Editor's Choice - Bypass versus Angioplasty for Severe Ischaemia of the Leg (BASIL) Prospective Cohort Study and the Generalisability of the BASIL-2 Randomised Controlled Trial.

OBJECTIVE: The Bypass versus Angioplasty in Severe Ischaemia of the Leg-2 (BASIL-2) randomised controlled trial has shown that, for patients with chronic limb threatening ischaemia (CLTI) who require an infrapopliteal (IP) revascularisation a vein bypass (VB) first revascularisation strategy led to a 35% increased risk of major amputation or death when compared with a best endovascular treatment (BET) first revascularisation strategy. The study aims are to place the BASIL-2 trial within the context of the CLTI patient population as a whole and to investigate the generalisability of the BASIL-2 outcome data. METHODS: This was an observational, single centre prospective cohort study. Between 24 June 2014 and 31 July 2018, the BASIL Prospective Cohort Study (PCS) was performed which used BASIL-2 trial case record forms to document the characteristics, initial and subsequent management, and outcomes of 471 consecutive CLTI patients admitted to an academic vascular centre. Ethical approval was obtained, and all patients provided fully informed written consent. Follow up data were censored on 14 December 2022. RESULTS: Of the 238 patients who required an infrainguinal revascularisation, 75 (32%) had either IP bypass (39 patients) or IP BET (36 patients) outside BASIL-2. Seventeen patients were initially randomised to BASIL-2. A further three patients who did not have an IP revascularisation as their initial management were later randomised in BASIL-2. Therefore, 95/471 (20%) of patients had IP revascularisation (16% outside, 4% inside BASIL-2). Differences in amputation free survival, overall survival, and limb salvage between IP bypass and IP BET performed outside BASIL-2 were not subject to hypothesis testing due to the small sample size. Reasons for non-randomisation into the trial were numerous, but often due to anatomical and technical considerations. CONCLUSION: CLTI patients who required an IP revascularisation procedure and were subsequently randomised into BASIL-2 accounted for a small subset of the CLTI population as a whole. For a wide range of patient, limb, anatomical and operational reasons, most patients in this cohort were deemed unsuitable for randomisation in BASIL-2. The results of BASIL-2 should be interpreted in this context.

Determining the affinities of high-affinity antibodies using KinExA and surface plasmon resonance.

Accurate and efficient affinity measurement techniques are essential for the biophysical characterization of therapeutic monoclonal antibodies, one of the fastest growing drug classes. Surface plasmon resonance (SPR) is widely used for determining antibody affinity, but does not perform well with extremely high affinity (low picomolar to femtomolar range) molecules. In this study, we compare the SPR-based Carterra LSA and the kinetic exclusion assay (KinExA) for measuring the affinities of 48 antibodies generated against the SARS-CoV-2 receptor-binding domain. These data reveal that high-affinity antibodies can be generated straight from selections using high-quality in vitro library platforms with 54% correspondence between affinities measured using LSA and KinExA. Generally, where there was a 2-fold or greater difference between LSA and KinExA, KinExA reported that affinities were tighter. We highlight the differences between LSA and KinExA, identifying the benefits and pitfalls of each in terms of dynamic range and throughput. Furthermore, we demonstrate for the first time that single-point screening with KinExA can significantly improve throughput while maintaining a strong correlation with full binding curve equilibrium measurements, enabling the accurate rank-ordering of clones with exceptionally tight binding properties.

Insights into next generation sequencing guided antibody selection strategies.

Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity. Our study establishes NGS guidelines for antibody drug discovery, demonstrating its advantages in expanding the number of unique HCDR3 clusters, broadening the number of high affinity antibodies, expanding the total number of antibodies recognizing different epitopes, and improving lead prioritization. Surprisingly, our investigation into the correlation between NGS-derived frequencies of CDRs and affinity revealed a lack of association, although this limitation could be moderately mitigated by leveraging NGS clustering, enrichment and/or relative abundance across different regions to enhance lead prioritization. This study highlights NGS benefits, offering insights, recommendations, and the most effective approach to leverage NGS in therapeutic antibody discovery.

High throughput purification of monoclonal recombinant antibodies using a Protein-A coated membrane plate system.

The therapeutic use of monoclonal antibodies (mAbs) ranges from cancer treatment to immune-mediated conditions, covering infectious and cardiovascular disorders, among others. The development of improved methods for therapeutic antibody discovery has accelerated the identification of numerous mAbs: a discovery campaign can be deeply mined, resulting in hundreds, even thousands, of potential antibody leads for a given target of interest. High throughput mAb expression and purification methods are required for the rapid validation of those leads. In this work, we describe the implementation of a Protein-A coated membrane plate system, the Purexa™ AHT membrane plate, for robust preparative purification of hundreds of recombinant mAbs, without the need for automation. The high efficiency (>80%) recovery generated sufficient mAb for downstream screening analyses such as ELISA and surface plasmon resonance (SPR). This new system allows the functional validation of hundreds of lead antibodies from discovery campaigns in a timely manner regardless of operational size.

A Systematic Review Network Meta-Analysis and Meta-Regression on Surgical and Endovenous Interventions for the Treatment of Lower Limb Venous Ulcer Disease.

BACKGROUND: Venous leg ulcer (VLU) disease constitutes the most severe form of chronic venous insufficiency. We performed a network meta-analysis and meta-regression to investigate the efficacy and safety of the various available interventions in the treatment of VLU. METHODS: We conducted a systematic research corresponding to the instructions by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement for studies reporting on surgical or endovenous interventions for the treatment of VLU. Following data extraction, we performed a Bayesian network meta-analysis and meta-regression. Primary endpoints included VLU healing and recurrence. The secondary endpoint was postintervention complications. RESULTS: Seventeen studies evaluating the impact of compression monotherapy, radiofrequency ablation (RFA), endovenous laser ablation (EVLA), sclerotherapy, and saphenous vein surgery on VLU treatment, consisting of 2156 patients (2186 VLU) were included. When compared to compression monotherapy, RFA was the only treatment displaying a statistically-significant impact on ulcer healing, odds ratio (OR) 5.80 (95% credibility interval (CI): 1.08-35.07), while EVLA, RR 0.06 (95% CI: 0.00-0.57), sclerotherapy, RR 0.07 (95% CI: 0.00-0.68) and RFA, RR 0.12 (95% CI: 0.01-0.91) were the 3 interventions reducing VLU recurrence. EVLA (SUCRA, 69.65) ranked as the most efficient intervention concerning ulcer recurrence reduction. Regarding postintervention complications, EVLA was the only intervention displaying a statistically-significant increased risk compared to compression monotherapy, RR 14.3 (95% CI: 2.03-172.56). Meta-regression analysis exploring the impact of perforator treatment on VLU failed to predict healing, ß = -0.27 (95% CI: -2.55 to 1.85), recurrence, ß = -0.02 (95% CI: -2.96 to 2.75) and complication outcomes, ß = -0.089 (95% CI: -3.13 to 2.85). During sensitivity analysis, RFA and sclerotherapy failed to sustain their effects on ulcer healing and ulcer recurrence, respectively. In addition, sclerotherapy displayed statistically-significant inferior outcomes compared to both EVLA and RFA regarding ulcer recurrence. CONCLUSION: This is the first network meta-analysis examining the effect of various interventions on VLU disease. While we have demonstrated the efficacy of RFA and ELVA in ulcer recurrence prevention, our results regarding the impact of RFA and sclerotherapy on VLU healing and recurrence, respectively, should be interpreted with caution. In addition, this review raises questions concerning the value of surgery and perforator interventions in the treatment of VLU. Further research through randomized controlled trials is required. CLINICAL IMPACT: Our review has revealed the significant efficacy of endovenous laser ablation (EVLA) and radiofrequency ablation (RFA) in effectively reducing the incidence of ulcer recurrence when compared to compression monotherapy. These outcomes hold the potential to provide relevant insights to both medical practitioners and patients, thereby informing a more prudent and enlightened decision-making approach. Such informed decisions, aimed at mitigating the recurring occurrence of venous leg ulcers, carry profound significance given the considerable socioeconomic implications associated with this medical condition.

A vein bypass first versus a best endovascular treatment first revascularisation strategy for patients with chronic limb threatening ischaemia who required an infra-popliteal, with or without an additional more proximal infra-inguinal revascularisation procedure to restore limb perfusion (BASIL-2): an open-label, randomised, multicentre, phase 3 trial.

BACKGROUND: Chronic limb-threatening ischaemia is the severest manifestation of peripheral arterial disease and presents with ischaemic pain at rest or tissue loss (ulceration, gangrene, or both), or both. We compared the effectiveness of a vein bypass first with a best endovascular treatment first revascularisation strategy in terms of preventing major amputation and death in patients with chronic limb threatening ischaemia who required an infra-popliteal, with or without an additional more proximal infra-inguinal, revascularisation procedure to restore limb perfusion. METHODS: Bypass versus Angioplasty for Severe Ischaemia of the Leg (BASIL)-2 was an open-label, pragmatic, multicentre, phase 3, randomised trial done at 41 vascular surgery units in the UK (n=39), Sweden (n=1), and Denmark (n=1). Eligible patients were those who presented to hospital-based vascular surgery units with chronic limb-threatening ischaemia due to atherosclerotic disease and who required an infra-popliteal, with or without an additional more proximal infra-inguinal, revascularisation procedure to restore limb perfusion. Participants were randomly assigned (1:1) to receive either vein bypass (vein bypass group) or best endovascular treatment (best endovascular treatment group) as their first revascularisation procedure through a secure online randomisation system. Participants were excluded if they had ischaemic pain or tissue loss considered not to be primarily due to atherosclerotic peripheral artery disease. Most vein bypasses used the great saphenous vein and originated from the common or superficial femoral arteries. Most endovascular interventions comprised plain balloon angioplasty with selective use of plain or drug eluting stents. Participants were followed up for a minimum of 2 years. Data were collected locally at participating centres. In England, Wales, and Sweden, centralised databases were used to collect information on amputations and deaths. Data were analysed centrally at the Birmingham Clinical Trials Unit. The primary outcome was amputation-free survival defined as time to first major (above the ankle) amputation or death from any cause measured in the intention-to-treat population. Safety was assessed by monitoring serious adverse events up to 30-days after first revascularisation. The trial is registered with the ISRCTN registry, ISRCTN27728689. FINDINGS: Between July 22, 2014, and Nov 30, 2020, 345 participants (65 [19%] women and 280 [81%] men; median age 72·5 years [62·7-79·3]) with chronic limb-threatening ischaemia were enrolled in the trial and randomly assigned: 172 (50%) to the vein bypass group and 173 (50%) to the best endovascular treatment group. Major amputation or death occurred in 108 (63%) of 172 patients in the vein bypass group and 92 (53%) of 173 patients in the best endovascular treatment group (adjusted hazard ratio [HR] 1·35 [95% CI 1·02-1·80]; p=0·037). 91 (53%) of 172 patients in the vein bypass group and 77 (45%) of 173 patients in the best endovascular treatment group died (adjusted HR 1·37 [95% CI 1·00-1·87]). In both groups the most common causes of morbidity and death, including that occurring within 30 days of their first revascularisation, were cardiovascular (61 deaths in the vein bypass group and 49 in the best endovascular treatment group) and respiratory events (25 deaths in the vein bypass group and 23 in the best endovascular treatment group; number of cardiovascular and respiratory deaths were not mutually exclusive). INTERPRETATION: In the BASIL-2 trial, a best endovascular treatment first revascularisation strategy was associated with a better amputation-free survival, which was largely driven by fewer deaths in the best endovascular treatment group. These data suggest that more patients with chronic limb-threatening ischaemia who required an infra-popliteal, with or without an additional more proximal infra-inguinal, revascularisation procedure to restore limb perfusion should be considered for a best endovascular treatment first revascularisation strategy. FUNDING: UK National Institute of Health Research Health Technology Programme.

Direct selection of functional fluorescent-protein antibody fusions by yeast display.

Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras. Here, we describe a high throughput method based on yeast display to directly select antibodies most suitable for conversion to fluorescent chimera. A library of scFv binders was converted to a fluorescent chimeric form, by cloning thermal green protein into the linker between VH and VL, and directly selecting for both binding and fluorescent functionality. This allowed us to directly identify antibodies functional in the single chain TGP format, that manifest higher protein expression, easier protein purification, and one-step binding assays.

Surgical implementation gap: an interrupted time series analysis with interviews examining the impact of surgical trials on surgical practice in England.

OBJECTIVES: Landmark studies published near the turn of the 21st century found an implementation gap concerning the effect of evidenced-based findings on clinical practice. The current study examines the uptake of six trials that produced actionable findings to describe the effects of evidence on practice and the reasons for those effects. DESIGN: A sequential, explanatory mixed methods study was conducted. First, a quantitative study assessed whether actionable findings from large, publicly funded elective surgical trials influenced practice. Subsequently, qualitative interviews were conducted to explain the quantitative findings. SETTING: Changes in NHS-funded practice were tracked across hospitals in England. Interviews were conducted online. DATA AND PARTICIPANTS: The six surgical trials were funded and published by England's National Institute for Health Research's Health Technology Assessment programme between 2006 and 2015. Quantitative time series analyses used data about the frequencies or proportions of relevant surgical procedures conducted in England between 2001 and 2020. Subsequently, qualitative interviews were conducted with 25 participants including study authors, surgeons and other healthcare staff in the supply chain. Transcripts were coded to identify major temporal events and Consolidated Framework for Implementation Research (CFIR) domains/constructs that could influence implementation. Findings were synthesised by clinical area. RESULTS: The quantitative analyses reveal that practice changed in accordance with findings for three trials. In one trial (percutaneous vs nasogastric tube feed after stroke), the change took a decade to occur. In another (patella resurfacing), change anticipated the trial findings. In the third (abdominal aortic aneurysm repair), changes tracked the evolving evidence base. In the remaining trials (two about varicose veins and one about gastric reflux), practice did not change in line with findings. For varicose veins, the results were superseded by a further trial. For gastric reflux, surgical referrals declined as medical treatment increased. The exploratory qualitative analysis informed by CFIR found that evidence from sources apart from the trial in question was mentioned as a reason for non-adoption in the three trials where evidence did not affect practice and in the trial where uptake was delayed. There were no other consistent patterns in the qualitative data. CONCLUSION: While practice does not always change in the direction indicated by clinical trials, our results suggest that individuals, official committees and professional societies do assimilate trial evidence. Decision-makers seem to respond to the totality of evidence such that there are often plausible reasons for not adopting the evidence of any one trial in isolation.

Evaluation of the Global Limb Anatomic Staging System in patients with chronic limb-threatening ischemia undergoing endovascular intervention for femoropopliteal disease.

BACKGROUND: The Global Limb Anatomic Staging System (GLASS) is a new method of quantifying the anatomic severity of infrainguinal disease in patients with chronic limb-threatening ischemia. However, because GLASS has undergone limited validation, its value as an aid to shared decision-making regarding the choice of revascularization strategy remains incompletely defined. Here we report the relationship between GLASS and outcomes in a contemporary series comprising all 309 patients who underwent an attempt at femoropopliteal and/or infrapopiteal endovascular therapy for chronic limb-threatening ischemia in our unit between 2009 and 2014. METHODS: Baseline patient characteristics and outcome data including immediate technical success (ITS), amputation-free survival (AFS), overall survival, limb salvage, freedom from reintervention (FF-R), and freedom from major adverse limb events (FF-MALE) were obtained from hospital databases. GLASS grades and stage were obtained from pre-endovascular therapy angiographic imaging. Outcome data were censored on May 31, 2017. RESULTS: Baseline patient characteristics were similar across different GLASS femoropopliteal and IP grades and overall limb stages. Worsening GLASS stage was associated with a significant reduction in ITS (97.5% vs 91.5% vs 84.0%; P = .029). At 72 months FF-R (hazard ratio, 2.00; 95% confidence interval, 1.11-3.57; P = .020) and FF-MALE (hazard ratio, 1.76, 95% confidence interval, 1.10-2.81; P = .019) were significant worse in GLASS stage 3 than in stage 2 limbs. CONCLUSIONS: In our study, there were significant differences in ITS, FF-R and FF-MALE between limbs with GLASS stage 2 and 3 disease. However, further GLASS refinement seems likely to be required if its usefulness in everyday clinical practice as an aid to shared decision-making regarding the choice of revascularization strategy is to be maximized.

Pandemic's silver lining.

The intense international focus on the COVID-19 pandemic has provided a unique opportunity to use a wide array of novel tools to carry out scientific studies on the SARS-CoV-2 virus. The value of these comparative studies extends far beyond their consequences for SARS-CoV-2, providing broad implications for health-related science. Here we specifically discuss the impacts of these comparisons on advances in vaccines, the analysis of host humoral immunity, and antibody discovery. As an extension, we also discuss potential synergies between these areas.Abbreviations: CoVIC: The Coronavirus Immunotherapeutic Consortium; EUA: Emergency Use Authorization.

Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs.

ABBREVIATIONS: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

A pandemic-enabled comparison of discovery platforms demonstrates a naïve antibody library can match the best immune-sourced antibodies.

As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies. Here, we show that it is possible to generate ultra-potent (IC50 < 2 ng/ml) human neutralizing antibodies directly from a unique semisynthetic naïve antibody library format with affinities, developability properties and neutralization activities comparable to the best from hyperimmune sources. This demonstrates that appropriately designed and constructed naïve antibody libraries can effectively compete with immunization to directly provide therapeutic antibodies against a viral pathogen, without the need for immune sources or downstream optimization.

Adverse neurological events after sodium tetradecyl sulfate foam sclerotherapy - A prospective, observational study of 8056 treatments.

BACKGROUND: Ultrasound guided foam sclerotherapy (UGFS) is a flexible and highly utilised tool in the treatment of varicose veins (VVs), both as a primary treatment and as an adjunct to other treatments. Concern remains regarding the risk of neurological adverse events (AEs) such as migraine, visual disturbance and serious adverse events (SAEs) such as cerebrovascular accident that have been reported after UGFS treatments. AIM: To determine the incidence of neurological AEs and SAEs after UGFS. METHODS: A prospective, multicentre, post-authorisation safety study across Europe (both private and government) was performed between January 2015-2020. Neurological adverse events after UGFS with Fibrovein® (Sodium Tetradecyl Sulfate) 1 and 3% physician generated foam. RESULTS: 8056 patients underwent treatment. There were 46 AE (including 5 SAEs), 30 (65%) SAEs were in female patients. Mean age was 55 years with mean body mass index (BMI) of 27. Univariable logistic regression demonstrate that UGFS only treatment (i.e. no adjunctive treatment), liquid-to-gas ratio, gas type and total foam volume (1% sodium tetradecyl sulfate, STS) were significantly associated with the odds of experiencing the outcome. Multivariable logistic regression model exhibits that migraine and total foam volume (1% STS) maintained statistical significance thus associated with the odds of adverse events. CONCLUSIONS: This study demonstrates that UGFS with Fibrovein is safe with a very low incidence of neurological AEs and SAEs. Past history of migraine, use of physiological gas (O2/CO2) and increasing volumes of 1% foam increase the risk of AEs.