Pharmacokinetics and pharmacodynamics of t-cell bispecifics in the tumour interstitial fluid

The goal of this study is to investigate the pharmacokinetics in plasma and tumour interstitial fluid of two T-cell bispecifics (TCBs) with different binding affinities to the tumour target and to assess the subsequent cytokine release in a tumour-bearing humanised mouse model. Pharmacokinetics (PK) as well as cytokine data were collected in humanised mice after iv injection of cibisatamab and CEACAM5-TCB which are binding with different binding affinities to the tumour antigen carcinoembryonic antigen (CEA). The PK data were modelled and coupled to a previously published physiologically based PK model. Corresponding cytokine release profiles were compared to in vitro data. The PK model provided a good fit to the data and precise estimation of key PK parameters. High tumour interstitial concentrations were observed for both TCBs, influenced by their respective target binding affinities. In conclusion, we developed a tailored experimental method to measure PK and cytokine release in plasma and at the site of drug action, namely in the tumour. Integrating those data into a mathematical model enabled to investigate the impact of target affinity on tumour accumulation and can have implications for the PKPD assessment of the therapeutic antibodies.publishedVersio

Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells.

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.This work was supported by Wellcome Trust Grant 091157, JDRF International Grant 9-2011-253, the National Institute for Health Research Cambridge Biomedical Research Centre, and the Medical Research Council Cusrow Wadia Fund. The Cambridge Institute for Medical Research (CIMR) is in receipt of a Wellcome Trust Strategic Award (100140). U.M.N. was the recipient of a Hoffmann-La Roche postdoctoral fellowship.This is thefinal version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S089684111400146

Tumor-activated lymph node fibroblasts suppress T cell function in diffuse large B cell lymphoma

Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of LN fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identified the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network expressing elevated fibroblast-activated protein (FAP). RNA-Seq analyses revealed that exposure to DLBCL reprogrammed key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen-presentation molecules. Functional assays showed that DLBCL-activated FRCs (DLBCL-FRCs) hindered optimal TIL and chimeric antigen receptor (CAR) T cell migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrated the potential to target inhibitory FRCs to rejuvenate interacting TILs. Cotreating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented antilymphoma TIL cytotoxicity. Our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis, and optimizing immunotherapy for patients

Engineering of protein glycosylation in Chinese hamster ovary cells

Improved versions of therapeutic glycoproteins may be produced by manipulation of their glycosylation patterns. Engineering of glycoform biosynthesis in CHO cells is studied here as a route to produce new variants of a recombinant, therapeutic protein and to maximize the proportion of beneficial glycoforms within the glycoform population. An anti-neuroblastoma monoclonal antibody (chCE7) was used as a model therapeutic glycoprotein, and the target glycoforms were those carrying bi-antennary complex Nlinked oligosaccharides modified with a bisecting N-acetylglucosamine (GlcNAc). A mathematical model of N-linked glycoform biosynthesis was constructed and used to simulate the qualitative effects of overexpression of GlcNAc-transferase III (GnTIII), the enzyme catalyzing the transfer of a bisecting GlcNAc to various oligosaccharide substrates. These simulations indicated a maximum level for bisected complex oligosaccharides, and accumulation of hybrid bisected oligosaccharides. To investigate the effects of GnTIII overexpression experimentally, a CHO cell line with tetracycline-regulated overexpression of a rat GnTIII cDNA was established. Expressed GnTIII was localized in the Goigi complex of CHO cells by means of immunoelectron microscopy using an antibody against a peptide epitope tag added to the carboxy-terminus of the enzyme. The enzyme concentrated on one side of the Golgi, mainly in cisternal compartments. Using the experimental system, it was found that overexpression of GnTIII to high levels led to growth inhibition and was toxic to the cells. Another CHO cell line with tetracycline-regulated overexpression of GnTV, a distinct glycosyltransferase, showed the same inhibitory effect, indicating that this may be a general feature of glycosyltransferase overexpression. The growth effect set an upper limit to glycosyltransferase overexpression and may therefore also limit the extent to which poorly accessible glycosylation sites can be modified by engineering of glycosylation pathways. A set of chCE7 mAb samples differing in their glycoform distributions was produced by controlling GnTIII expression in a range between basal and toxic levels. Measurement of the ADCC activity of these samples showed an optimal range of GnTIII expression for maximal chCE7 in vitro biological activity. The activity correlated with the level of Fc-associated bisected, complex oligosaccharides. Expression of GnTIII within the biotechnologically practical range, i.e., where no significant growth inhibition and toxicity are observed, led to a consumption of more than 90% of non-bisected, non-galactosylated bi-antennary complex oligosaccharides, but, at most, 50% was converted to the target bisected, complex structures for this set of chCE7 samples. The pattern of oligosaccharide peaks in MALDUTOF-mass spectrometric analysis of samples produced at high levels of GnTIII, indicate that a significant proportion of potential GnTIII substrates is diverted to bisected hybrid oligosaccharide by-products. Minimization of these by-products by further engineering of the pathway could therefore be valuable. The new optimized variants of chCE7 are promising candidate reagents for the treatment of neuroblastoma. The strategy presented here may also be applicable to other therapeutic IgGs

Measuring the Impact of Shared Value. A Business Case

Shared Value is a concept that since its inception in 2011 has attracted the interest of academics, governments, businessmen and decision-makers in all areas. It is through this first exploratory study that is illustrated as a sample of three Central American companies create this value and how important this creation is for companies and society when is measured. Traditionally, the creation of value in the three dimensions of sustainability has not been quantified at the enterprise level, although it is mentioned in the reports of those that use the GRI but without trying to estimate a measure of this intrinsic value in the concept of sustainability. The liberal use of concepts of Sustainable Development, Sustainability and Corporate Social Responsibility, sometimes confusing, does not propose its measurement with the exception of the Corporate Sustainability model that only records the total financial bottom line without intending to separate it into its three dimensions. The study is exploratory and descriptive with a focus on measuring the phenomenon under study using a meta-matrix created based on the theory of Shared Value. The results of the research are represented in the matrix, which allows to open the pattern of continuing to measure more cases, has been raised in the Harvard Business School in Global Impact Council in December 2019 from this three cases from three different Central American countries: El Salvador, Honduras and Nicaragua. Finally, this research offers a discussion and proposal to study about Measuring Shared Value and the Ratio Social Value - Private Valu

Hypothyroidism is a Risk Factor for Atrial Fibrillation after Coronary Artery Bypass Graft

Abstract Introduction: Few reports in the world have shown a differential effect of hypothyroidism in relation to morbidity and mortality following cardiac surgery. Objective: To determine the association between preoperative hypothyroidism, composite and disaggregated outcomes of mortality and complications in patients undergoing first-time isolated myocardial revascularization surgery. Methods: Historical cohort of patients undergoing myocardial revascularization between January 2008 and December 2014, with 626 patients included for evaluation of the composite and disaggregated outcomes of in-hospital mortality and complications (atrial fibrillation, surgical site infection and reoperation due to bleeding). A logistic regression model was used to determine the association between hypothyroidism and the onset of those outcomes. Results: Cohort of 1696 eligible patients for the study, with 1.8 mortality. Median age, female gender and prevalence of arterial hypertension were all significantly higher among hypothyroid patients. No differences were found in other preoperative or intraoperative characteristics. Hypothyroidism was associated with the presence of the composite outcome, RR 1.6 (1.04-2.4) and atrial fibrillation 1.9 (1.05-3.8). No association with mortality, infections or reoperation due to bleeding was found. Conclusion: Hypothyroidism is a disease that affects females predominantly and does not determine the presence of other comorbidities. Hypothyroidism is a risk factor for the onset of postoperative fibrillation in patients undergoing myocardial revascularization surgery. Postoperative care protocols focused on the prevention of these complications in this type of patients must be instituted

Preclinical studies on the mechanism of action and the anti-lymphoma activity of the novel anti-CD20 antibody GA101.

International audienceGA101 is a novel glycoengineered Type II CD20 monoclonal antibody. When compared with rituximab, it mediates less complement-dependent cytotoxicity (CDC). As expected for a Type II antibody, GA101 appears not to act through CDC and is more potent than the Type I antibody rituximab in inducing cell death via nonclassical induction of apoptosis cytotoxicity, with more direct cytotoxicity and more antibody-dependent cell-mediated cytotoxicity. We evaluated the antitumor activity of GA101 against the human-transformed follicular lymphoma RL model in vivo in severe combined immunodeficient mice (SCID) mice. GA101 induced stronger inhibition of tumor growth than rituximab. Combination of GA101 with cyclophosphamide in vivo confirmed the superiority of GA101 over rituximab. Neutralizing the complement system with cobra venom factor partially impaired the antitumor activity of rituximab, but had no impact on the efficacy of GA101. In vitro GA101 more potently induced cell death of RL cells than rituximab. The expression of a limited number of genes was found to be induced by both antibodies after exposure in vitro. Among these, early growth response 1 and activation transcription factor 3 were confirmed to be increased at the protein level, suggesting a possible role of these proteins in the apoptotic signalling of anti-CD20 antibodies. These data imply that GA101 is superior to rituximab not only as a single agent, but also in combination with chemotherapy. These data suggest the presence of novel signalization pathways activated after exposure to anti-CD20 antibodies