Blockade of SIRPα-CD47 axis by anti-SIRPα antibody enhances anti-tumor activity of DXd antibody-drug conjugates.

Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells, which binds to CD47, a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells, therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer, several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However, the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited, suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end, DS-1103a, a newly developed anti-human SIRPα antibody (Ab), was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd), DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2, respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model, vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab, implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore, in syngeneic mouse models, both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together, this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs.

A novel T cell-redirecting anti-GPRC5D × CD3 bispecific antibody with potent antitumor activity in multiple myeloma preclinical models.

G-protein-coupled receptor class 5 member D (GPRC5D) is detected in malignant plasma cells in approximately 90% of patients diagnosed with multiple myeloma (MM). Here, we constructed BsAb5003, a novel humanized bispecific monoclonal antibody targeting CD3 and GPRC5D, and evaluated its therapeutic impact on MM. BsAb5003 induced specific cytotoxicity of GPRC5D-positive MM cells with concomitant T cell activation and cytokine release. The efficacy of BsAb5003 was associated with GPRC5D expression levels in MM cell lines. Flow cytometry analysis of bone marrow mononuclear cells (BMMNCs) from 49 MM patients revealed that GPRC5D was expressed in a wide population of MM patients, including heavily treated and high-risk patients. In ex vivo assays using BMMNCs, BsAb5003 induced potent efficacy against CD138 + MM cells in both newly diagnosed and relapsed/refractory patient samples in a GPRC5D expression-dependent manner. BsAb5003 significantly enhanced T cell activation and cytokine production in combination with immunomodulatory drugs (IMiDs) against MM cell lines. BsAb5003 also demonstrated significant inhibition of in vivo tumor growth by recruiting T cells. Taken together, these results suggest that T cell-redirecting bispecific antibody targeting GPRC5D as monotherapy and combination therapy with IMiDs could be a highly potent and effective treatment approach for a wide population of MM patients.

A novel [5.2.1]bicyclic amine is a potent analgesic without µ opioid activity.

We identified (5R)-6-methyl-5-phenyl-1,3,4,5,6,7-hexahydro-2,5-methano-2,6-benzodiazonine (DS21980956: 4-(R)) as a novel [5.2.1]bicyclic basic compound. The scaffold was inspired by fentanyl or pethidine, which possess potent analgesic activities. DS21980956 had potent analgesic activity in the mouse acetic acid writhing test or tail flick test without agonistic activity at the µ opioid receptor (MOR). The mechanism of analgesic action of DS21980956 was considered to differ from a biased ligand, for example, TRV-130 (3, oliceridine).

Oncolytic Virus Therapy with HSV-1 for Hematological Malignancies.

Oncolytic herpes simplex virus type 1 (HSV-1) has been investigated to expand its application to various malignancies. Because hematopoietic cells are resistant to HSV-1, its application to hematological malignancies has been rare. Here, we show that the third generation oncolytic HSV-1, T-01, infected and killed 18 of 26 human cell lines and 8 of 15 primary cells derived from various lineages of hematological malignancies. T-01 replicated at low levels in the cell lines. Viral entry and the oncolytic effect were positively correlated with the expression level of nectin-1 and to a lesser extent 3-O-sulfated heparan sulfate, receptors for glycoprotein D of HSV-1, on tumor cells. Transfection of nectin-1 into nectin-1-negative tumor cells made them susceptible to T-01. The oncolytic effects did not appear to correlate with the expression or phosphorylation of antiviral molecules in the cyclic GMP-AMP (cGAS)-stimulator of interferon genes (STING) and PKR-eIF2α pathways. In an immunocompetent mouse model, intratumoral injection of T-01 into lymphoma induced regression of injected, as well as non-injected, contralateral tumors accompanied by abundant infiltration of antigen-specific CD8+ T cells. These data suggest that intratumoral injection of oncolytic HSV-1 may be applicable to systemic hematological malignancies. Nectin-1 expression may be the most useful biomarker for optimal efficacy.

Association of Protein and Endotoxin in Outdoor Air with Emergency Department Visits for Children and Adults with Asthma in Fukuoka, Japan.

We examined the association of biological components in airborne particles, i.e., proteins and endotoxins, in outdoor air with asthma exacerbation in the Fukuoka metropolitan area, Fukuoka, Japan. Data on emergency department (ED) visits for asthma in children (age, 0-14 years) and adults (age, 15-64 years) were collected at a medical center from December 2014 to November 2015. One hundred eighty-one children and 143 adults visited the ED for asthma, and the weekly number of ED visits in children increased in autumn, i.e., September (second week) to November (first week). Fine (aerodynamic diameter ≤2.5 µm) and coarse (≥2.5 µm) particles were collected for 3 or 4 weeks per month, and protein and endotoxin concentrations were analyzed. Protein was largely prevalent in fine particles (0.34-7.33 µg/m3), and concentrations were high in April, May, June, and October. In contrast, endotoxin was mainly included in coarse particles (0.0010-0.0246 EU/m3), and concentrations were high in September (third week), October (first, second, and fourth weeks), February (fourth week), and July (first week). The results of a Poisson regression analysis indicated that endotoxin (in fine and coarse particles alike) was a significant factor for ED visits related to asthma in children, even after adjusting for meteorological factors, i.e., temperature, relative humidity, and wind speed. However, there was no association between environmental factors and ED visits for asthma in adults. These results suggest that endotoxin in outdoor air is significantly associated with an increased risk of asthma exacerbation in children.

Anti-inflammatory modulation of human myeloid-derived dendritic cell subsets by lenalidomide.

Although immunomodulatory drugs (IMiDs) were originally developed as anti-inflammatory drugs, they are effective for multiple myeloma. In order to gain further insights into the immunomodulatory mechanisms of IMiDs for the treatment of inflammatory disorders and myeloma, we investigated the influence of a representative IMiD, lenalidomide, on human primary dendritic cell (DC) subsets: myeloid-derived CD1c+ DCs, CD141+ DCs, and plasmacytoid DCs. Lenalidomide did not affect the viability or expression of costimulatory molecules, but it potently suppressed the production of the key inflammatory cytokines IL-12 and IL-23, and enhanced the production of the anti-inflammatory cytokine IL-10 by CD1c+ DCs. Lenalidomide also suppressed the production of IFN-α by CD141+ DCs but not that by plasmacytoid DCs. Lenalidomide likely targets pathways downstream of the nuclear translocation of the transcription factors nuclear factor κB (NF-κB) and IFN regulatory 5 (IRF5) in CD1c+ DCs. Consistent with the direct immunomodulatory effects on DCs, lenalidomide decreased the capacity of CD1c+ DCs to induce differentiation of naïve CD4+ T cells into effector cells producing immune activating and myeloma-promoting cytokines. This study demonstrated that lenalidomide has anti-inflammatory effects via the modulation of cytokine production by human myeloid-derived DCs. Such effects on DCs may allow for beneficial immunomodulation aiding in the treatment of inflammatory disorders and multiple myeloma.

Seasonal Fluctuation of Endotoxin and Protein Concentrations in Outdoor Air in Sasebo, Japan.

To determine the levels of endotoxin, which is a major component of outer membrane of Gram-negative bacteria, and protein in the atmosphere in Sasebo, Japan, we measured these biological materials in fine (aerodynamic diameter ≤2.5 µm) and coarse (≥2.5 µm) particles collected for 81 weeks (September 2014 to May 2016). The monthly concentrations (i.e., the mean value of weekly concentrations for each month) of endotoxin were higher in coarse particles than in fine particles. Fluctuations in monthly endotoxin concentrations were large in both fine (0.0005-0.0208 EU/m3) and coarse (0.0032-0.1164 EU/m3) particles. Furthermore, the endotoxin concentrations in coarse particles were highest in October 2014 and 2015 as well as September 2014 (0.0407-0.1164 EU/m3). However, the monthly protein concentrations were higher in fine particles than in coarse particles. Compared to the endotoxin concentrations, the fluctuations in the monthly protein concentrations were smaller in both coarse and fine particles. To our knowledge, this study is the first to report long-term atmospheric concentrations of endotoxin and protein in Japan. Since the endotoxin concentrations in coarse particles were positively associated with the concentrations of Na+ and Cl-, it suggests the involvement of Gram-negative bacteria from seawater to the endotoxin levels in the atmosphere. For fine particles, the protein concentrations were positively associated with the concentrations of particles, NO3- and SO42-. These results suggest that combustion of organic materials, such as biomass burning, may be a contributor to atmospheric protein during this study period.

Discovery and SAR of a novel series of Natriuretic Peptide Receptor-A (NPR-A) agonists.

Novel thienopyrimidine compounds 2 and 3 were discovered from high-throughput screening as Natriuretic Peptide Receptor A (NPR-A) agonists. Scaffold hopping of a thienopyrimidine ring to a quinazoline ring, introduction of the basic functional group and optimization of the substituent on the 6-position of the benzene ring of quinazoline led to improved agonistic activity. We discovered compound 48, which showed potent agonistic activity for NPR-A with an EC50 value of 0.073µM, indicating 350-fold potency compared to the hit compound 3.

Synthesis and SAR studies of benzyl ether derivatives as potent orally active S1P1 agonists.

We report herein the synthesis and structure-activity relationships (SAR) of a series of benzyl ether compounds as an S1P1 receptor modulator. From our SAR studies, the installation of substituents onto the central benzene ring of 2a was revealed to potently influence the S1P1 and S1P3 agonistic activities, in particular, an ethyl group on the 2-position afforded satisfactory S1P1/S1P3 selectivity. These changes of the S1P1 and S1P3 agonistic activities caused by the alteration of substituents on the 2-position were reasonably explained by a docking study using an S1P1 X-ray crystal structure and S1P3 homology modeling. We found that compounds 2b and 2e had a potent in vivo immunosuppressive efficacy along with acceptable S1P1/S1P3 selectivity, and confirmed that these compounds had less in vivo bradycardia risk through the evaluation of heart rate change after oral administration of the compounds (30 mg/kg, p.o.) in rats.

Synthesis and SAR of 1,3-thiazolyl thiophene and pyridine derivatives as potent, orally active and S1P3-sparing S1P1 agonists.

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P(3)-sparing S1P(1) agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC(50) value of 3.4 nM for human S1P(1), and over 5800-fold selectivity against S1P(3). In rat on host versus graft reaction (HvGR), the ID(50) value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.

Synthesis and evaluation of CS-2100, a potent, orally active and S1P(3)- sparing S1P(1) agonist.

Modulators of sphingosine phosphate receptor-1 (S1P(1)) have recently been focused as a suppressant of autoimmunity. We have discovered a 4-ethylthiophene-based S1P(1) agonist 1-({4-Ethyl-5-[5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl]-2-thienyl}methyl)azetidine-3-carboxylic acid (CS-2100, 8) showing potent S1P(1) agonist activity against S1P(3) and an excellent in vivo potency. We report herein the synthesis of CS-2100 (8) and pharmacological effects such as S1P(1) and S1P(3) agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effects on adjuvant-induced arthritis and experimental autoimmune encephalomyelitis (EAE) in animal models. The pharmacokinetic data were also reported. CS-2100 (8) had >5000-fold greater agonist activity for human S1P(1) (EC(50); 4.0 nM) relative to S1P(3) (EC(50); >20,000 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-2100 (8) in rats, lymphocyte counts decreased significantly, with a nadir at 8 and/or 12 h post-dose and recovery to vehicle control levels by 24-48 h post-dose. CS-2100 (8) is efficacious in the adjuvant-induced arthritis model in rats (ID(50); 0.44 mg/kg). In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for 0.3 and 1 mg/kg CS-2100 (8) groups in mice. While CS-2100 (8) showed potent efficacy in various animal disease models, it was also revealed that the central 1,2,4-oxadiazole ring of CS-2100 (8) was decomposed by enterobacteria in intestine of rats and monkeys, implicating the latent concern about an external susceptibility in its metabolic process in the upcoming clinical studies.

Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist.

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.

Discovery of CS-0777: A Potent, Selective, and Orally Active S1P1 Agonist.

CS-0777 (3) is phosphorylated in vivo, and the phosphate of CS-0777 (CS-0777-P) (4) acts as a selective S1P receptor-1 (S1P1) modulator. We report herein the synthesis of CS-0777 and CS-0777-P, pharmacological effects such as S1P1 and S1P3 agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effect on experimental autoimmune encephalomyelitis (EAE), and also the pharmacokinetics in rats. CS-0777-P had ∼320-fold greater agonist activity for human S1P1 (EC50; 1.1 nM) relative to S1P3 (EC50; 350 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-0777 in rats, lymphocyte counts decreased significantly, with a nadir at 12 h postdose and recovery to vehicle control levels by 5 days postdose. In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for the 0.1 and 1 mg/kg CS-0777 groups in rats. CS-0777 is currently in clinical trials for the treatment of multiple sclerosis (MS).

RS-1748, a novel CC chemokine receptor 4 antagonist, inhibits ovalbumin-induced airway inflammation in guinea pigs.

CC chemokine receptor 4 (CCR4) is generally recognized as a preferential marker for T helper 2 cells, and we have previously reported morpholine-derivative CCR4 antagonists, RS-1154 and RS-1269. Here, we investigate the pharmacological profiles of a novel pyrimidine-derivative CCR4 antagonist, 2-{4-[2-(diethylamino)ethoxy]phenyl}-N-(2,4-difluorobenzyl)-5-fluoropyrimidin-4-amine (RS-1748), which showed potency to inhibit the bindings of [(125)I]CCL17 and [(35)S]GTPgammaS to human CCR4-expressing Chinese hamster ovary (CHO) cells with IC(50) values of 59.9 nM and 18.4 nM, respectively. Furthermore, RS-1748 inhibited ovalbumin-induced airway inflammation in guinea pigs at a dose of 10 mg/kg. These results indicate that RS-1748 would be a promising lead compound for developing a therapeutic agent against asthma.

Involvement of sphingosine-1-phosphate and S1P1 in angiogenesis: analyses using a new S1P1 antagonist of non-sphingosine-1-phosphate analog.

Chemical lead 2 (CL2) is the first non-sphingosine-1-phosphate (Sph-1-P) analog type antagonist of endothelial differentiation gene-1 (Edg-1/S1P(1)), which is a member of the Sph-1-P receptor family. CL2 inhibits [(3)H]Sph-1-P/S1P(1) binding and shows concentration-dependent inhibition activity against both intracellular cAMP concentration decrease and cell invasion induced by the Sph-1-P/S1P(1) pathway. It also inhibits normal tube formation in an angiogenesis culture model, indicating that CL2 has anti-angiogenesis activity. This compound improved the disease conditions in two angiogenic models in vivo. It significantly inhibited angiogenesis induced by vascular endothelial growth factor in a rabbit cornea model as well as the swelling of mouse feet in an anti-type II collagen antibody-induced arthritis model. These results indicate that the Sph-1-P/S1P(1) pathway would have an important role in disease-related angiogenesis, especially in the processes of migration/invasion and tube formation. In addition, CL2 would be a powerful tool for the pharmacological study of the mechanisms of the Sph-1-P/S1P(1) pathway in rheumatoid arthritis, diabetes retinopathy, and solid tumor growth processes.