Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. However, novel immunotherapeutics with these dual functions are not frequently reported. ...Here we describe the creation of a heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology. This complex comprises extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin-15 (IL-15)/IL-15 receptor α complex. HCW9218 can be readily expressed in CHO cells and purified using antibody-based affinity chromatography in a large-scale manufacturing setting. HCW9218 potently activates mouse natural killer (NK) cells and CD8+ T cells in vitro and in vivo to enhance cell proliferation, metabolism, and antitumor cytotoxic activities. Similarly, human immune cells become activated with increased cytotoxicity following incubation with HCW9218. This fusion complex also exhibits TGF-β neutralizing activity in vitro and sequesters plasma TGF-β in vivo. In a syngeneic B16F10 melanoma model, HCW9218 displayed strong antitumor activity mediated by NK cells and CD8+ T cells and increased their infiltration into tumors. Repeat-dose subcutaneous administration of HCW9218 was well tolerated by mice, with a half-life sufficient to provide long-lasting biological activity. Thus, HCW9218 may serve as a novel therapeutic to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors.
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A bifunctional fusion protein, HCW9218, was created using tissue factor-based scaffold technology. HCW9218 comprises the extracellular domains of human transforming growth factor-β receptor II and human interleukin-15 (IL-15)/IL-15 receptor α. This fusion protein represents a potent anti-cancer immunotherapeutic to simultaneously provide immune stimulation and lessen immunosuppressive activities associated with tumors.
Atherosclerosis is a chronic inflammatory disease caused by deposition of oxidative low-density lipoprotein (LDL) in the arterial intima which triggers the innate immune response through myeloid ...cells such as macrophages. Regulatory T cells (Tregs) play an important role in controlling the progression or regression of atherosclerosis by resolving macrophage-mediated inflammatory functions. Interleukin-2 (IL-2) signaling is essential for homeostasis of Tregs. Since recombinant IL-2 has an unfavorable pharmacokinetic profile limiting its therapeutic use, we constructed a fusion protein, designated HCW9302, containing two IL-2 domains linked by an extracellular tissue factor domain. We found that HCW9302 exhibited a longer serum half-life with an approximately 1000-fold higher affinity for the IL-2Rα than IL-2. HCW9302 could be administered to mice at a dosing range that expanded and activated Tregs but not CD4
effector T cells. In an
mouse model, HCW9302 treatment curtailed the progression of atherosclerosis through Treg activation and expansion, M2 macrophage polarization and myeloid-derived suppressor cell induction. HCW9302 treatment also lessened inflammatory responses in the aorta. Thus, HCW9302 is a potential therapeutic agent to expand and activate Tregs for treatment of inflammatory and autoimmune diseases.
Accumulation of senescent cells (SNCs) with a senescence‐associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age‐related ...pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF‐β and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet β cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro‐inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single‐nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell‐cycle, and senescence‐associated pathways in naturally aged mice. Long‐term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence‐associated diseases.
Bifunctional immunotherapeutic HCW9218 functions as a novel SNC‐reducing and senomorphic agent in mice. Subcutaneous administration of HCW9218 activates NK, innate lymphoid group‐1, and CD8+ T cells, and neutralizes TGF‐ß to reduce senescent cells (SNC‐reducing) and SASP (senomorphic) leading to lower chronic inflammation and restored tissue homeostasis.
Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells ...with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.
Infectious diseases have been responsible for an increasing number of deaths worldwide. Staphylococcus aureus has been recognized as one of the most notable causative agents of severe infections, ...while efflux pump (EP) expression is one of the main mechanisms associated with S. aureus resistance to antibiotics.
This study aimed to investigate the potential of α-pinene as an efflux pump inhibitor in species of S. aureus carrying the TetK and MrsA proteins.
The minimum inhibitory concentrations (MIC) of α-pinene and other efflux pump inhibitors were assessed using serial dilutions of each compound at an initial concentration above 1024 μg/mL. Solutions containing culture medium and bacterial inoculums were prepared in test tubes and subsequently transferred to 96-well microdilution plates. The modulation of ethidium bromide (EtBr) and antibiotics (tetracycline and erythromycin) was investigated through analysis of the modification in their MICs in the presence of a subinhibitory concentration of α-pinene (MIC/8). Wells containing only culture medium and bacterial inoculums were used as negative control. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control.
The MIC of ethidium bromide against S. aureus strains RN-4220 and IS-58 was reduced by association with α-pinene. This monoterpene potentiated the effect of tetracycline against the IS-58 strain but failed in modulating the antibacterial effect of erythromycin against RN-4220, suggesting a selective inhibitory effect on the TetK EP by α- pinene.
In conclusion, α-pinene has promising effects against S.aureus strains, which should be useful in the combat of antibacterial resistance associated with EP expression. Nevertheless, further research is required to fully characterize its molecular mechanism of action as an EP inhibitor.
This study aimed to investigate the potential of limonene as an efflux pump (EP) inhibitor in Staphylococcus aureus strains, RN-4220 and IS-58, which carry EPs for erythromycin (MrsA) and ...tetracycline (TetK), respectively.
The evolution of bacterial resistance mechanisms over time has impaired the action of most classes of antibiotics. Staphylococcus aureus is a notable bacterium, with high pathogenic potential and demonstrated resistance to conventional antibiotics. Considering the importance of discovering novel compounds to combat antibiotic resistance, our group previously demonstrated the antibacterial properties of limonene, a compound present in the essential oils of several plant species.
This study aimed to investigate the potential of limonene as an efflux pump (EP) inhibitor in Staphylococcus aureus strains RN-4220 and IS-58, which carry EPs for erythromycin (MrsA) and tetracycline (TetK), respectively.
The minimum inhibitory concentrations (MIC) of limonene and other efflux pump inhibitors were determined through the broth microdilution method. A reduction in the MIC of ethidium bromide was used as a parameter of EP inhibition.
While limonene was not shown to exhibit direct antibacterial effects against EP-carrying strains, in association with ethidium bromide and antibiotics, this compound demonstrated enhanced antibacterial activity, indicating the inhibition of the MrsA and TetK pumps.
In conclusion, this pioneering study demonstrated the effectiveness of limonene as an EP inhibitor in S. aureus strains, RN-4220 and IS-58. Nevertheless, further studies are required to characterize the molecular mechanisms associated with limonene-mediated EP inhibition.
Previous studies have shown that children and adolescents with COVID-19 generally have mild disease. Children and adolescents with cancer, however, can have severe disease when infected with ...respiratory viruses. In this study, we aimed to understand the clinical course and outcomes of SARS-CoV-2 infection in children and adolescents with cancer.
We did a cohort study with data from 131 institutions in 45 countries. We created the Global Registry of COVID-19 in Childhood Cancer to capture de-identified data pertaining to laboratory-confirmed SARS-CoV-2 infections in children and adolescents (<19 years) with cancer or having received a haematopoietic stem-cell transplantation. There were no centre-specific exclusion criteria. The registry was disseminated through professional networks through email and conferences and health-care providers were invited to submit all qualifying cases. Data for demographics, oncological diagnosis, clinical course, and cancer therapy details were collected. Primary outcomes were disease severity and modification to cancer-directed therapy. The registry remains open to data collection.
Of 1520 submitted episodes, 1500 patients were included in the study between April 15, 2020, and Feb 1, 2021. 1319 patients had complete 30-day follow-up. 259 (19·9%) of 1301 patients had a severe or critical infection, and 50 (3·8%) of 1319 died with the cause attributed to COVID-19 infection. Modifications to cancer-directed therapy occurred in 609 (55·8%) of 1092 patients receiving active oncological treatment. Multivariable analysis revealed several factors associated with severe or critical illness, including World Bank low-income or lower-middle-income (odds ratio OR 5·8 95% CI 3·8–8·8; p<0·0001) and upper-middle-income (1·6 1·2–2·2; p=0·0024) country status; age 15–18 years (1·6 1·1–2·2; p=0·013); absolute lymphocyte count of 300 or less cells per mm3 (2·5 1·8–3·4; p<0·0001), absolute neutrophil count of 500 or less cells per mm3 (1·8 1·3–2·4; p=0·0001), and intensive treatment (1·8 1·3–2·3; p=0·0005). Factors associated with treatment modification included upper-middle-income country status (OR 0·5 95% CI 0·3–0·7; p=0·0004), primary diagnosis of other haematological malignancies (0·5 0·3–0·8; p=0·0088), the presence of one of more COVID-19 symptoms at the time of presentation (1·8 1·3–2·4; p=0·0002), and the presence of one or more comorbidities (1·6 1·1–2·3; p=0·020).
In this global cohort of children and adolescents with cancer and COVID-19, severe and critical illness occurred in one fifth of patients and deaths occurred in a higher proportion than is reported in the literature in the general paediatric population. Additionally, we found that variables associated with treatment modification were not the same as those associated with greater disease severity. These data could inform clinical practice guidelines and raise awareness globally that children and adolescents with cancer are at high-risk of developing severe COVID-19 illness.
American Lebanese Syrian Associated Charities and the National Cancer Institute.
Melanized fungi and black yeasts in the family Herpotrichiellaceae (order Chaetothyriales) are important agents of human and animal infectious diseases such as chromoblastomycosis and ...phaeohyphomycosis. The oligotrophic nature of these fungi enables them to survive in adverse environments where common saprobes are absent. Due to their slow growth, they lose competition with common saprobes, and therefore isolation studies yielded low frequencies of clinically relevant species in environmental habitats from which humans are thought to be infected. This problem can be solved with metagenomic techniques which allow recognition of microorganisms independent from culture. The present study aimed to identify species of the family Herpotrichiellaceae that are known to occur in Brazil by the use of molecular markers to screen public environmental metagenomic datasets from Brazil available in the Sequence Read Archive (SRA). Species characterization was performed with the BLAST comparison of previously described barcodes and padlock probe sequences. A total of 18,329 sequences was collected comprising the genera Cladophialophora, Exophiala, Fonsecaea, Rhinocladiella and Veronaea, with a focus on species related to the chromoblastomycosis. The data obtained in this study demonstrated presence of these opportunists in the investigated datasets. The used techniques contribute to our understanding of environmental occurrence and epidemiology of black fungi.
In this paper, we report the design, synthesis and pharmacological evaluation of a series of pyrazine N-acylhydrazone (NAH) derivatives (2a–s), planned by molecular simplification of prototype ...LASSBio-1018 (1). The series (2a–s) was evaluated in several animal models of pain and inflammation. All compounds presented important antinociceptive and anti-inflammatory profiles, especially compound 2o (LASSBio-1181), presenting a trimethoxylated phenyl moiety. This derivative 2o has shown better pharmacological profile than prototype 1, being also active in adjuvant-induced arthritis test in rats.
In this paper, we report the synthesis and pharmacological evaluation of pyrazine N-acylhydrazone (NAH) derivatives (2a–s) designed as novel analgesic and anti-inflammatory drug candidates. This series was planned by molecular simplification of prototype 1 (LASSBio-1018), previously described as a non-selective cyclooxygenase inhibitor. Derivatives 2a–s were evaluated in several animal models of pain and inflammation, standing-out compound 2o (2-N′-(E)-(3,4,5-trimethoxyphenyl) methylidene-2-pyrazinecarbohydrazide; LASSBio-1181), that was also active in a murine model of chronic inflammation (i.e., adjuvant-induced arthritis test in rats) and can be considered a new analgesic and anti-inflammatory lead for drug development.
Cobalt-ceria catalysts (5 wt% in Co) were prepared using two different approaches: (i) a one-step synthesis through the EDTA-citrate complexing method, and (ii) via a traditional wet impregnation ...method. Materials were characterized by XRD, Raman, N
2
-adsorption, EDS, SEM, TEM, XPS, H
2
-TPR and O
2
-TPO. The catalytic properties in the methanol decomposition (MD) reaction were studied in a conventional flow reactor coupled to a GC, while in-situ DRIFTS studies followed the methanol surface reactivity. Moreover, DFT calculations on the methanol interaction with the Co/CeO
2
system were performed to elucidate the mechanisms behind the MD. Catalytic tests showed that both cobalt-ceria catalysts exhibited excellent stability and significantly high hydrogen yields regardless of the synthesis procedure. Besides, during long-term operations (68 h on-stream), the impregnated sample (Co/CeO
2
-WI) exhibited an enhanced performance, attributed to the close interaction of cobalt species over the surface of the ceria support. In this regard, the DFT results showed that the Co dispersion on the ceria surface plays a dominant role in forming the methoxy group, exhibiting an evident selectivity to achieve the reaction. The DRIFTS study revealed an increased population of adsorbed carbonate species over the surface of the on-step catalyst (Co/CeO
2
-OS), probably blocking the active sites during long-term operations.
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