The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants

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)

Implementation of immuno-assays for the early diagnosis of hypothyroidism

Le diagnostic précoce de l'hypothyroïdie permet d'initier le traitement au plus tôt et ainsi de préserver la santé du patient. Le bénéfice du traitement de l'hypothyroïdie franche a été depuis longtemps établi, mais les critères de prise en charge des patients en hypothyroïdie fruste sont encore difficiles à définir. En effet, les symptômes ne sont pas toujours présents et leur appréciation est subjective. Afin d'établir le diagnostic et la prise en charge, le médecin s'appuie sur le dosage de la thyréostimuline (TSH) dans le sang, qui peut éventuellement être complété par le dosage des hormones thyroïdiennes. Le dosage de la TSH, très sensible, peut présenter sur un même échantillon sanguin d'importantes variations qui rendent d'autant plus difficiles la décision du médecin et le suivi du patient. Le polymorphisme naturel de la TSH peut expliquer en partie ces variations. La TSH appartient en effet à la famille des hormones glycoprotéiques et sa glycosylation peut constituer jusqu'à 30% de son poids. Dans le cas de l'hypothyroïdie en particulier, ces glycanes sont modifiés et présentent une plus grande quantité d'acides sialiques terminaux. Ainsi, certaines variations entre les dosages de la TSH, qui freinent actuellement leur harmonisation, peuvent être dues à des différences de reconnaissance de glycoformes par les anticorps utilisés dans les dosages. Dans ce contexte, l'objectif de de ces travaux était de contribuer à la construction de dosages plus performants que ceux actuellement utilisés dans le diagnostic de l'hypothyroïdie. Un nouveau calibrateur recombinant sialylé plus proche de la TSH circulante dans l'hypothyroïdie a alors été produit. De nouvelles associations d'anticorps monoclonaux ont été utilisées pour construire des dosages. Les nouveaux dosages sélectionnés ont ensuite été calibrés avec la TSH sialylée produite et le calibrateur de référence international. Ils ont alors servi à doser plusieurs séries de sérums de patients. Ces travaux ont donc validé l'utilisation d'un nouveau calibrateur d'origine recombinante pour les dosages de la TSH, ce qui devrait à l'harmonisation des dosages existants.If iodine deficiency is the first cause of low thyroid hormone levels in the world, there are also other etiologies to thyroid disorders. Diagnosis of those allow an early treatment to preserve patient's health. Although there is a general agreement concerning treatment of overt hypothyroidism, treatment of subclinical hypothyroidism is still under debate. In these cases, symptoms are, by definition, not always present. In order to establish diagnosis, the clinicians rely on the measurement of circulating thyroid stimulating hormone (TSH, potentially completed with thyroid hormones measurement). TSH assays are now very sensitive, but can present important between assays variations. The diagnosis and follow up of the patient are consequently complicated. Natural polymorphism of TSH can explain a part of this variability. TSH belongs to the glycoprotein hormones family and its glycans can count for more than 30% of its weight. In hypothyroidism, these glycans are subject of modulation and present higher levels of terminal sialylation. Variation in immuno-assays can be explained by these modifications of sialylation if recognition by antibodies used in immuno-assays is glycosylation dependent. In this context, the aim of this work was to contribute to the construction of new immuno-assays, more reliable in the early diagnosis of subclinical hypothyroidism. During this thesis a new recombinant standard closer to circulating TSH was produced. The total level of sialylation was higher and better mimic the circulating forms in hypothyroidism. In order to select the best antibodies associations in immuno-assays, new antibodies were obtained and associated with commercially available antibodies. New immuno assays improvement is based on the following two approaches: the first one is the use of a new standard which presents glycoformes closer to the circulating TSH and the second one consists in an appropriate selection of antibodies involved in the assays. The new assays were used to measure TSH concentration in blood samples. These studies associated with validation steps allow us to select four assays and constitute a proof of concept for the use of a new sialylated recombinant standard for TSH assays. This can contribute to the needed harmonization of TSH assays

The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging DARPin (designed ankyrin repeat protein) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells co-expressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mouse models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity towards LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057)