation of olefins, ethers, thioethers, and glycols into the alkyl side chain afforded an opportunity to reduce the projected human T1/2 and improve the formation of the active phosphate metabolite while maintaining efficacy as well as the improved safety profile. These efforts led to the discovery of 12 and 24, each of which are highly potent, biased agonists of S1P1. These compounds not only exhibited shorter in vivo T1/2 in multiple species but are also projected to have significantly shorter T1/2 values in humans when compared to our first clinical candidate. In models of arthritis, treatment with 12 and 24 demonstrated robust efficacy.

Population Pharmacokinetic Analysis of BMS-986166, a Novel Selective Sphingosine-1-Phosphate-1 Receptor Modulator, and Exposure-Response Assessment of Lymphocyte Counts and Heart Rate in Healthy Participants.

Sphingosine-1-phosphate (S1P) binding to the S1P-1 receptor (S1P1R) controls the egress of lymphocytes from lymphoid organs and targets modulation of immune responses in autoimmune diseases. Pharmacologic modulation of S1P receptors has been linked to heart rate reduction. BMS-986166, a prodrug of the active phosphorylated metabolite BMS-986166-P, presents an improved cardiac safety profile in preclinical studies compared to other S1P1R modulators. The pharmacokinetics, safety, and pharmacodynamics of BMS-986166 versus placebo after single (0.75-5.0 mg) and repeated (0.25-1.5 mg/day) oral administration were assessed in healthy participants after a 1-day lead-in placebo period. A population model was developed to jointly describe BMS-986166 and BMS-986166-P pharmacokinetics and predict individual exposures. Inhibitory sigmoid models described the relationships between average daily BMS-986166-P concentrations and nadir of time-matched (day -1) placebo-corrected heart rate on day 1 (nDDHR, where DD represents ∆∆) and nadir of absolute lymphocyte count (nALC). Predicted decreases in nDDHR and nALC were 9 bpm and 20% following placebo, with maximum decreases of 10 bpm in nDDHR due to drug effect, and approximately 80% in nALC due to drug and placebo. A 0.5-mg/day dose regimen achieves the target 65% reduction in nALC associated with a 2-bpm decrease in nDDHR over placebo.

Novel Tricyclic Pyroglutamide Derivatives as Potent RORγt Inverse Agonists Identified using a Virtual Screening Approach.

Employing a virtual screening approach, we identified the pyroglutamide moiety as a nonacid replacement for the cyclohexanecarboxylic acid group which, when coupled to our previously reported conformationally locked tricyclic core, provided potent and selective RORγt inverse agonists. Structure-activity relationship optimization of the pyroglutamide moiety led to the identification of compound 18 as a potent and selective RORγt inverse agonist, albeit with poor aqueous solubility. We took advantage of the tertiary carbinol group in 18 to synthesize a phosphate prodrug, which provided good solubility, excellent exposures in mouse PK studies, and significant efficacy in a mouse model of psoriasis.

Biologic-like In Vivo Efficacy with Small Molecule Inhibitors of TNFα Identified Using Scaffold Hopping and Structure-Based Drug Design Approaches.

Scaffold hopping and structure-based drug design were employed to identify substituted 4-aminoquinolines and 4-aminonaphthyridines as potent, small molecule inhibitors of tumor necrosis factor alpha (TNFα). Structure-activity relationships in both the quinoline and naphthyridine series leading to the identification of compound 42 with excellent potency and pharmacokinetic profile are discussed. X-ray co-crystal structure analysis and ultracentrifugation experiments clearly demonstrate that these inhibitors distort the TNFα trimer upon binding, leading to aberrant signaling when the trimer binds to TNF receptor 1 (TNFR1). Pharmacokinetic-pharmacodynamic activity of compound 42 in a TNF-induced IL-6 mouse model and in vivo activity in a collagen antibody-induced arthritis model, where it showed biologic-like in vivo efficacy, will be discussed.

Tricyclic sulfones as potent, selective and efficacious RORγt inverse agonists - Exploring C6 and C8 SAR using late-stage functionalization.

In order to rapidly develop C6 and C8 SAR of our reported tricyclic sulfone series of RORγt inverse agonists, a late-stage bromination was employed. Although not regioselective, the bromination protocol allowed us to explore new substitution patterns/vectors that otherwise would have to be incorporated at the very beginning of the synthesis. Based on the SAR obtained from this exercise, compound 15 bearing a C8 fluorine was developed as a very potent and selective RORγt inverse agonist. This analog's in vitro profile, pharmacokinetic (PK) data and efficacy in an IL-23 induced mouse acanthosis model will be discussed.

Aryl Ether-Derived Sphingosine-1-Phosphate Receptor (S1P1) Modulators: Optimization of the PK, PD, and Safety Profiles.

Efforts aimed at increasing the in vivo potency and reducing the elimination half-life of 1 and 2 led to the identification of aryl ether and thioether-derived bicyclic S1P1 differentiated modulators 3-6. The effects of analogs 3-6 on lymphocyte reduction in the rat (desired pharmacology) along with pulmonary- and cardiovascular-related effects (undesired pharmacology) are described. Optimization of the overall properties in the aryl ether series yielded 3d, and the predicted margin of safety against the cardiovascular effects of 3d would be large enough for human studies. Importantly, compared to 1 and 2, compound 3d had a better profile in both potency (ED50 < 0.05 mg/kg) and predicted human half-life (t 1/2 ∼ 5 days).

Persistent IL-2 Receptor Signaling by IL-2/CD25 Fusion Protein Controls Diabetes in NOD Mice by Multiple Mechanisms.

Low-dose interleukin-2 (IL-2) represents a new therapeutic approach to regulate immune homeostasis to promote immune tolerance in patients with autoimmune diseases, including type 1 diabetes. We have developed a new IL-2-based biologic, an IL-2/CD25 fusion protein, with greatly improved pharmacokinetics and pharmacodynamics when compared with recombinant IL-2 to enhance this type of immunotherapy. In this study, we show that low-dose mouse IL-2/CD25 (mIL-2/CD25), but not an equivalent amount of IL-2, prevents the onset of diabetes in NOD mice and controls diabetes in hyperglycemic mice. mIL-2/CD25 acts not only to expand regulatory T cells (Tregs) but also to increase their activation and migration into lymphoid tissues and the pancreas. Lower incidence of diabetes is associated with increased serum levels of IL-10, a cytokine readily produced by activated Tregs. These effects likely act in concert to lower islet inflammation while increasing Tregs in the remaining inflamed islets. mIL-2/CD25 treatment is also associated with lower anti-insulin autoantibody levels in part by inhibition of T follicular helper cells. Thus, long-acting mIL-2/CD25 represents an improved IL-2 analog that persistently elevates Tregs to maintain a favorable Treg/effector T cell ratio that limits diabetes by expansion of activated Tregs that readily migrate into lymphoid tissues and the pancreas while inhibiting autoantibodies.

Discovery of BMS-986251: A Clinically Viable, Potent, and Selective RORγt Inverse Agonist.

Novel tricyclic analogues were designed, synthesized, and evaluated as RORγt inverse agonists. Several of these compounds were potent in an IL-17 human whole blood assay and exhibited excellent oral bioavailability in mouse pharmacokinetic studies. This led to the identification of compound 5, which displayed dose-dependent inhibition of IL-17F production in a mouse IL-2/IL-23 stimulated pharmacodynamic model. In addition, compound 5 was studied in mouse acanthosis and imiquimod-induced models of skin inflammation, where it demonstrated robust efficacy comparable to a positive control. As a result of this excellent overall profile, compound 5 (BMS-986251) was selected as a clinically viable developmental candidate.

The safety and pharmacokinetics of a novel, selective S1P1R modulator in healthy participants.

Background: Safety, pharmacokinetics and pharmacodynamics of BMS-986166, a novel sphingosine-1-phosphate receptor 1 modulator, were assessed.Research design and methods: Two double-blind, placebo-controlled, randomized Phase l studies were conducted in healthy participants. In the single ascending dose study (N = 70), BMS-986166 was administered as a single dose, upwardly titrated daily doses or a single dose in participants who were fed, fasted or administered famotidine. In the multiple ascending dose study (N = 32), BMS-986166 was administered daily for 28 days. Safety, pharmacokinetics and pharmacodynamics (absolute lymphocyte count [ALC]) were assessed.Results: BMS-986166 was generally well tolerated; treatment-related adverse events were mild. Dose-related, clinically insignificant reductions in time-matched heart rate were recorded versus placebo. Pharmacokinetics were linear and stationary with approximately dose-related increases in blood exposure of BMS-986166. Decreases in ALC percent change from baseline with multiple doses of BMS-986166 versus placebo were dose-related. Between Day 0 and 35, median nadir lymphocyte reductions were 53.7%, 75.9% and 81.9% with 0.25-, 0.75- and 1.5-mg BMS-986166 doses. ALC recovery began 14, 14-21 and 7 days after last dose of 0.25, 0.75 and 1.5 mg.Conclusions: BMS-986166 was generally well tolerated in this population and warrants further investigation.Trial registration: ClinicalTrials.gov: NCT02790125, NCT03038711.

Rationally Designed, Conformationally Constrained Inverse Agonists of RORγt-Identification of a Potent, Selective Series with Biologic-Like in Vivo Efficacy.

RORγt is an important nuclear receptor that regulates the production of several pro-inflammatory cytokines such as IL-17 and IL-22. As a result, RORγt has been identified as a potential target for the treatment of various immunological disorders such as psoriasis, psoriatic arthritis, and inflammatory bowel diseases. Structure and computer-assisted drug design led to the identification of a novel series of tricyclic RORγt inverse agonists with significantly improved in vitro activity in the reporter (Gal4) and human whole blood assays compared to our previous chemotype. Through careful structure activity relationship, several potent and selective RORγt inverse agonists have been identified. Pharmacokinetic studies allowed the identification of the lead molecule 32 with a low peak-to-trough ratio. This molecule showed excellent activity in an IL-2/IL-23-induced mouse pharmacodynamic study and demonstrated biologic-like efficacy in an IL-23-induced preclinical model of psoriasis.

Autoimmune pathways in mice and humans are blocked by pharmacological stabilization of the TYK2 pseudokinase domain.

TYK2 is a nonreceptor tyrosine kinase involved in adaptive and innate immune responses. A deactivating coding variant has previously been shown to prevent receptor-stimulated activation of this kinase and provides high protection from several common autoimmune diseases but without immunodeficiency. An agent that recapitulates the phenotype of this deactivating coding variant may therefore represent an important advancement in the treatment of autoimmunity. BMS-986165 is a potent oral agent that similarly blocks receptor-stimulated activation of TYK2 allosterically and with high selectivity and potency afforded through optimized binding to a regulatory domain of the protein. Signaling and functional responses in human TH17, TH1, B cells, and myeloid cells integral to autoimmunity were blocked by BMS-986165, both in vitro and in vivo in a phase 1 clinical trial. BMS-986165 demonstrated robust efficacy, consistent with blockade of multiple autoimmune pathways, in murine models of lupus nephritis and inflammatory bowel disease, supporting its therapeutic potential for multiple immune-mediated diseases.

Structure-based Discovery of Phenyl (3-Phenylpyrrolidin-3-yl)sulfones as Selective, Orally Active RORγt Inverse Agonists.

A new phenyl (3-phenylpyrrolidin-3-yl)sulfone series of RORγt inverse agonists was discovered utilizing the binding conformation of previously reported bicyclic sulfonamide 1. Through a combination of structure-based design and structure-activity relationship studies, a polar set of amides at N1-position of the pyrrolidine ring and perfluoroisopropyl group at para-position of the 3-phenyl group were identified as critical structural elements to achieve high selectivity against PXR, LXRα, and LXRß. Further optimization led to the discovery of (1R,4r)-4-((R)-3-((4-fluorophenyl)sulfonyl)-3-(4-(perfluoropropan-2-yl)phenyl)pyrrolidine-1-carbonyl)cyclohexane-1-carboxylic acid (26), which displayed excellent selectivity, desirable liability and pharmacokinetic properties in vitro, and a good pharmacokinetic profile in mouse. Oral administration of 26 demonstrated dose-dependent inhibition of IL-17 production in a mouse IL-2/IL-23-induced pharmacodynamic model and biologic-like efficacy in an IL-23-induced mouse acanthosis model.

Critical role of kinase activity of hematopoietic progenitor kinase 1 in anti-tumor immune surveillance.

Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of-function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens.

Identification and Preclinical Pharmacology of ((1 R,3 S)-1-Amino-3-(( S)-6-(2-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (BMS-986166): A Differentiated Sphingosine-1-phosphate Receptor 1 (S1P1) Modulator Advanced into Clinical Trials.

Recently, our research group reported the identification of BMS-986104 (2) as a differentiated S1P1 receptor modulator. In comparison to fingolimod (1), a full agonist of S1P1 currently marketed for the treatment of relapse remitting multiple sclerosis (RRMS), 2 offers several potential advantages having demonstrated improved safety multiples in preclinical evaluations against undesired pulmonary and cardiovascular effects. In clinical trials, 2 was found to exhibit a pharmacokinetic half-life ( T1/2) longer than that of 1, as well as a reduced formation of the phosphate metabolite that is required for activity against S1P1. Herein, we describe our efforts to discover highly potent, partial agonists of S1P1 with a shorter T1/2 and increased in vivo phosphate metabolite formation. These efforts culminated in the discovery of BMS-986166 (14a), which was advanced to human clinical evaluation. The pharmacokinetic/pharmacodynamic (PK/PD) relationship as well as pulmonary and cardiovascular safety assessments are discussed. Furthermore, efficacy of 14a in multiple preclinical models of autoimmune diseases are presented.