Differential roles of CTP synthetases CTPS1 and CTPS2 in cell proliferation.

The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and CTPS2) that catalyze the conversion of UTP into CTP. CTP synthetase activity is high in proliferating cells including cancer cells; however, the respective roles of CTPS1 and CTPS2 in cell proliferation are not known. By inactivation of CTPS1 and/or CTPS2 and complementation experiments, we showed that both CTPS1 and CTPS2 are differentially required for cell proliferation. CTPS1 was more efficient in promoting proliferation than CTPS2, in association with a higher intrinsic enzymatic activity that was more resistant to inhibition by 3-deaza-uridine, an UTP analog. The contribution of CTPS2 to cell proliferation was modest when CTPS1 was expressed but essential in absence of CTPS1. Public databases analysis of more than 1,000 inactivated cancer cell lines for CTPS1 or CTPS2 confirmed that cell growth is highly dependent of CTPS1 but less or not of CTPS2. Therefore, our results demonstrate that CTPS1 is the main contributor to cell proliferation.

Target occupancy biomarker assay development using a conformation-selective antibody against small-molecule-bound TNF.

Background: An antibody specific to small-molecule inhibitor-bound TNF has enabled the development of target occupancy biomarker assays to support the development of novel treatments for autoimmune disorders. Materials & methods: ELISAs were developed for inhibitor-bound and total TNF to determine the percentage of TNF occupancy in samples from stimulated blood. Inhibitor-saturated samples allowed measurement of total and inhibitor-bound TNF in a single electrochemiluminescence immunoassay. Results: TNF occupancy was proportional to inhibitor concentration in plasma samples. An electrochemiluminescence method for inhibitor-bound TNF was validated for use as a potential clinical occupancy biomarker assay. Conclusion: Development of these assays has allowed measurement of a target occupancy biomarker, which has supported progression of the first small-molecule inhibitors of TNF.

An orally available small molecule that targets soluble TNF to deliver anti-TNF biologic-like efficacy in rheumatoid arthritis.

Tumor necrosis factor (TNF) is a pleiotropic cytokine belonging to a family of trimeric proteins with both proinflammatory and immunoregulatory functions. TNF is a key mediator in autoimmune diseases and during the last couple of decades several biologic drugs have delivered new therapeutic options for patients suffering from chronic autoimmune diseases such as rheumatoid arthritis and chronic inflammatory bowel disease. Attempts to design small molecule therapies directed to this cytokine have not led to approved products yet. Here we report the discovery and development of a potent small molecule inhibitor of TNF that was recently moved into phase 1 clinical trials. The molecule, SAR441566, stabilizes an asymmetrical form of the soluble TNF trimer, compromises downstream signaling and inhibits the functions of TNF in vitro and in vivo. With SAR441566 being studied in healthy volunteers we hope to deliver a more convenient orally bioavailable and effective treatment option for patients suffering with chronic autoimmune diseases compared to established biologic drugs targeting TNF.

Transitioning EPMA applications in a large multisite teaching hospital.

INTRODUCTION: University Hospitals of Leicester (UHL) has co-developed and deployed a novel Electronic Prescribing and Medicines Administration (EPMA) application as part of the trust electronic patient record (EPR) programme that meets specific clinical demands and interoperability standards of the National Health Service (NHS) despite clinical pressures from the COVID-19 pandemic. METHODS: Following an initial limited pilot deployment, a big-bang whole site-based approach allowed transition of 1844 acute adult inpatients beds from an existing standalone EMPA to the new system. This project used a frontline driven and agile management strategy. Clinical risk was managed using a combination of standard risk logs, robust clinical prototyping and robust disaster recovery plans. Early engagement with clinical teams allowed for advanced product configuration before live deployment and reduced the need for sustained transition support for clinical staff. RESULTS: An iterative, well-governed approach, led by a combination of information technology (IT) and clinical staff with a responsive vendor, enabled a complex new EPMA system in a large acute NHS trust to be deployed with limited resources despite the ongoing COVID-19 pandemic. DISCUSSION: The development and deployment of EMPA and EPR systems across NHS trusts is a key enabler for better healthcare delivery. This case study shows it is possible to deploy a new clinical IT system at scale without interruption of clinical services and with a relatively modest deployment team. Sustainability of the project was also ensured through a clear clinically led governance structure to manage risk quickly and carry lessons learnt onto new developments.

Codeveloping an effective EMPA to maturity in an acute NHS Trust: an implementer report.

INTRODUCTION: University Hospitals Leicester has codeveloped, with Nervecentre, an Electronic Prescribing and Medicines Administration System that meets specific clinical and interoperability demands of the National Health Service (NHS). METHODS: The system was developed through a frontline-led and agile approach with a project team consisting of clinicians, Information Technology (IT) specialists and the vendor's representatives over an 18-month period. RESULTS: The system was deployed successfully with more than a thousand transcriptions during roll-out. Despite the high caseload and novelty of the system, there was no increase in error rates within the first 3 months of roll-out. Healthcare professionals perceived the new system as efficient with improved clinical workflow, and safe through an integrated medication alert system. DISCUSSION: This case study demonstrates how NHS trusts can successfully co-develop, with vendors, new IT systems which meet interoperability standards such as Fast Healthcare Interoperability Resources, while improving front line clinical experience. CONCLUSION: Alternative methods to the 'big bang' deployment of IT projects, such as 'gradual implementation', must be demonstrated and evaluated for their ability to deliver digital transformation projects in the NHS successfully.

MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function.

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

Peri-operative Variables Associated With Prolonged Intensive Care Stay Following Cytoreductive Surgery for Ovarian Cancer.

BACKGROUND: Peri-operative variables associated with prolonged Intensive Care Unit (ICU) admission following cytoreductive surgery for ovarian cancer were investigated. PATIENTS AND METHODS: A retrospective review was carried out of patients admitted to the ICU following cytoreductive surgery for ovarian cancer in a single tertiary referral centre from 2015-2019. Patients were categorized according to length of ICU stay (<48 h and ≥48 h), and peri-operative variables were compared across the two groups. RESULTS: A total of 56 patients were admitted to the ICU post-operatively, 37 for <48 h and 19 for ≥48 h (range=3-11 days). Greater duration of procedure and estimated blood loss, bowel resection, higher post-operative lactate level, lower post-operative albumin level and requirement for post-operative blood products were associated with prolonged ICU stay. Increased intraoperative fluid requirement was an independent predictor of extended ICU stay. CONCLUSION: Utilizing identified intra-operative risk factors to perform individualized risk assessments might improve planning of ICU resources. Optimizing intraoperative fluid management may improve short-term patient outcomes.

Structural insights into the disruption of TNF-TNFR1 signalling by small molecules stabilising a distorted TNF.

Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors.

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF.

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.

Impaired lymphocyte function and differentiation in CTPS1-deficient patients result from a hypomorphic homozygous mutation.

Cytidine triphosphate (CTP) synthetase 1 (CTPS1) deficiency is caused by a unique homozygous frameshift splice mutation (c.1692-1G>C, p.T566Dfs26X). CTPS1-deficient patients display severe bacterial and viral infections. CTPS1 is responsible for CTP nucleotide de novo production involved in DNA/RNA synthesis. Herein, we characterized in depth lymphocyte defects associated with CTPS1 deficiency. Immune phenotyping performed in 7 patients showed absence or low numbers of mucosal-associated T cells, invariant NKT cells, memory B cells, and NK cells, whereas other subsets were normal. Proliferation and IL-2 secretion by T cells in response to TCR activation were markedly decreased in all patients, while other T cell effector functions were preserved. The CTPS1T566Dfs26X mutant protein was found to be hypomorphic, resulting in 80%-90% reduction of protein expression and CTPS activity in cells of patients. Inactivation of CTPS1 in a T cell leukemia fully abolished cell proliferation. Expression of CTPS1T566Dfs26X failed to restore proliferation of CTPS1-deficient leukemia cells to normal, except when forcing its expression to a level comparable to that of WT CTPS1. This indicates that CTPS1T566Dfs26X retained normal CTPS activity, and thus the loss of function of CTPS1T566Dfs26X is completely attributable to protein instability. This study supports that CTPS1 represents an attractive therapeutic target to selectively inhibit pathological T cell proliferation, including lymphoma.

Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer.

Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn's disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions.

Efficacy of an Inhaled IL-13 Antibody Fragment in a Model of Chronic Asthma.

RATIONALE: IL-13 is an important cytokine implicated in the pathogenesis of allergic asthma and is an attractive target for an inhaled therapeutic. OBJECTIVE: To investigate the efficacy of CDP7766, a nebulized inhaled anti-IL-13 monoclonal antibody Fab fragment, in a model of allergic asthma in cynomolgus macaques naturally sensitized to Ascaris suum. METHODS: CDP7766 was nebulized using a vibrating-membrane nebulizer on the basis of eFlow technology. The aerosol generated was analyzed to determine the particle size profile and the biophysical and functional properties of CDP7766. Nebulized CDP7766 (0.1-60 mg/animal, once daily for 5 d) was delivered via the inhaled route. MEASUREMENTS AND MAIN RESULTS: The investigational eFlow nebulizer used in this study generated a respirable aerosol of CDP7766 with no evidence of degradation, loss of potency, aggregation, or formation of particulates. Inhaled CDP7766 was well tolerated in the model (no adverse effects related to local irritation) and significantly inhibited BAL allergen-induced cytokine and chemokine upregulation (60 mg vs. vehicle: eotaxin-3, P < 0.0008; MIP [macrophage inflammatory protein]-1ß, IL-8, IFN-γ, P ≤ 0.01). CDP7766 significantly inhibited the increase in pulmonary resistance stimulated by inhaled allergen, measured 15 minutes and 24 hours after allergen challenge. CONCLUSION: Inhaled CDP7766 potently inhibited the function of IL-13 generated during the airway response to inhaled allergen in cynomolgus macaques, demonstrating the potential of inhaled anti-IL-13 therapeutics for the treatment of allergic asthma.

Collagen II antibody-induced arthritis in Tg1278TNFko mice: optimization of a novel model to assess treatments targeting human TNFα in rheumatoid arthritis.

BACKGROUND: Novel molecules that specifically target human TNFα in rheumatoid arthritis pose problems for preclinical assessment of efficacy. In this study collagen antibody-induced arthritis (CAIA) has been induced in human TNFα transgenic mice to provide a novel model that has been optimised for the evaluation of molecules targeting human TNFα. METHODS: Tg1278TNFko mice lack murine TNFα and are heterozygous for multiple copies of the human TNFα transgene that is expressed under normal physiological control. To establish CAIA, a collagen II monoclonal antibody cocktail (CAb) at 2, 4 or 8 mg was injected i.p. on Day 0 followed by a lipopolysaccharide (LPS) boost (10 or 100 µg) i.p. on Day 1 or Day 4. Animals were assessed for arthritis symptoms using a clinical score, cytokine levels (human TNFα, IL-1ß and IL-6) in sera and joints, and histopathology. The dependence of the model on human TNFα was determined by dosing animals with etanercept. RESULTS: Tg1278TNFko animals treated with 2, 4 or 8 mg CAb on Day 0, with 100 µg LPS on Day 4, had more severe arthritis and earlier symptoms than wild type animals at all doses of CAb tested. Subsequently it was found that the transgenic model did not require LPS at all for arthritis development but a lower dose of LPS (10 µg) was found necessary for reproducible and robust disease (close to 100% incidence, well-synchronised, with high arthritis scores). Furthermore the LPS challenge could be brought forward to Day 1 so that its' actions to facilitate disease could be separated temporally from the arthritis phase (beginning about Day 4). Etanercept, administered immediately after the serum spike of cytokines associated with LPS had subsided, was able to dose-dependently inhibit arthritis development and this was associated with a marked protection of the joints histologically on Day 14. Etanercept was also able to reverse the signs of arthritis when given therapeutically allowing animals to be matched for disease burden before dosing begins. CONCLUSIONS: The features of CAIA in Tg1278TNFko animals make the model well-suited to testing the next generation of therapeutics that will target human TNFα in rheumatoid arthritis.

Discovery and characterization of olokizumab: a humanized antibody targeting interleukin-6 and neutralizing gp130-signaling.

Interleukin-6 (IL-6) is a critical regulator of the immune system and has been widely implicated in autoimmune disease. Here, we describe the discovery and characterization of olokizumab, a humanized antibody to IL-6. Data from structural biology, cell biology and primate pharmacology demonstrate the therapeutic potential of targeting IL-6 at "Site 3", blocking the interaction with the signaling co-receptor gp130.

The discovery, engineering and characterisation of a highly potent anti-human IL-13 fab fragment designed for administration by inhalation.

We describe the discovery, engineering and characterisation of a highly potent anti-human interleukin (IL)-13 Fab fragment designed for administration by inhalation. The lead candidate molecule was generated via a novel antibody discovery process, and the selected IgG variable region genes were successfully humanised and reformatted as a human IgG γ1 Fab fragment. Evaluation of the biophysical properties of a selection of humanised Fab fragments in a number of assays allowed us to select the molecule with the optimal stability profile. The resulting lead candidate, CA652.g2 Fab, was shown to have comparable activity to the parental IgG molecule in a range of in vitro assays and was highly stable. Following nebulisation using a mesh nebuliser, CA652.g2 Fab retained full binding affinity, functional neutralisation potency and structural integrity. Epitope mapping using solution nuclear magnetic resonance confirmed that the antibody bound to the region of human IL-13 implicated in the interaction with IL-13Rα1 and IL-13Rα2. The work described here resulted in the discovery and design of CA652.g2 human γ1 Fab, a highly stable and potent anti-IL-13 molecule suitable for delivery via inhalation.

The use of surrogate antibodies to evaluate the developmental and reproductive toxicity potential of an anti-TNFalpha PEGylated Fab' monoclonal antibody.

Certolizumab pegol (CZP) is a PEGylated Fab' fragment of a humanized monoclonal immunoglobulin G (IgG)1 antibody that binds to human tumor necrosis factor alpha (TNFα) with high affinity. As for many monoclonal antibodies (mAbs), nonclinical safety assessment of CZP has been constrained because of its limited species cross-reactivity and recognition of only nonhuman primate and human TNFα, which presents particular challenges for assessing reproductive and developmental safety. To comprehensively assess the potential liability of TNFα suppression on reproductive and developmental processes, a PEGylated Fab' anti-rat TNFα antibody surrogate (cTN3 PF) has been developed and evaluated for reproductive toxicity. Conventional rat fertility and early embryonic development, embryo-fetal toxicity and pre- and postnatal development studies have been shown to be free of maternal, reproductive, or development toxicity effects, following sustained TNFα inhibition with cTN3 PF. Importantly, these studies have also shown that in marked contrast to a whole IgG anti-TNFα antibody, the PEGylated Fab' antibody cTN3 PF homologous to certolizumab pegol demonstrated negligible fetal exposure following maternal administration during the period of organogenesis. In addition to minimal placental transmission, transfer to milk was lower and fetal absorption negligible compared with the whole IgG antibody cTN3 γ1, resulting in little or no detectable antibody in the plasma of lactating pups.

A PEGylated Fab' fragment against tumor necrosis factor for the treatment of Crohn disease: exploring a new mechanism of action.

Antibodies, having a high specificity for their particular target, are increasingly being used as therapeutic agents with functions including agonist, antagonist, and targeted drug delivery. The use of many biologic therapies, including antibody fragments, is generally limited by their rapid clearance from plasma. A commonly used approach to extend exposure to biologic therapies is the attachment of polyethylene glycol.Tumor necrosis factor (TNF)-alpha is a multifunctional cytokine involved in the regulation of immune responses. Elevated levels of TNFalpha are found in a wide range of diseases, including the chronic inflammatory conditions rheumatoid arthritis, psoriasis, and Crohn disease (CD). Anti-TNFalpha antibodies have proved highly efficacious in the treatment of these conditions. In addition, they have proved invaluable for investigating the role of TNFalpha in disease etiology. Based on evidence showing that neutralizing antibodies to TNFalpha were effective in animal models of CD, anti-TNFalpha antibody treatments were assessed in clinical trials. Interestingly, the anti-TNFalpha antibody etanercept proved ineffective at achieving remission in active CD despite potently neutralizing soluble TNFalpha. This indicated that an additional mode of action is also involved in the efficacy of the anti-TNFalpha agents adalimumab, certolizumab pegol, and infliximab in CD; one suggestion was apoptosis. However, etanercept, like adalimumab and infliximab, can induce apoptosis. Furthermore, certolizumab pegol (which has demonstrated efficacy in CD) does not cause complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, apoptosis, or necrosis of neutrophils, all measured in vitro. These functional differences observed with certolizumab pegol stem from its unique structure that does not include the crystallizable fragment (Fc) portion present in the other anti-TNFalpha agents, and the way in which it signals through membrane TNF. It is well established that bacteria are a major part of the inflammatory process in CD. The property identified that reflected the efficacies of the anti-TNFalpha agents etanercept, adalimumab, certolizumab pegol, and infliximab in CD was the ability to inhibit the cytokine production by monocytes that is induced by bacterial lipopolysaccharide. It may therefore be the case that this mode of action is important for efficacy in CD.

Mechanism of action of certolizumab pegol (CDP870): in vitro comparison with other anti-tumor necrosis factor alpha agents.

BACKGROUND: Inhibitors of tumor necrosis factor alpha (TNFalpha) have demonstrated significant efficacy in chronic inflammatory diseases, including Crohn's disease (CD). To further elucidate the mechanisms of action of these agents, we compared the anti-TNFalpha agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems. METHODS: The ability of each anti-TNFalpha agent to neutralize soluble and membrane-bound TNFalpha; mediate cytotoxicity, affect apoptosis of activated human peripheral blood lymphocytes and monocytes; induce degranulation of human peripheral blood granulocytes, and modulate lipopolysaccharide (LPS)-induced interleukin (IL)-1beta production by human monocytes was measured in vitro. RESULTS: All 4 agents neutralized soluble TNFalpha and bound to and neutralized membrane TNFalpha. Infliximab and adalimumab were comparable in their ability to mediate complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and to increase the proportion of cells undergoing apoptosis and the level of granulocyte degranulation. Etanercept generally mediated these effects to a lesser degree, while certolizumab pegol gave similar results to the control reagents. LPS-induced IL-1beta production was inhibited by certolizumab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept. CONCLUSIONS: In contrast to the other anti-TNFalpha agents tested, certolizumab pegol did not mediate increased levels of apoptosis in any of the in vitro assays used, suggesting that these mechanisms are not essential for the efficacy of anti-TNFalpha agents in CD. As certolizumab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS-induced IL-1beta release from monocytes, inhibition of cytokine production may be important for efficacy of anti-TNFalpha agents in CD.

Essential role for CD103 in the T cell-mediated regulation of experimental colitis.

The integrin CD103 is highly expressed at mucosal sites, but its role in mucosal immune regulation remains poorly understood. We have analyzed the functional role of CD103 in intestinal immune regulation using the T cell transfer model of colitis. Our results show no mandatory role for CD103 expression on T cells for either the development or CD4+CD25+ regulatory T (T reg) cell-mediated control of colitis. However, wild-type CD4+CD25+ T cells were unable to prevent colitis in immune-deficient recipients lacking CD103, demonstrating a nonredundant functional role for CD103 on host cells in T reg cell-mediated intestinal immune regulation. Non-T cell expression of CD103 is restricted primarily to CD11c(high)MHC class II(high) dendritic cells (DCs). This DC population is present at a high frequency in the gut-associated lymphoid tissue and appears to mediate a distinct functional role. Thus, CD103+ DCs, but not their CD103- counterparts, promoted expression of the gut-homing receptor CCR9 on T cells. Conversely, CD103- DCs promoted the differentiation of IFN-gamma-producing T cells. Collectively, these data suggest that CD103+ and CD103- DCs represent functionally distinct subsets and that CD103 expression on DCs influences the balance between effector and regulatory T cell activity in the intestine.