Interleukin (IL)-7 is broadly active on T-cell populations, and modified versions have been clinically evaluated for a variety of therapeutic applications, including cancer, lymphopenia, and ...infectious diseases; and found to be relatively well-tolerated and biologically active. Here we describe novel IL-7R agonists that are unrelated in structure to IL-7, bind to the receptor subunits differently from IL-7, but closely emulate IL-7 biology. The small size, low structural complexity, and the natural amino acid composition of the pharmacologically active peptide MDK1472 allows facile incorporation into protein structures, such as the IgG2-Fc fusion MDK-703. This molecule possesses properties potentially better suited to therapeutic applications than native IL-7 or its derivatives. We compared these compounds with IL-7 for immune cell selectivity, induction of IL-7R signaling, receptor-mediated internalization, proliferation, and generation of immune cell phenotypes in human and non-human primate (NHP) peripheral blood cells in vitro; and found them to be similar in biological activity to IL-7. In cynomolgus macaques, MDK-703 exhibits a circulating half-life of 46 hr and produces sustained T-cell expansion characteristic of IL-7 treatment. In the huCD34.sup.+ -engrafted NSG mouse model of the human immune system, MDK-703 induces an immune cell profile very similar to that generated by IL-7-derived compounds; including the pronounced expansion of memory T-cells, particularly the population of stem-like memory T-cells (Tscm) which may be important for anti-tumor activities reported with IL-7 treatment. Clinical administration of IL-7 and modified variants has been reported to induce anti-drug antibodies (ADAs), including IL-7 neutralizing antibodies. The novel peptide agonist reported here scores very low in predicted immunogenicity, and because the peptide lacks sequence similarity with IL-7, the problematic immunogenic neutralization of endogenous cytokine should not occur. The properties we report here implicate MDK-703 as a candidate for clinical evaluation in oncology, anti-viral and other infectious disease, vaccine enhancement, and treatment of lymphopenia.
The endocrine hormone FGF21 has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to the native cytokine, we generated bispecific Avimer ...polypeptides that bind with high affinity and specificity to one of the receptor and coreceptor pairs used by FGF21, FGFR1c and β-Klotho. These Avimers exhibit FGF21-like activity in in vitro assays with potency greater than FGF21. In a study conducted in obese male cynomolgus monkeys, animals treated with an FGFR1c/β-Klotho bispecific Avimer showed improved metabolic parameters and reduced body weight comparable to the effects seen with FGF21. These results not only demonstrate the essential roles of FGFR1c and β-Klotho in mediating the metabolic effects of FGF21, they also describe a first bispecific activator of this unique receptor complex and provide validation for a novel therapeutic approach to target this potentially important pathway for treating diabetes and obesity.
BackgroundRecent reports demonstrate that directing a “non-alpha” IL-2 mutant to a PD-1high CD8+ stem-like population induces proliferation, and differentiation into a highly functional cytotoxic ...phenotype. We previously reported small synthetic peptides, unrelated to IL-2 or IL-15, that bind IL-2/15Rβγc to induce receptor signaling. These peptides do not bind IL-2Rα and are therefore IL-2/15Rβγc-restricted agonists. We now describe fusion of potency-attenuated peptide agonists to an anti-PD-1 antibody (α-PD-1) to achieve selective targeting to PD-1high lymphocytes, and enhanced potency of IL-2R agonists acting in Cis with α-PD-1 binding.MethodsPeptidyl IL-2/15Rβγc agonists with attenuated potency due to weakened binding to either IL-2/15Rβ or γc were fused to the C-termini of both heavy chains of an α-PD-1 IgG and expressed in CHO cells. The fusion proteins retained PD-1 binding affinity comparable to the α-PD-1; and were evaluated for potency of IL-2Rβγc-dependent STAT5 phosphorylation in TF-1β cells (with undetectable PD-1 expression), and in TF-1β-derived lines expressing varying levels of PD-1. The fusion proteins were also assessed for Rβγc stimulation of CD8+ cells treated with anti-CD3 and anti-CD28 to induce elevated PD-1 expression.ResultsAn analysis of pembrolizumab (Pem) fused to MDK1169, a potent IL-2Rβγc agonist, showed a 15-fold increase in potency in TF-1β/PD-1+ cells. This served as an initial demonstration of the PD-1-directed, cis-acting mechanism; but the potency of MDK1169 in this construct (~500pM, EC50 pSTAT5 induction) is too high (relative to the affinity of Pem for PD-1) to achieve a more substantial selectivity for PD-1+ cells. To improve selectivity, fusions of α-PD-1 to peptide agonists with potencies as weak as 1uM on TF-1β cells were constructed. Some of these fusion proteins exhibited up to 100-fold increase in potency when tested on TF-1β/PD-1high compared to parental TF-1β cells; and addition of an excess of α-PD-1 blocked this gain in potency in the PD-1high cells. When tested on CD8+ cells activated to express elevated PD-1 levels, potency of the PD-1-directed agonists correlated with PD-1 expression.ConclusionsThe malleability of peptidyl agonists makes them useful for optimizing antibody-targeted cis-acting agonists designed to produce minimal activity on non-targeted cells and high potency at targeted cells. IL-2/15Rβγc agonists directed by PD-1 binding to a stem-like highly cytotoxic tumor infiltrating CD8+ population may have useful anti-tumor applications.
BackgroundIL-7 receptor activation is essential for the proper development and homeostasis of T-cell subpopulations, and maintenance of the TCR clonal repertoire. Emerging evidence indicates ...potential clinical utility of IL-7 for immunotherapy of lymphopenia, oncology, and other indications. Here we report the discovery of MDK1319, a small novel peptidyl agonist of IL-7R. This peptide is structurally unrelated to IL-7, with a MW less than 5000D. To improve in vivo properties, we fused MDK1319 to an IgG-Fc-domain to construct MDK-701, which exhibits biological properties similar to those of IL-7 in vitro, and when administered to non-human primates.MethodsPeptides were selected from peptide libraries by screens designed to identify molecules binding simultaneously to the Rα and γc subunits of the human IL-7 receptor. Synthetic peptides, and peptides fused to IgG Fc-domains were evaluated for efficacy, potency, and quality of signaling in IL-7-responsive cell lines and human lymphocytes. PK/PD properties in non-human primates were also determined.ResultsMDK1319 and MDK-701 activate the major IL-7R signaling pathways, JAK-STAT (pSTAT5), and PI3K (pAKT), and induce proliferation in human PBMCs, exhibiting lymphocyte subpopulation selectivity, kinetics, efficacy, and potency similar to those of IL-7. Agonism is attributable to direct activation of IL-7R, as shown by dependence on the presence of the IL-7Rα subunit for response in test cells, and insensitivity to IL-7 neutralizing antibodies. MDK1319 and MDK-701 do not activate nor inhibit any other (off target) Rγc family receptors at concentrations 100-fold greater than required for maximal IL-7R activation. MDK-701 administered to cynomologous macaques (single dose, IV at 1 mg/kg) exhibits a circulating terminal half life of ~32 hr; and induces peripheral lymphocyte profiles similar to IL-7 treatment, including initial reduction (tissue migration), followed by sustained elevation of peripheral lymphocytes remaining above baseline for 29 days, with no observed adverse effects.ConclusionsIn addition to the utility of Fc-fusion MDK-701 for monotherapy, the small peptidyl nature of the active peptide MDK1319, fusable to recombinant protein partners, offers opportunities for incorporation into bispecific molecules, linking IL-7 activity to a variety of useful functions. These include synergistic cytokine activities, checkpoint blockade, and tissue targeting. Cells engineered to secrete MDK1319 display autocrine stimulation potentially useful in T-cell therapeutics. The structural novelty of MDK1319 substantially decreases risk of cross reactivity of any anti-drug immune response with endogenous IL-7, and may provide a safer alternative to modified forms of IL-7 reported to produce significant anti-IL-7 immunogenicity.Ethics ApprovalAnimal studies were performed by Envol Biomedical or Charles Rivers Laboratories, as approved by the Institution Ethics Boards with the following study and approval numbers:Envol Biomedical 7037-20 MDK2002; 7037-20: PK/PD Cynomolgus monkeysCharles Rivers Laboratories 20200121001K; US19001: PK Mouse The use of human PBMC in this study was authorized under Minimal Risk Research Related Activities at Stanford Blood Center (SQL 79075)
BackgroundEfforts to modify IL-2 for immuno-oncology applications focus on modifying the receptor selectivity of IL-2 to bias effects on immune cells; in particular, to reduce Rα interaction via ...mutation, chemical modification, complexation with antibodies, or fusion to the Rα-ectodomain. IL-2/15Rβγc-biased agonists also incorporate PK enhancement to extend duration of action, and reduce side effects associated with peak drug levels. We previously reported discovery of small synthetic peptides, unrelated to IL-2 or IL-15, that simultaneously bind IL-2Rβ and γc subunits to induce IL-2/15R signaling. These peptides do not bind IL-2Rα, and are therefore IL-2/15Rβγc-selective agonists with MW less than 5000D. We now describe properties of an IL-2/15Rβγc agonist peptide fused to an Fc-domain (MDK-202).MethodsPeptides were selected from recombinant peptide libraries to identify molecules binding simultaneously to the β and γc subunits of IL-2/15R. Active peptides were fused to Fc-domains to evaluate efficacy, potency, and quality of signaling upon activating IL-2/15Rβγc in cell lines and human lymphocytes. PK and PD properties in mice and NHP were also determined.ResultsMDK-202 exhibits in vitro potency similar to the synthetic peptide (MDK1169). MDK-202 does not bind IL-2Rα, activates the major IL-2/15R signaling pathways: JAK-STAT(pSTAT5), MAPK (pERK1/2), PI3K (pAKT), and induces proliferation (Ki-67) in human PBMCs, with kinetics and efficacy similar to IL-2. Agonism is attributable to direct activation of IL-2/15Rβγc as shown by dependence on Rβ expression in test cells, and insensitivity to blockade by neutralizing antibodies against IL-2 and IL-15. At concentrations greatly exceeding that required for maximum IL-2/15R activation in vitro, MDK-202 does not interfere with the activities of other Rγc family receptors. The predicted immunogenicity potential for MDK-202 is very low, and in the unlikely event of MDK-202-induced ADA, neutralization of endogenous IL-2 or IL-15 would not be expected. MDK-202 is highly stable in human serum, showing no significant degradation after 4 days at 37C. In ex vivo human PBMC and in vivo studies in normal mice, hPBMC-engrafted NCG mice, and non-human primates, MDK-202 exhibited extended half-life, and activation, proliferation, and population dynamics of lymphocytes similar to those induced by ‘non-Rα’ variants of IL-2.ConclusionsMDK-202 is an attractive alternative to IL-2/15 variants for use in immuno-oncology therapy. Constructed without reference or similarity to cytokine or receptor structures or contacts, the peptidyl agonist component (MDK1169) is completely unique, and shown to be active when fused to other proteins such as anti-PD-1 antibodies and other cytokine receptor agonists.Ethics ApprovalAnimal studies were performed by Charles Rivers Laboratories, as approved by the CRL Instuition Ethics Board with the following study and approval numbers:CRL-220007; 20222440 : PK Cynomolgus Monkeys: BA-e451;BA-e451: PD NCG mice BA-e411; BA-e411:PD NCG miceKey 2152; US19001: PK mice: The use of human PBMC in this study was authorized under Minimal Risk Research Related Activities at Stanford Blood Center (SQL 79075).
BackgroundActivation of IL-2/15Rβγc or IL-7R on immune cells using modified versions of IL-2 or IL-7 is under investigation as monotherapy, or in combination with checkpoint inhibitors, engineered T ...or NK cells, or neo-antigen vaccines. We previously described small synthetic peptides, unrelated to IL-2, IL-15, or IL-7, that selectively activate either IL-2/15Rβγc or IL-7R. IL-2/15Rβγc and IL-7R activation exhibit complementary effects on immune cells that when combined may offer benefits over each independent mechanism. We now report the creation of an Fc-fusion protein that incorporates both IL-2/15Rβγc and IL-7R agonist peptides, and characterize its properties in cell lines and human (PBMC) lymphocyte subpopulations.MethodsPeptide agonists of IL-2/15Rβγc (MDK1169) and IL-7R (MDK1319) were separately fused to each chain of obligate heterodimeric (asymmetric) Fc molecules. The Fc-fusion was purified by protein-A and size exclusion chromatography, and characterized by LC-MS. Receptor-mediated signaling, proliferation, and cell-surface receptor expression in cell lines, PBMCs, mixed and isolated lymphocyte subpopulations were determined by flow cytometry and ELISA to evaluate effects of IL-2, IL-2v, IL-7, MDK-202 or MDK-701 (Fc-fusions of MDK1169 and MDK1319, respectively), and combinations (mixtures) of these molecules, in comparison with the dual agonist MDK-271.ResultsLC-MS analysis indicates MDK-271 is a heterodimeric molecule containing one copy each of MDK1169 (IL-2/15Rβγc-biased agonist) and MDK1319 (IL-7R agonist) fused to individual Fc-chains. Cell-based assay of MDK-271 demonstrates potent, fully efficacious phosphorylation of STAT5 in TF-1 cells expressing Rγc, and engineered to express either IL-2/15Rβ or IL-7Rα. PBMCs exposed ex vivo to MDK-271 exhibit additive, complementary, and synergistic effects among various lymphocyte subpopulations: CD4+Tn, Teff, Treg, Tmem; C8+Tn, Teff, Tmem; and NK cells, in this analysis. The mono-specific agonists MDK-202 and MDK-701 produce proliferative effects and signaling patterns in responsive cell lines and lymphocyte subsets similar to those induced by IL-2v (an IL-2/15Rβγc-biased mutant of IL-2) and IL-7, respectively. Combining both activities in MDK-271 induces response profiles that differ in some T-cell subsets from those of mono-specific agonists of the two receptors. Animal studies designed to understand the effects of these differences are underway.ConclusionsIL-2/15Rβγc and IL-7R are both currently undergoing extensive scrutiny as potential immuno-oncology therapeutic targets. The biology of these cytokines is both overlapping and complementary in stimulating and supporting T-cell populations; and some recent evidence suggests possible superiority of the combination. Based on in vitro properties, the Fc-peptide fusion reported here, exhibiting both IL-2/15Rβγc-biased agonist and Il-7Rαγc agonist activities, could be valuable in anti-tumor therapeutic applications.Ethics ApprovalThe use of human PBMC in this study was authorized under Minimal Risk Research Related Activities at Stanford Blood Center (SQL 79075)
This study investigated mental health- and work-related problems of 67 rescue workers (police officers and medical emergency drivers) caused by the accumulation of critical incidents during their ...career. Using Hobfoll's theory of conservation of resources, this is one of the first studies in The Netherlands that tries to shed some light at the number of critical incidents experienced by rescue workers. The moderation effect of social support from colleagues and supervisors was also studied. Although no effects were found for the health questionnaires, a significant effect was found for the number of critical incidents on the experience of workload. No significant moderation effects of social support were found. Implications for police practice, such as using assessment tools for critical incidents and further research are discussed.
This study explores how shared decision-making (SDM) is integrated in undergraduate nursing and medical education.
A dual-method design was applied. The integration of SDM in medicine and nursing ...education programs (i.e. SDM on paper) was explored through document analyses; the integration of SDM in curricula (i.e. SDM in class) through interviews with teachers and curriculum coordinators (N = 19).
A majority of the education programs featured SDM, mostly non-explicit. In curricula SDM was generally implicitly featured in compulsory courses across all study years. SDM was often integrated into preexisting theories and models and taught through various methods and materials. Generally, teachers and supervisors were not trained in SDM themselves. They assessed students’ competence in SDM in a summative manner.
Overall, SDM was featured in undergraduate nursing and medical education, however, very implicitly.
•Shared decision-making is featured in undergraduate nursing and medical education•Nonetheless, shared decision-making is variedly and mostly implicitly integrated•Teachers and supervisors often are not trained in shared decision-making•The competency of taking the patient’s perspective into account should receive more attention
E-selectin is an inducible cell adhesion molecule which mediates rolling of neutrophils on the endothelium, an early event in the development of an inflammatory response. Inhibition of ...selectin-mediated rolling is a possible means for controlling inflammation-induced diseases, and several classes of compounds have been tested for this use. We describe here the use of recombinant peptide library screening for identification and optimization of novel ligands which bind to E-selectin. Several of these peptides bind with Kd values in the low nanomolar range and block E-selectin-mediated adhesion of neutrophils in static and flow-cell assays. Administration of the peptide to mice undergoing an acute inflammatory response reduced the extent of neutrophil transmigration to the site of inflammation, demonstrating the utility of this compound as a potential therapeutic. The identification of a peptide ligand for E-selectin suggests that the complete natural ligand for this adhesion molecule may include protein as well as carbohydrate moieties.
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