Modulation of the high concentration viscosity of IgG1 antibodies using clinically validated Fc mutations.

The self-association of therapeutic antibodies can result in elevated viscosity and create problems in manufacturing and formulation, as well as limit delivery by subcutaneous injection. The high concentration viscosity of some antibodies has been reduced by variable domain mutations or by the addition of formulation excipients. In contrast, the impact of Fc mutations on antibody viscosity has been minimally explored. Here, we studied the effect of a panel of common and clinically validated Fc mutations on the viscosity of two closely related humanized IgG1, κ antibodies, omalizumab (anti-IgE) and trastuzumab (anti-HER2). Data presented here suggest that both Fab-Fab and Fab-Fc interactions contribute to the high viscosity of omalizumab, in a four-contact model of self-association. Most strikingly, the high viscosity of omalizumab (176 cP) was reduced 10.7- and 2.2-fold by Fc modifications for half-life extension (M252Y:S254T:T256E) and aglycosylation (N297G), respectively. Related single mutations (S254T and T256E) each reduced the viscosity of omalizumab by ~6-fold. An alternative half-life extension Fc mutant (M428L:N434S) had the opposite effect in increasing the viscosity of omalizumab by 1.5-fold. The low viscosity of trastuzumab (8.6 cP) was unchanged or increased by ≤2-fold by the different Fc variants. Molecular dynamics simulations provided mechanistic insight into the impact of Fc mutations in modulating electrostatic and hydrophobic surface properties as well as conformational stability of the Fc. This study demonstrates that high viscosity of some IgG1 antibodies can be mitigated by Fc mutations, and thereby offers an additional tool to help design future antibody therapeutics potentially suitable for subcutaneous delivery.

Tissue-free liquid biopsies combining genomic and methylation signals for minimal residual disease detection in patients with early colorectal cancer from the UK TRACC Part B study.

PURPOSE: Absence of post-operative circulating tumour DNA (ctDNA) identifies resected colorectal cancer (CRC) patients with low recurrence risk for adjuvant chemotherapy (ACT) de-escalation. We present the largest resected CRC cohort to date with tissue-free minimal residual disease (MRD) detection. DESIGN: TRACC included stage I-III resectable CRC patients. Prospective longitudinal plasma collection for ctDNA occurred pre- and post-surgery, post-ACT, every 3m for year 1 and every 6m in years 2 and 3 with imaging annually. The Guardant Reveal assay evaluated genomic and methylation signals. The primary endpoint was 2yr recurrence free survival (RFS) by post-operative ctDNA detection. (NCT04050345) Results: Between December 2016 and August 2022, 1203 were patients enrolled. Plasma samples (n=997) from 214 patients were analysed. 143 patients were evaluable for the primary endpoint; 92 (64.3%) colon, 51 (35.7%) rectal; 2 (1.4%) stage I, 64 (44.8%) stage II, 77 (53.8%) stage III. Median follow-up was 30.3m (95% CI: 29.5-31.3). 2yr RFS was 91.1% in patients with ctDNA not detected post-operatively and 50.4% in those with ctDNA detected (HR 6.5 [2.96-14.5] p<0.0001). Landmark negative predictive value (NPV) was 91.2% (95% CI 83.9-95.9). Longitudinal sensitivity and specificity were 62.1% (95% CI 42.2-79.3) and 85.9% (95% CI 78.9-91.3) respectively. Median lead-time from ctDNA detection to radiological recurrence was 7.3m (IQR 3.3-12.5; n=9). CONCLUSIONS: Tissue-free MRD detection with longitudinal sampling predicts recurrence in stage I-III CRC without need for tissue sequencing. NPV is high supporting ACT de-escalation in patients with ctDNA not detected post-operatively, now being investigated in the UK TRACC Part C study.

Nonclinical immunogenicity risk assessment for knobs-into-holes bispecific IgG1 antibodies.

Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.

Gene Electrotransfer via Conductivity-Clamped Electric Field Focusing Pivots Sensori-Motor DNA Therapeutics: "A Spoonful of Sugar Helps the Medicine Go Down".

Viral vectors and lipofection-based gene therapies have dispersion-dependent transduction/transfection profiles that thwart precise targeting. The study describes the development of focused close-field gene electrotransfer (GET) technology, refining spatial control of gene expression. Integration of fluidics for precise delivery of "naked" plasmid deoxyribonucleic acid (DNA) in sucrose carrier within the focused electric field enables negative biasing of near-field conductivity ("conductivity-clamping"-CC), increasing the efficiency of plasma membrane molecular translocation. This enables titratable gene delivery with unprecedently low charge transfer. The clinic-ready bionics-derived CC-GET device achieved neurotrophin-encoding miniplasmid DNA delivery to the cochlea to promote auditory nerve regeneration; validated in deafened guinea pig and cat models, leading to improved central auditory tuning with bionics-based hearing. The performance of CC-GET is evaluated in the brain, an organ problematic for pulsed electric field-based plasmid DNA delivery, due to high required currents causing Joule-heating and damaging electroporation. Here CC-GET enables safe precision targeting of gene expression. In the guinea pig, reporter expression is enabled in physiologically critical brainstem regions, and in the striatum (globus pallidus region) delivery of a red-shifted channelrhodopsin and a genetically-encoded Ca2+ sensor, achieved photoactivated neuromodulation relevant to the treatment of Parkinson's Disease and other focal brain disorders.

Novel Injectable Hydrogel Formulations and Gas Chromatography Analysis of the Residual Crosslinker in Formulations Intended for Pharmaceutical and Cosmetic Applications.

Novel hyaluronic acid (HA) crosslinked with pentaerythritol tetra-acrylate (PT) injectable hydrogels was invented. These injectable hydrogel/dermal filler formulations were synthesised using HA and the acrylate PT as a crosslinker under basic pH conditions using thermal crosslinking methods (oven heating), which provides a simple, safe, and eco-friendly method for crosslinking in 4 h under 45 °C. Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) analyses were conducted to represent the difference between the formulations in terms of peak formation and pore size, respectively. The crosslinking was partial as is considered to be typical for dermal injectable fillers. The rheological properties of these formulations showed that these novel dermal injectables are highly promising, and the newly developed fillers could be used with better results for dermal anti-wrinkle corrections, shaping, and volumising reasons. Furthermore, crosslinker (PT) residual analysis was carried out to state the formulations that are valid and acceptable for intradermal usage. The results from the GC method validation revealed it was a suitable method for this study. The GC analysis of all five injectable hydrogel/filler formulations demonstrated the formulations HA-PT 1, 2, 3 and 4 were formulated using (0.05-0.1)% w/w PT containing residual PT monomers within the safe limits that were determined to be below (0.008% w/w). This work has shown the development of a novel injectable hydrogel/filler formulation for pharmaceutical and cosmetic applications can be prepared in a more sustainable and simple way using pentaerythritol tetra-acrylate as a crosslinker agent, which holds great promise for the industry's future advancement.

Deciphering platinum dissolution in neural stimulation electrodes: Electrochemistry or biology?

Platinum (Pt) is the metal of choice for electrodes in implantable neural prostheses like the cochlear implants, deep brain stimulating devices, and brain-computer interfacing technologies. However, it is well known since the 1970s that Pt dissolution occurs with electrical stimulation. More recent clinical and in vivo studies have shown signs of corrosion in explanted electrode arrays and the presence of Pt-containing particulates in tissue samples. The process of degradation and release of metallic ions and particles can significantly impact on device performance. Moreover, the effects of Pt dissolution products on tissue health and function are still largely unknown. This is due to the highly complex chemistry underlying the dissolution process and the difficulty in decoupling electrical and chemical effects on biological responses. Understanding the mechanisms and effects of Pt dissolution proves challenging as the dissolution process can be influenced by electrical, chemical, physical, and biological factors, all of them highly variable between experimental settings. By evaluating comprehensive findings on Pt dissolution mechanisms reported in the fuel cell field, this review presents a critical analysis of the possible mechanisms that drive Pt dissolution in neural stimulation in vitro and in vivo. Stimulation parameters, such as aggregate charge, charge density, and electrochemical potential can all impact the levels of dissolved Pt. However, chemical factors such as electrolyte types, dissolved gases, and pH can all influence dissolution, confounding the findings of in vitro studies with multiple variables. Biological factors, such as proteins, have been documented to exhibit a mitigating effect on the dissolution process. Other biological factors like cells and fibro-proliferative responses, such as fibrosis and gliosis, impact on electrode properties and are suspected to impact on Pt dissolution. However, the relationship between electrical properties of stimulating electrodes and Pt dissolution remains contentious. Host responses to Pt degradation products are also controversial due to the unknown chemistry of Pt compounds formed and the lack of understanding of Pt distribution in clinical scenarios. The cytotoxicity of Pt produced via electrical stimulation appears similar to Pt-based compounds, including hexachloroplatinates and chemotherapeutic agents like cisplatin. While the levels of Pt produced under clinical and acute stimulation regimes were typically an order of magnitude lower than toxic concentrations observed in vitro, further research is needed to accurately assess the mass balance and type of Pt produced during long-term stimulation and its impact on tissue response. Finally, approaches to mitigating the dissolution process are reviewed. A wide variety of approaches, including stimulation strategies, coating electrode materials, and surface modification techniques to avoid excess charge during stimulation and minimise tissue response, may ultimately support long-term and safe operation of neural stimulating devices.

Millimeter wave-based patient setup verification and motion tracking during radiotherapy.

BACKGROUND: Position verification and motion monitoring are critical for safe and precise radiotherapy (RT). Existing approaches to these tasks based on visible light or x-ray are suboptimal either because they cannot penetrate obstructions to the patient's skin or introduce additional radiation exposure. The low-cost mmWave radar is an ideal solution for these tasks as it can monitor patient position and motion continuously throughout the treatment delivery. PURPOSE: To develop and validate frequency-modulated continuous wave (FMCW) mmWave radars for position verification and motion tracking during RT delivery. METHODS: A 77 GHz FMCW mmWave module was used in this study. Chirp Z Transform-based (CZT) algorithm was developed to process the intermediate frequency (IF) signals. Absolute distances to flat Solid Water slabs and human shape phantoms were measured. The accuracy of absolute distance and relative displacement were evaluated. RESULTS: Without obstruction, mmWave based on the CZT algorithm was able to detect absolute distance within 1 mm for a Solid Water slab that simulated the reflectivity of the human body. Through obstructive materials, the mmWave device was able to detect absolute distance within 5 mm in the worst case and within 3.5 mm in most cases. The CZT algorithm significantly improved the accuracy of absolute distance measurement compared with Fast Fourier Transform (FFT) algorithm and was able to achieve submillimeter displacement accuracy with and without obstructions. The surface-to-skin distance (SSD) measurement accuracy was within 8 mm in the anterior of the phantom. CONCLUSIONS: With the CZT signal processing algorithm, the mmWave radar is able to measure the absolute distance to a flat surface within 1 mm. But the absolute distance measurement to a human shape phantom is as large as 8 mm at some angles. Further improvement is necessary to improve the accuracy of SSD measurement to uneven surfaces by the mmWave radar.

Variable domain mutational analysis to probe the molecular mechanisms of high viscosity of an IgG1 antibody.

Subcutaneous injection is the preferred route of administration for many antibody therapeutics for reasons that include its speed and convenience. However, the small volume limit (typically ≤2 mL) for subcutaneous delivery often necessitates antibody formulations at high concentrations (commonly ≥100 mg/mL), which may lead to physicochemical problems. For example, antibodies with large hydrophobic or charged patches can be prone to self-interaction giving rise to high viscosity. Here, we combined X-ray crystallography with computational modeling to predict regions of an anti-glucagon receptor (GCGR) IgG1 antibody prone to self-interaction. An extensive mutational analysis was undertaken of the complementarity-determining region residues residing in hydrophobic surface patches predicted by spatial aggregation propensity, in conjunction with residue-level solvent accessibility, averaged over conformational ensembles from molecular dynamics simulations. Dynamic light scattering (DLS) was used as a medium throughput screen for self-interaction of ~ 200 anti-GCGR IgG1 variants. A negative correlation was found between the viscosity determined at high concentration (180 mg/mL) and the DLS interaction parameter measured at low concentration (2-10 mg/mL). Additionally, anti-GCGR variants were readily identified with reduced viscosity and antigen-binding affinity within a few fold of the parent antibody, with no identified impact on overall developability. The methods described here may be useful in the optimization of other antibodies to facilitate their therapeutic administration at high concentration.

The introduction of a Surgical Care Practitioner training programme to an acute National Health Service trust and an exploration of the interrelationships between Surgical Care Practitioners, their trainers and surgical trainees.

This study aimed to explore the impact of the introduction of a Surgical Care Practitioner programme on junior surgical training within an acute National Health Service trust. A qualitative methodology of semi-structured interviews was used to gather information from eight Surgical Care Practitioners, eight surgical trainees and eight consultant grade trainers. The authors found an overall positive and mutually beneficial outcome of the training programme, with surgical trainees unanimous that the presence of the Surgical Care Practitioners freed them up for more time to be spent in theatre, as well as acting as highly experienced surgical assistants when the trainees were operating on their own. This study found significant mutual benefits to surgical trainees and Surgical Care Practitioners, as well as smoother running of the wards, theatres and the clinical firms through the addition of a highly skilled and versatile Surgical Care Practitioner workforce.

Inline dynamometry provides reliable measurements of quadriceps strength in healthy and ACL-reconstructed individuals and is a valid substitute for isometric electromechanical dynamometry following ACL reconstruction.

BACKGROUND: Quadriceps strength testing is recommended to guide rehabilitation and mitigate the risk of second injury following anterior cruciate ligament (ACL) reconstruction. Hand-held dynamometry is a practical alternative to electromechanical dynamometry but demonstrates insufficient reliability and criterion validity in healthy and ACL-reconstructed participants respectively. The purpose of this study is to investigate the reliability and concurrent validity of inline dynamometry for measuring quadriceps strength. The hypotheses are that intra-class correlation coefficient (ICC) values will be >0.90 for reliability and concurrent validity. METHODS: This was a cross sectional study using a within-participant, repeated measures design. Isometric quadriceps testing was performed at 60° knee flexion in 50 healthy and 52 ACL-reconstructed participants. Interrater reliability, intrarater reliability, and concurrent validity of inline dynamometry was investigated through calculation of ICCs, Bland-Altman analysis, linear regression, standard error of measurement (SEM) and minimal detectable change (MDC). RESULTS: The lower bounds of the 95% confidence intervals were >0.90 for all reliability and validity ICCs in healthy and ACL-reconstructed participants, except for intrarater reliability in healthy participants using absolute scores (ICC = 0.936 [95% CI 0.890-0.963]). In ACL-reconstructed participants, Bland-Altman bias was 0.01 Nm/kg for absolute and average scores, limits of agreement were -11.74% to 12.59% for absolute scores, the SEM was 0.13Nm/kg (95% CI 0.10-0.17) and the MDC was 0.36Nm/kg (95% CI 0.28 - 0.47). CONCLUSION: Inline dynamometry is a reliable and economical alternative to electromechanical dynamometry for the assessment of quadriceps strength following ACL-reconstruction. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT05109871).

Sex-Specific Reproductive Factors Augment Cardiovascular Disease Risk in Women: A Mendelian Randomization Study.

Background Observational studies suggest that reproductive factors are associated with cardiovascular disease, but these are liable to influence by residual confounding. This study explores the causal relevance of reproductive factors on cardiovascular disease in women using Mendelian randomization. Methods and Results Uncorrelated (r2<0.001), genome-wide significant (P<5×10-8) single-nucleotide polymorphisms were extracted from sex-specific genome-wide association studies of age at first birth, number of live births, age at menarche, and age at menopause. Inverse-variance weighted Mendelian randomization was used for primary analyses on outcomes of atrial fibrillation, coronary artery disease, heart failure, ischemic stroke, and stroke. Earlier genetically predicted age at first birth increased risk of coronary artery disease (odds ratio [OR] per year, 1.49 [95% CI, 1.28-1.74], P=3.72×10-7) heart failure (OR, 1.27 [95% CI, 1.06-1.53], P=0.009), and stroke (OR, 1.25 [95% CI, 1.00-1.56], P=0.048), with partial mediation through body mass index, type 2 diabetes, blood pressure, and cholesterol traits. Higher genetically predicted number of live births increased risk of atrial fibrillation (OR for <2, versus 2, versus >2 live births, 2.91 [95% CI, 1.16-7.29], P=0.023), heart failure (OR, 1.90 [95% CI, 1.28-2.82], P=0.001), ischemic stroke (OR, 1.86 [95% CI, 1.03-3.37], P=0.039), and stroke (OR, 2.07 [95% CI, 1.22-3.52], P=0.007). Earlier genetically predicted age at menarche increased risk of coronary artery disease (OR per year, 1.10 [95% CI, 1.06-1.14], P=1.68×10-6) and heart failure (OR, 1.12 [95% CI, 1.07-1.17], P=5.06×10-7); both associations were at least partly mediated by body mass index. Conclusions These results support a causal role of a number of reproductive factors on cardiovascular disease in women and identify multiple modifiable mediators amenable to clinical intervention.

Molecular Characterization of Circulating Tumor DNA in Pediatric Rhabdomyosarcoma: A Feasibility Study.

PURPOSE: Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS: We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS: Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION: Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.

Reimagining antibody-dependent cellular cytotoxicity in cancer: the potential of natural killer cell engagers.

Bi-, tri- and multispecific antibodies have enabled the development of targeted cancer immunotherapies redirecting immune effector cells to eliminate malignantly transformed cells. These antibodies allow for simultaneous binding of surface antigens on malignant cells and activating receptors on innate immune cells, such as natural killer (NK) cells, macrophages, and neutrophils. Significant progress with such antibodies has been achieved, particularly in hematological malignancies. Nevertheless, several major challenges remain, including increasing their immunotherapeutic efficacy in a greater proportion of patients, particularly in those harboring solid tumors, and overcoming dose-limiting toxicities and immunogenicity. Here, we discuss novel antibody-engineering developments designed to maximize the potential of NK cells by NK cell engagers mediating antibody-dependent cellular cytotoxicity (ADCC), thereby expanding the armamentarium for cancer immunotherapy.

Coffee consumption and cancer risk: a Mendelian randomisation study.

BACKGROUND: Coffee contains many bioactive chemicals and associations with cancer have been reported in observational studies. In this Mendelian randomisation (MR) study we investigated the causal associations of coffee consumption with a broad range of cancers. MATERIALS AND METHODS: Twelve independent genetic variants proxied coffee consumption. Genetically-predicted risk of any cancer (59,647 cases) and 22 site-specific cancers was estimated in European-descent individuals in UK Biobank. Univariable and multivariable MR analyses were conducted. RESULTS: Genetically-predicted coffee consumption was not associated with risk of any cancer in the main analysis (OR 1.05, 95% CI 0.98-1.14, p = 0.183) but was associated with an increased risk of digestive system cancer (OR 1.28, 95% CI 1.09-1.51, p = 0.003), driven by a strong association with oesophageal cancer (OR 2.79, 95% CI 1.73-4.50, p = 2.5×10-5). This association was consistent after adjustment for genetically-predicted body mass index, smoking and alcohol consumption. There was no strong evidence supporting a causal relationship between genetically-predicted coffee consumption and the majority of cancers studied. However, genetically-predicted coffee consumption was associated with increased risk of multiple myeloma (OR 2.25, 95% CI 1.30-3.89, p = 0.004) and reduced ovarian cancer risk (OR 0.63, 95% CI 0.43-0.93, p = 0.020). CONCLUSIONS: This MR study provides strong support for a causal association of coffee consumption with oesophageal cancer, but not for the majority of cancer types, and the underlying mechanisms require investigation.

Circulating vitamin C and digestive system cancers: Mendelian randomization study.

BACKGROUND & AIMS: Vitamin C is an antioxidant with a potential role in the prevention of digestive system cancers, but there is yet no consensus whether vitamin C has a causal role in these cancers. The aim of this study was to utilize Mendelian randomization to decipher the potential causal associations of vitamin C with risk of digestive system cancers. METHODS: Ten genetic variants previously found to be significantly associated with circulating vitamin C were used as instrumental variables. Effect size estimates for the genetic associations of the vitamin C-associated genetic variants with six major malignancies of digestive system were obtained from the FinnGen (N = 309 154) and UK Biobank (N = 367 542) studies. Results from the two studies were combined using meta-analysis. RESULTS: Genetically predicted higher circulating vitamin C showed a suggestive association with lower risk of small intestine and colorectal cancer after accounting for multiple testing. The odds ratio per 1 standard deviation increment in circulating vitamin C was 0.55 (95% confidence interval 0.32-0.94; P = 0.029) for small intestine cancer and 0.84 (95% confidence interval 0.73-0.96; P = 0.013) for colorectal cancer. There was a suggestive association between genetically predicted higher circulating vitamin C with lower risk of liver cancer in FinnGen but no association in the meta-analysis (odds ratio 0.69; 95% CI 0.36-1.32; P = 0.265). Genetically predicted circulating vitamin C was not associated with cancers of the esophagus, stomach, or pancreas. CONCLUSION: This Mendelian randomization study indicates that vitamin C might play a role in the prevention of small intestine and colorectal cancer.

Genome-wide analyses of 200,453 individuals yield new insights into the causes and consequences of clonal hematopoiesis.

Clonal hematopoiesis (CH), the clonal expansion of a blood stem cell and its progeny driven by somatic driver mutations, affects over a third of people, yet remains poorly understood. Here we analyze genetic data from 200,453 UK Biobank participants to map the landscape of inherited predisposition to CH, increasing the number of germline associations with CH in European-ancestry populations from 4 to 14. Genes at new loci implicate DNA damage repair (PARP1, ATM, CHEK2), hematopoietic stem cell migration/homing (CD164) and myeloid oncogenesis (SETBP1). Several associations were CH-subtype-specific including variants at TCL1A and CD164 that had opposite associations with DNMT3A- versus TET2-mutant CH, the two most common CH subtypes, proposing key roles for these two loci in CH development. Mendelian randomization analyses showed that smoking and longer leukocyte telomere length are causal risk factors for CH and that genetic predisposition to CH increases risks of myeloproliferative neoplasia, nonhematological malignancies, atrial fibrillation and blood epigenetic ageing.

Designing antibodies as therapeutics.

Antibody therapeutics are a large and rapidly expanding drug class providing major health benefits. We provide a snapshot of current antibody therapeutics including their formats, common targets, therapeutic areas, and routes of administration. Our focus is on selected emerging directions in antibody design where progress may provide a broad benefit. These topics include enhancing antibodies for cancer, antibody delivery to organs such as the brain, gastrointestinal tract, and lungs, plus antibody developability challenges including immunogenicity risk assessment and mitigation and subcutaneous delivery. Machine learning has the potential, albeit as yet largely unrealized, for a transformative future impact on antibody discovery and engineering.

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells.

BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Genetic Liability to Rheumatoid Arthritis in Relation to Coronary Artery Disease and Stroke Risk.

OBJECTIVE: To assess the causality of the associations of rheumatoid arthritis (RA) with coronary artery disease (CAD) and stroke using the Mendelian randomization approach. METHODS: Independent single-nucleotide polymorphisms strongly associated with RA (n = 70) were selected as instrumental variables from a genome-wide association meta-analysis including 14,361 RA patients and 43,923 controls of European ancestry. Summary-level data for CAD, all stroke, any ischemic stroke and its subtypes, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage were obtained from meta-analyses of genetic studies, international genetic consortia, the UK Biobank, and the FinnGen consortium. We obtained summary-level data for common cardiovascular risk factors and related inflammatory biomarkers to assess possible mechanisms. RESULTS: Genetic liability to RA was associated with an increased risk of CAD and ICH. For a 1-unit increase in log odds of RA, the combined odds ratios were 1.02 (95% confidence interval [1.01, 1.03]; P = 0.003) for CAD and 1.05 (95% confidence interval [1.02, 1.08]; P = 0.001) for ICH. Genetic liability to RA was associated with increased levels of tumor necrosis factor and C-reactive protein (CRP). The association with CAD was attenuated after adjustment for genetically predicted CRP levels. There were no associations of genetic liability to RA with the other studied outcomes. CONCLUSION: This study found that genetic liability to RA was associated with an increased risk of CAD and ICH and that the association with CAD might be mediated by CRP. The heightened cardiovascular risk should be actively monitored and managed in RA patients, and this may include dampening systemic inflammation.

Promoting innovation while controlling cost: The UK's approach to health technology assessment.

New technologies, including pharmaceuticals and medical devices, can improve treatment options in healthcare but also bring concerns about rising healthcare costs. We undertake a narrative review of the United Kingdom's (UK) approach to appraising new health technologies. We find that the National Institute for Health and Care Excellence (NICE), Scottish Medicines Consortium (SMC) and All Wales Medicines Strategy Group (AWMSG) have contributed to the UK's robust and transparent approach towards the evaluation of new health technologies using the cost per QALY approach. However, there are limitations to this approach including several external benefits not captured, bias against less treatable diseases, and deciding the appropriate level of the threshold. NICE, SMC, and AWMSG have attempted to overcome some of these limitations by considering additional factors such as end-of-life criteria, highly specialised treatments, and populations that experience unmet need. Looking to the future, the advent of 'personalised' and 'genomic' medicine, will likely mean the UK has to accommodate an increasing number of 'step-change' and 'highly specialised' technologies as well as respond to changes in pharmaceutical licensing and increasing use of real-world evidence.