Abstract
A long systemic half-life is key for therapeutic proteins. To that end we have generated serum albumin-binding designed ankyrin repeat domains. These domains bind serum albumin of different ...species with nanomolar affinities, and have significantly improved pharmacokinetic properties both in mouse and cynomolgus monkey compared to non-serum albumin-binding DARPin® domains. In addition, they exhibit high thermal stability and long storage stability, which is an essential feature for their use in drug development. Covalently linking a serum albumin-binding DARPin® domain to domains with other target specificities results in improvements of multiple orders of magnitude in exposure and terminal half-life, both in mouse and cynomolgus monkey. Pharmacokinetic assessment of such constructs revealed terminal half-life values ranging from 27 h to 80 h in mouse, and from 2.6 days to 20 days in cynomolgus monkey. Extrapolation by allometric scaling on these findings suggests terminal half-life values of 5-50 days in human, indicating that pharmacokinetic properties in the range of monoclonal antibodies can be achieved with DARPin® drug candidates. Such serum albumin-binding DARPin® domains are thus valuable tools for the generation of multi-functional drugs with an extended in vivo half-life.
Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels is essential for T-cell activation and proliferation. Recently, it has been shown that 3,5-bistrifluoromethyl pyrazole ...(BTP) derivatives are specific inhibitors of Ca2+-dependent transcriptional activity in T-cells (Trevillyan, J. M., Chiou, X. G., Chen, Y. W., Ballaron, S. J., Sheets, M. P., Smith, M. L., Wiedeman, P. E., Warrior, U., Wilkins, J., Gubbins, E. J., Gagne, G. D., Fagerland, J., Carter, G. W., Luly, J. R., Mollison, K. W., and Djuric, S. W. (2001) J. Biol. Chem. 276, 48118-48126). Whereas inhibition of Ca2+ signals was reported for BTP2 (Ishikawa, J., Ohga, K., Yoshino, T., Takezawa, R., Ichikawa, A., Kubota, H., and Yamada, T. (2003) J. Immunol. 170, 4441-4449), it was not found for BTP3 (Chen, Y., Smith, M. L., Chiou, G. X., Ballaron, S., Sheets, M. P., Gubbins, E., Warrior, U., Wilkins, J., Surowy, C., Nakane, M., Carter, G. W., Trevillyan, J. M., Mollison, K., and Djuric, S. W. (2002) Cell. Immunol. 220, 134-142). We show that BTP2 specifically inhibits CRAC channels in T-cells with an IC50 of ∼10 nm. It does not interfere with other mechanisms important for Ca2+ signals in T-cells, including Ca2+ pumps, mitochondrial Ca2+ signaling, endoplasmic reticulum Ca2+ release, and K+ channels. BTP2 inhibits Ca2+ signals in peripheral blood T-lymphocytes (in particular in CD4+ T-cells) and in human Jurkat T-cells. Inhibition of Ca2+ signals is independent of the stimulation method as Ca2+ entry was blocked following stimulation with anti-CD3, which activates the T-cell receptor, and also following stimulation with thapsigargin or inositol 1,4,5-trisphosphate. BTP2 also inhibited Ca2+-dependent gene expression (interleukins 2 and 5 and interferon γ) and proliferation of T-lymphocytes with similar IC50 values. BTP2 is the first potent and specific inhibitor of CRAC channels in primary T-lymphocytes. The inhibition of CRAC channels as well as Ca2+-dependent signal transduction with similar IC50 values in T-lymphocytes emphasizes the importance of CRAC channel activity during T-cell activation. Furthermore, BTP2 could prove to be a tool to finally unmask the molecular identity of CRAC channels.
An early key event in the activation of neutrophil granulocytes is Ca(2+) influx. Members of the transient receptor potential (TRP) channel family may be held responsible for this. The aim of the ...present study is to analyse the expression pattern of TRP mRNA and identify characteristic currents unambiguously attributable to particular TRP channels. mRNA was extracted from human neutrophils, isolated by gradient centrifugation and also by magnetically labelled CD15 antibodies. The presence of mRNA was demonstrated using reverse transcriptase-PCR in neutrophils (controlled to be CD5-negative) as well as in human leukaemic cell line 60 (HL-60) cells, for the following TRP species: the long TRPC2 (LTRPC2), the vanilloid receptor 1, the vanilloid receptor-like protein 1 and epithelial Ca(2+) channels 1 and 2. TRPC6 was specific for neutrophils, whereas only in HL-60 cells were TRPC1, TRPC2, TRPC3, melastatin 1 and melastatin-related 1 found. Patch-clamp measurements in neutrophils revealed non-selective cation currents evoked by intracellular ADP-ribose and by NAD(+). Both these modes of activation have been found to be characteristic of LTRPC2. Furthermore, single-channel activity was resolved in neutrophils and it was indistinguishable from that in LTRPC2-transfected HEK-293 cells. The results provide evidence that LTRPC2 in neutrophil granulocytes forms an entry pathway for Na(+) and Ca(2+), which is regulated by ADP-ribose and the redox state.
Stimulation of membrane receptors linked to a phospholipase C and the subsequent production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (InsP(3)) is a signaling pathway ...of fundamental importance in eukaryotic cells. Signaling downstream of these initial steps involves mobilization of Ca(2+) from intracellular stores and Ca(2+) influx through the plasma membrane. For this influx, several contrasting mechanisms may be responsible but particular relevance is attributed to the induction of Ca(2+) influx as consequence of depletion of intracellular calcium stores. This phenomenon (frequently named store-operated calcium entry, SOCE), in turn, may be brought about by various signals, including soluble cytosolic factors, interaction of proteins of the endoplasmic reticulum with ion channels in the plasma membrane, and a secretion-like coupling involving translocation of channels to the plasma membrane. Experimental approaches to analyze these mechanisms have been considerably advanced by the discovery of mammalian homologs of the Drosophila cation channel transient receptor potential (TRP). Some members of the TRP family can be expressed to Ca(2+)-permeable channels that enable SOCE; other members form channels activated independently of stores. TRP proteins may be an essential part of endogenous Ca(2+) entry channels but so far expression of most TRP cDNAs has not resulted in restitution of channels found in any mammalian cells, suggesting the requirement for further unknown subunits. A major exception is CaT1, a TRP channel demonstrated to provide Ca(2+)-selective, store-operated currents identical to those characterized in several cell types. Ongoing and future research on TRP channels will be crucial to understand the molecular basis of receptor-mediated Ca(2+) entry, with respect to the structure of the entry channels as well as to the mechanisms of its activation and regulation.
Depletion of intracellular calcium stores generates a signal that activates Ca2+-permeable channels in the plasma membrane. We have identified a human cDNA, TRPC1A, from a human fetal brain cDNA ...library. TRPC1A is homologous to the cation channels trp and trpl in Drosophila and is a splice variant of the recently identified cDNA Htrp-1. Expression of TRPC1A in CHO cells induced nonselective cation currents with similar permeabilities for Na+, Ca2+, and Cs+. The currents were activated by intracellular infusion of myo inositol 1,4,5-trisphosphate or thapsigargin. Expression of TRPC1A significantly enhanced increases in the intracellular free calcium concentration induced by Ca2+ restitution after prolonged depletion. Similar results were obtained in Sf9 cells. We conclude that TRPC1A encodes a Ca2+-permeable cation channel activated by depletion of intracellular calcium stores.
Aims
This study aimed to assess safety, tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) effects of ensovibep, a designed ankyrin repeat protein antiviral being evaluated as a COVID‐19 ...treatment, in healthy volunteers in a first‐in‐human ascending single‐dose study.
Methods
Subjects were dosed intravenously, in a randomized double‐blinded manner, with either ensovibep at 3, 9 or 20 mg/kg or with placebo, and followed until Day 100. PK and safety were assessed throughout the study duration. Immunogenicity and PD via viral neutralization in serum were also assessed.
Results
All adverse events were of mild to moderate severity, and no serious adverse events were observed. One subject who received the 20‐mg/kg dose presented with moderate hypersensitivity vasculitis 3 weeks after infusion, which fully resolved using standard procedures. In most subjects ensovibep showed expected mono‐exponential decline with a half‐life of around 2 weeks. Anti‐drug antibodies were detected in 15 of 17 subjects, with the earliest onset detected on Day 29. Viral neutralization assays on subject serum showed effective viral neutralization over the first 3 weeks following dosing with titre values in a dose dependent manner.
Conclusion
Ensovibep proved safe in this first‐in‐human safety study and exhibited PK and PD parameters consistent with the expected treatment period required for acute COVID‐19 infection.
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad ...variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).
A first-in-human study was performed with MP0250, a DARPin drug candidate. MP0250 specifically inhibits both vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) with the aim ...of disrupting the tumor microenvironment.
A multicenter, open-label, repeated-dose, phase I study was conducted to assess the safety, tolerability, and pharmacokinetics of MP0250 in 45 patients with advanced solid tumors. In the dose-escalation part, 24 patients received MP0250 as a 3-hour infusion once every 2 weeks at five different dose levels (0.5-12 mg/kg). Once the maximum tolerated dose (MTD) was established, 21 patients were treated with a 1-hour infusion (n = 13, 8 mg/kg, once every 2 weeks and n = 8, 12 mg/kg, once every 3 weeks) of MP0250 in the dose confirmation cohorts.
In the dose-escalation cohort, patients treated with 12 mg/kg MP0250 once every 2 weeks experienced dose-limiting toxicities. Therefore, MTD was 8 mg/kg once every 2 weeks or 12 mg/kg once every 3 weeks. The most common adverse events (AEs) were hypertension (69%), proteinuria (51%), and diarrhea and nausea (both 36%); hypoalbuminemia was reported in 24% of patients. Most AEs were consistent with inhibition of the VEGF and HGF pathways. Exposure was dose-proportional and sustained throughout the dosing period for all patients (up to 15 months). The half-life was about 2 weeks. Signs of single-agent antitumor activity were observed: 1 unconfirmed partial response with a time to progression of 23 weeks and 24 patients with stable disease, with the longest duration of 72 weeks and a median duration of 18 weeks.
MP0250 is a first-in-class DARPin drug candidate with suitable tolerability and appropriate pharmacokinetic properties for further development in combination with other anticancer therapies.
Abstract
CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor receptor superfamily which can activate both innate and adaptive immune system, making it an interesting target for ...tumor immunotherapy. Systemic administration of agonistic CD40 antibodies (Ab) has shown signs of activity in cancer patients, but dose-limiting toxicity impaired the clinical efficacy. New approaches are therefore needed to increase the therapeutic index of CD40-targeting molecules and achieve better clinical outcomes. Here, we report an alternative approach designed to activate CD40 specifically in the tumor microenvironment (TME), and not systemically, in order to increase efficacy and reduce systemic toxicity. This novel approach is based on a bispecific DARPin® molecule, targeting CD40 and fibroblast activation protein (FAP) alpha, intended to induce immune activation only when clustered by binding to FAP-expressing cells in the TME. The bispecific FAP x CD40 DARPin®molecule was tested in a reporter assay and in additional cell assays using primary human 1) B cells, 2) macrophages and 3) dendritic cells. These studies demonstrated CD40 activation only in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x CD40) was generated and tested in similar in vitro assays and showed FAP-dependent activation of CD40 and comparable results as the human construct. In vivo experiments, performed in tumor-free mice, showed a comparable half-life between mFAP x CD40 and an anti-mouse CD40 Ab (clone FGK45). However, mFAP x CD40, in contrast to the FGK45 Ab, did not increase the serum level of IL-6, supporting a mode of action that is dependent on FAP-mediated crosslinking of CD40 receptor. In additional studies mFAP x CD40 was active and inhibited the growth of FAP+ tumors. There were no signs of toxicity with mFAP x CD40 in contrast to the FGK45 Ab which resulted in body weight loss. In conclusion, we have generated bispecific agonist FAP x CD40 DARPin® molecules able to activate the CD40 pathway with a targeting (FAP)-dependent mechanism of action.
Citation Format: Nicolo Rigamonti, Anja Schlegel, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Christof Zitt, Niina Veitonmäki, Victor Levitsky, Clara Metz. Fibroblast activation protein (FAP)-selective delivery of CD40 agonistic DARPin®molecule for tumor-localized immune activation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3251.
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