Sediment samples from a large physical-model aquifer and laboratory-generated samples were used to systematically assess the effects of whole-sample freezing on the integrity of biomolecules relevant to bioremediation. Impacts of freezing on DNA and RNA were assessed using quantitative polymerase chain reaction (PCR) as well as the community fingerprinting method, PCR single-strand conformation polymorphism (PCR-SSCP). We did not observe any significant degradation of a suite of genes and gene transcripts, including short-lived mRNA transcripts, from P. putida F1 or from B. subtilis JH642 in single-species samples, or from archaea in enrichment culture samples that also contained members of diverse bacterial phyla. Similarly, freezing did not change the relative abundance of dominant phylotypes in enrichment culture samples as measured by PCR-SSCP of bacterial 16S rDNA. Additionally, freezing and storage for 5 months at −80 °C did not affect the microbial community composition of samples from the model aquifer. Of even greater significance is that freezing and storage did not affect the relative abundance of 16S rRNA phylotypes, since in vivo rRNA content is often correlated with cellular growth rate. Thus, we conclude that cryogenic preservation and storage of intact sediment samples can be used for accurate molecular characterization of microbial populations and may facilitate high-resolution capture of biogeochemical interfaces important to bioremediation.
Introduction: Antibodies targeting the immune checkpoint PD1/PD-L1 are emerging as effective cancer immunotherapies, but most cancers fail to respond to these as single agents. Tumor cells and the tumor microenvironment (TME) frequently express transforming growth factor-β (TGF-β) to either drive immune cell dysfunction in the microenvironment or to exclude immune cell infiltration into tumors. Concurrently targeting the immunosuppressive/immune-excluding TGF-β pathway represents a rational and promising strategy to enhance PD1/PD-L1-based immunotherapies. ASKG843 is a bifunctional molecule designed to simultaneously block PD1/PD-L1 pathway and disable all three TGF-β isoforms in the TME. Here we report the functional evaluation of ASKG843 in vitro, anti-tumor activity in vivo, pharmacokinetic (PK) and pharmacodynamic (PD) properties in cynomolgus monkeys as well as its safety profiles following 5-repeat weekly dosing in NHPs. Methods: ASKG843 was constructed comprising αhPD1 Ab with the C-terminus of the heavy chain fused to the ECD of human TGF-βRII via flexible linkers. Bispecific binding properties were evaluated by Fortebio and bispecific ELISA. Respective signaling blockade for PD1 and TGF-β were assessed using either PD1/NFAT-luciferase reporter system or TGF-β/SMAD-luciferase reporter cell line. The anti-tumor activity of ASKG843 was determined in human PBMC-engrafted NSG mice-bearing A375 xenografts. The PK/PD and safety profiles of ASKG843 were assessed in NHPs. Results: Fortebio and bispecific ELISA analyses demonstrated the ability of ASKG843 to simultaneously bind both human PD1 and TGF-β1-3. ASKG843 not only dose-dependently blocked PD1/PD-L1 inhibitory axis with similar potency to parental αPD1 Ab in PD1 bioassay, but also thwarted TGF-β canonical signaling with IC50 =14.7 ng/mL in TGF-β bioassay. In human PBMC-reconstituted A375 xenograft model ASKG843 promoted strong anti-tumor response with efficacy comparable to the reference molecule (PD-L1/TGF-βRII) at the same dose/dosing frequency. In cynomolgus monkeys ASKG843 demonstrated superior PK to PD-L1/TGF-βRII reference molecule resulting in sustained depletion of TGF-β in peripheral blood for at least 10 days following single injection at 5 mg/kg dose, and no toxicity-related sign observed which contrasted with the reference molecule. Most encouragingly ASKG843 maintained very good safety profiles in cynomolgus monkeys following multiple injections at dose up to 100 mg/kg. Conclusions: ASKG843 demonstrated potent bi-functional activities in vitro and in vivo endowed by its design. Superior PK to the reference molecule yet maintaining better safety profiles in NHPs support further development of ASKG843 as an effective immunotherapeutic candidate. Citation Format: Liqin Liu, Shiwen Zhang, Kurt Shanebeck, Mouzhong Xu, Yunxia Yuan, Ming Li, Lu Li, Ray Chuang, Yong Wen, Yuefeng Lu, Jeff Lu. ASKG843, a bifunctional fusion protein of PD1/TGF-βRII, demonstrates potent in vitro/in vivo activities, and excellent pharmacokinetic properties in non-human primates (NHPs) with good safety profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 693.
Introduction: Cytokines are potent molecules, yet their broad application as therapeutics has been significantly hampered by several limitations. First, cytokines typically have very short half-lives in vivo due to “PK sink”, leading to limited exposure and poor efficacy yet dose-dependent systemic toxicities. Secondly, “homing” of cytokines to the disease sites is often challenging even when the cytokines were fused to disease specific targeting antibodies, because the cytokine molecules dominantly control the destinations of the fusion molecules. In addition, cytokines can activate counter-regulatory pathways thus impair their potential efficacy. To fully harness the therapeutic potentials of cytokine molecules, AskGene created a novel cytokine prodrug platform (Smartkine®), wherein cytokine molecules can avoid the “PK sink” at systemic level and be activated at a disease site. ASKG215β and ASKG215γ are the first two programs from the prodrug platform. Methods: The in vitro activities of ASKG215β and ASKG215γ were evaluated in NK92 cell proliferation assay and primary T cell proliferation assay. The PK/PD properties and safety profiles of ASKG215β and ASKG215γ were assessed in non-human primates (NHPs) following three weekly IV injections at 1 mg/kg for ASKG215β and 2 mg/kg for ASKG215γ. The in vivo immune activities were evaluated in a GvHD model with human PBMC-engrafted NSG mice. The anti-tumor activities are currently being tested in human PBMC-engrafted tumor xenograft models. Results: Both ASKG215β and ASKG215γ showed significantly enhanced activity in vitro after protease-dependent activation. In NHPs, ASKG215β and ASKG215γ demonstrated prolonged and antibody-like PK profiles, which were well maintained throughout the study. More importantly both molecules were well tolerated in cynomolgus monkeys with acceptable safety profiles, with no cytokine release syndrome (CRS) observed, and no immune reaction at injection sites reported. Some immune cell activation and expansion were detected in the circulation potentially due to low levels of non-specific activation in the circulation. At high dosages, both molecules showed immune-stimulating activities in the GvHD model, including Interferon γ release and proliferations of CD8+ T cells and NK cells, potentially due non-specific activation in mice serum. The anti-tumor efficacy of in human PBMC-engrafted tumor xenograft models are expected to be presented at the conference. Conclusions: We established a novel cytokine prodrug platform. The early programs ASKG215β and ASKG215γ showed extended antibody-like PK in NHPs with acceptable safety profiles. To our knowledge this is the first report showing that IL-15 fusion molecules were able to avoid the “PK sink” usually associated IL-15 molecules. This would enable the targeted delivery of cytokine molecules to a disease site. Citation Format: chunxiao Yu, Kurt Shanebeck, Shiwen Zhang, Jeanine Ruiz, Ray Chuang, Yuanxia Yuan, Yong Wen, Tobin Streamland, Lu Li, Ming Li, Lynwel Cunanan, Mouzhong Xu, Hung-yen Lee, Jeff Lu, Liqin Liu, Yuefeng Lu. Activatable Fc-IL-15 and anti-PD1 -IL-15 fusion molecules with extended half-life [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1742.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.