Synthego (United States)

Poultry meat and its processed products are a key factor in the transmission of this pathogen to humans. Therefore, monitoring the level of Salmonella contamination in livestock products is a critical task in ensuring food safety. The aim of this study was to develop and validate a method for the quantitative determination of Salmonella spp. in meat products. The developed method is based on a combination of microbiological and molecular biological methods, namely, the use of polymerase chain reaction (PCR) with a preliminary stage of target culture accumulation, in combination with the most probable number (MPN) method. This study also included a comparative evaluation of the effectiveness of the proposed method and the classical culture method. In addition, a comprehensive validation of the developed method was performed, including determination of its analytical sensitivity, specificity, and accuracy on poultry meat samples. The results of the comprehensive validation of the developed method, conducted in accordance with international requirements for evaluating the analytical performance of microbiological tests, are presented. The method for the quantitative determination of Salmonella spp . bacteria in meat products was validated at the Cherkizovo Research and Testing Center in Moscow. The developed method demonstrated high efficiency, reducing the analysis time to 24 hours compared to the traditional method (up to 78 hours) and ensuring high specificity in detecting low pathogen concentrations with a quantification limit of 101 CFU/g and in the presence of accompanying microflora.

Background: Bispecific antibodies have emerged as a promising class of therapeutics, enabling simultaneous targeting of two distinct antigens. Single-domain antibodies (sdAbs) comprising camelid variable heavy chains (VHHs) provide a compact and adaptable platform for bispecific antibody design due to their small size and ease of linkage. Methods: Here we investigate structure-activity relationship of VHH-based cytokine surrogates by combining cell signaling and functional assays with x-ray crystallography and other biophysical techniques. Results: We describe crystal structures of four unique bispecific VHHs that engage and activate the cytokine receptor pairs IL-18Rα/IL-18Rβ and IL-2Rβ/IL-2Rγ. These bispecific VHH molecules, referred to as surrogate cytokine agonists (SCAs), create unique cytokine signals that can be tuned by linker engineering. Our structural analysis reveals multiple dimeric conformations for these bispecific SCAs, where the two VHH domains can interact to form a compact structure. We demonstrate that the dimeric conformation can be enforced via engineering of a non-native disulfide bond between the VHH subunits, thus enhancing molecular thermostability. Conclusion: Our findings have important implications for the design and engineering of bispecific VHHs or sdAbs, offering a novel strategy for tuning their activity and increasing their stability.

BackgroundInterleukin-12 (IL-12) is a pro-inflammatory cytokine produced by antigen-presenting cells which activates NK cells and cytotoxic CD8+ T cells and drives Th1 polarization in CD4+ T cells. In murine tumor models, IL-12 has potent T cell-mediated anti-tumor effects,1 2 but induces a systemic cytokine release syndrome (CRS) primarily via NK cell activation.2 3 In patients, use of wildtype IL-12 is hampered by early dose-limiting toxicities, including CRS, hepatotoxicity, lymphopenia (especially NK cytopenia), neutropenia, and rapid tachyphylaxis on repeat dosing.4–6STK-026 is an Fc-fused human IL-12 partial agonist with diminished binding to IL-12Rb1, leading to decreased sensitivity to NK cells and increased selectivity towards T cells, which strongly upregulate IL-12Rb1 upon antigen activation. Previously, mouse STK-026 demonstrated strong anti-tumor immunity while avoiding acute NK activation and toxicity seen with WT mouse IL-12.2 Similarly, a single dose of human STK-026 in monkeys avoided early NK activation yet induced T cell proliferation.2 Here, the results from a repeat-dose GLP toxicity study of STK-026 in cynomolgus monkeys are described.MethodsCynomolgus macaques were intravenously administered 0.5, 1.5 or 5 mg/kg STK-026, or vehicle, every other week for 3 cycles. Toxicity was evaluated by monitoring clinical symptoms, body weight, respiratory rate, cardiac function, clinical chemistry, and hematology. Immunological responses were assessed by blood immunophenotyping.ResultsAll doses of STK-026 were well-tolerated with no drug-related clinical symptoms. Body weights and weekly peripheral lymphocyte, NK cell, platelet, and neutrophil counts remained stable upon treatment. Minor increases (<3-fold) in alanine aminotransferase and aspartate transaminase were observed 2 weeks after the first cycle of STK-026, while bilirubin remained stable, suggesting minimal liver-related toxicities. Total NK and T cell frequencies remained stable throughout the dosing. STK-026 induced transient NK cell proliferation only after the first dose. In contrast, STK-026 specifically, robustly and repeatedly increased and maintained CD38+Ki67+ and GranzymeB+Ki67+ memory CD8+ T cell activation at all dose levels without inducing PD-1. Similarly, STK-026 induced and maintained proliferation of CD4+ effector memory T cells. Lastly, STK-026 increased MHC-I and CD64 on monocytes and granulocytes, consistent with IFNγ pathway activation.ConclusionsSTK-026 is well-tolerated with minimal cytopenias or transaminase changes in cynomolgus macaques over 3 cycles of treatment up to 5 mg/kg. STK-026 induced transient NK cell proliferation, but sustained memory CD8+ T cell responses. These data indicate that STK-026 is a biased IL-12 partial agonist that could retain T cell responses while avoiding NK-mediated toxicities.ReferencesBrunda MJ, Luistro L, Warrier, et al. Gately MK. Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J Exp Med 1993 Oct 1;178(4):1223–30.Koliesnik Z, Totagrande M, Burgess R, et al. Preclinical pharmacodynamic characterization of STK-026: a novel IL-12 partial agonist for cancer with maintained CD8 T cell activity, reduced NK-mediated toxicity and an improved therapeutic window. J ImmunoTher Cancer 2023;11:1053.Carson W, Yu H, Dierkshiede J, et al. A fatal cytokine-induced systemic inflammatory response reveals a critical role for NK cells. J Immunol 1999;162(8):4943–51.Atkins MB, Robertson MJ, Gordon M, et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 1997;3(3):409–17.Portielje J, Lamers C, Kruit V, et al. Repeated Administrations of Interleukin-12 Are Associated with Persistently Elevated Plasma Levels of IL-10 and Declining IFNγ, TNFα, IL-6 and IL-8 responses. Clin Canc Res 2003;9:76–83.Portielje J, Kruit V, Eerenberg A. Subcutaneous injection of interleukin 12 induces systemic inflammatory responses in humans; implications for the use of IL-12 as a vaccine adjuvant. Cancer Immunol Immunother 2005;54:37–43.Ethics ApprovalAll animal experiments were carried out in accordance with local government and institutional guidelines and regulations. All in vivo protocols were approved by a local Institutional Animal Care and Use Committee, or other appropriate review board, before study execution. All animals were housed in accordance with local regulations.

BackgroundInterleukin-12 (IL-12) is a pro-inflammatory cytokine composed of p35 and p40 subunits produced by antigen-presenting cells to stimulate Th1 cells, cytotoxic CD8 T cells, and NK cells. IL-12 has potent anti-tumor properties in multiple preclinical models, however clinical applications of wild type IL-12 (IL-12wt) or IL-12wt-Fc fusions are hampered by severe dose-limiting toxicities.1 Preclinically, IL-12 toxicity is mediated by NK cell activation.2Here we report on a novel human IL-12 partial agonist (STK-026) with diminished binding to IL-12Rb1. STK-026 was designed to increase selectivity towards antigen activated T cells, which strongly upregulate IL-12Rb1 upon activation, and to reduce stimulation of NK cells or resting T cells, which express modest levels of IL-12Rb1.MethodsTo understand the preclinical pharmacology of STK-026, we generated a half-life extended mouse surrogate of the IL-12 partial agonist (mSTK-026) for pharmacokinetic (PK) and pharmacodynamic (PD) assessment in healthy and tumor bearing mice. Further, PK and PD parameters of human STK-026 were analyzed in healthy cynomolgus macaques.ResultsAt efficacious doses, systemic administration of a half-life extended version of wild type mouse IL-12 (mIL-12wt-Fc) induced significant weight loss and lethality with early proinflammatory cytokine release and systemic NK cell activation. Conversely, mSTK-026 was well tolerated, and avoided rapid NK cell activation seen with mIL-12wt-Fc. Both mSTK-026 and mIL-12wt-Fc showed robust single-agent anti-tumor efficacy in syngeneic tumor models however mSTK-026 demonstrated a >10-fold higher therapeutic index than mIL-12wt-Fc. Depletion of NK cells did not diminish anti-tumor efficacy of either IL-12 and efficacy of mSTK-026 was associated with intratumoral CD8 T cell activation and myeloid cell activation. In cynomolgus macaques, human STK-026 demonstrated antibody like PK, was well-tolerated at all doses tested (highest: 5mg/kg), and induced no detectable systemic IL-6 or TNFα. Compared to hIL-12wt-Fc, STK-026 avoided early spikes in NK cell activity and systemic chemokine levels, reduced lymphocyte activation in peripheral tissues, and reduced ALT/AST induction at doses activating effector and memory CD8 T cells.ConclusionsOverall, mSTK-026 retained anti-tumor efficacy without spikes of early NK activation and induction of severe toxicities seen with mIL-12wt-Fc. Similarly, human STK-026 avoided early spikes of NK activity in cynomolgus macaques and showed reduced signs of systemic toxicities compared to hIL-12wt-Fc. These data suggest that STK-026 is a novel immunotherapy approach with the potential to maintain anti-tumor efficacy while avoiding dose limiting toxicities classically associated with IL-12 therapy.ReferencesAtkins M, et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clinical Cancer Research. 1997;3(3):409–17.Carson W, et al. A fatal cytokine-induced systemic inflammatory response reveals a critical role for NK cells. J Immunology. 1999;162(8):4943–4951.Ethics ApprovalAll animal experiments were carried out in accordance with local government and institutional guidelines and regulations. All in vivo protocols were approved by a local Institutional Animal Care and Use Committee, or other appropriate review board, before study execution. All animals were housed in accordance with local regulations.

Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.

Abstract High-dose Interleukin-2 (IL-2) monotherapy induces complete responses in cancer patients but its use is limited by acute vascular toxicities including capillary leak syndrome and severe hypotension 1,2. IL-2 activates lymphocytes and NK-cells through the intermediate-affinity dimeric IL-2 receptor, IL-2Rβ/γ (CD122/CD132), while antigen activated T-cells and regulatory T-cells (Tregs) have increased sensitivity to IL-2 by expressing the high-affinity trimeric IL-2 receptor, IL-2Rα/β/γ (CD25/CD122/CD132)3. CD25-independent IL-2s (“non-α-IL-2s”) aim to increase the therapeutic efficacy of IL2 in cancer patients by avoiding Treg activation through selective binding to IL-2Rβ/γ 4. However, those molecules still are reported to induce fever and hypotension and have limited efficacy as a monotherapy or in combination with anti-PD-1 5,6. Here we show that a novel α/β-IL-2 agonist that was designed to preferentially bind to the IL-2Rα/β/γ receptor highly upregulated on antigen activated T-cells can greatly improve on the efficacy of IL-2 while avoiding the vascular toxicity commonly associated with IL-2 treatment. In syngeneic tumor models, this α/β-IL-2 agonist significantly reduced exhaustion of tumor infiltrating T cells compared to WT-IL-2 or a non-α-IL-2 leading to improved expansion of tumor antigen specific CD25+PD-1+CD8+ T cells systemically and in the tumor microenvironment. This resulted in complete responses and tumor immune memory with α/β-IL-2 monotherapy as well as improved outcomes in combination with anti-PD-1 therapy in PD-1 refractory syngeneic tumors. In contrast, WT-IL-2 reduced T cell exhaustion and drove antigen specific T cell responses to a lesser degree, resulting in reduced combinatorial efficacy with anti-PD-1, while the non-α-IL-2 failed to do either. Furthermore, the α/β-IL-2 agonist reduced intratumoral Tregs compared to treatment with WT-IL-2 or PBS improving the intratumoral CD8 to Treg ratio. In non-human primates and mice, WT-IL-2 and a non-α-IL-2 led to broad extravasation of lymphocytes and NK cells and activation of intra-pulmonal T cells resulting in systemic tissue inflammation and NK cell-mediated lethal capillary leak syndrome whereas the α/β-IL-2 agonist, which avoids binding the dimeric IL-2Rβ/γ expressed on NK cells, avoided systemic lymphocyte activation which facilitated continuous treatment without acute vascular toxicities. Overall, through selective engagement of CD25+ T cells, this α/β-IL-2 agonist demonstrated improved efficacy and tolerability of IL-2 in preclinical tumor models. Clinical trials with STK-012, a human α/β-IL-2 agonist, are in progress.1 Atkins, et al.; JCO 1999, 2 Dutcher, et al.; JITC 2014, 3 Liao, et al.; Immunity 2013, 4 Levin, et al.; Nature 2012, 5 Janku, et al.; Cancer Research 2021; 6 Diab, et al.; Cancer Disc. 2020 Citation Format: Martin Oft, Navneet Ratti, Sandro Vivona, Jan Emmerich, Romina Riener, Ievgen O. Koliesnik, Scott McCauley, Michele Bauer, Marie Semana, Deepti Rokkam, Bhargavi Jayaraman, Rene de Waal Malefyt, Paul-Joseph Aspuria, Michael Totagrande, Anita Mehta-Damani, Patrick J. Lupardus, Rob A. Kastelein. STK-012, an a/b-selective IL-2 activates tumor antigen specific CD25+ CD8 T cells to reject tumors without acute vascular toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1801.

ABSTRACTA series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic—Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

This review provides a brief history of the advances of cellular analysis tools focusing on instrumentation, detection probes, and data analysis tools. The interplay of technological advancement and a deeper understanding of cellular biology are emphasized. The relevance of this topic to drug development is that the evaluation of cellular biomarkers has become a critical component of the development strategy for novel immune therapies, cell therapies, gene therapies, antiviral therapies, and vaccines. Moreover, recent technological advances in single-cell analysis are providing more robust cellular measurements and thus accelerating the advancement of novel therapies.Graphical abstract