Modular design of bi- and multi-specific knob domain fusions.

Introduction: The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively. Methods: Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights. Results: Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference. Discussion: Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.

Discovery of ginisortamab, a potent and novel anti-gremlin-1 antibody in clinical development for the treatment of cancer.

Gremlin-1, a high-affinity antagonist of bone morphogenetic proteins (BMP)-2, -4, and -7, is implicated in tumor initiation and progression. Increased gremlin-1 expression, and therefore suppressed BMP signaling, correlates with poor prognosis in a range of cancer types. A lack of published work using therapeutic modalities has precluded the testing of the hypothesis that blocking the gremlin-1/BMP interaction will provide benefits to patients. To address this shortfall, we developed ginisortamab (UCB6114), a first-in-class clinical anti-human gremlin-1 antibody, currently in clinical development for the treatment of cancer, along with its murine analog antibody Ab7326 mouse immunoglobulin G1 (mIgG1). Surface plasmon resonance assays revealed that ginisortamab and Ab7326 mIgG1 had similar affinities for human and mouse gremlin-1, with mean equilibrium dissociation constants of 87 pM and 61 pM, respectively. The gremlin-1/Ab7326 antigen-binding fragment (Fab) crystal structure revealed a gremlin-1 dimer with a Fab molecule bound to each monomer that blocked BMP binding. In cell culture experiments, ginisortamab fully blocked the activity of recombinant human gremlin-1, and restored BMP signaling pathways in human colorectal cancer (CRC) cell lines. Furthermore, in a human CRC - fibroblast co-culture system where gremlin-1 is produced by the fibroblasts, ginisortamab restored BMP signaling in both the CRC cells and fibroblasts, demonstrating its activity in a relevant human tumor microenvironment model. The safety and efficacy of ginisortamab are currently being evaluated in a Phase 1/2 clinical trial in patients with advanced solid tumors (NCT04393298).

Phage Display of Bovine Ultralong CDRH3.

Phage display is an in vitro technique used in the discovery of monoclonal antibodies that has been used successfully in the discovery of both camelid VHH and shark variable new antigen receptor domains (VNAR). Bovines also contain a unique "ultralong CDRH3" with a conserved structural motif, comprising a knob domain and ß-stalk. When removed from the antibody scaffold, either the entire ultralong CDRH3 or the knob domain alone, is typically capable of binding an antigen, to produce antibody fragments that are smaller than both VHH and VNAR. By extracting immune material from bovine animals and specifically amplifying knob domain DNA sequences by PCR, knob domain sequences can be cloned into a phagemid vector producing knob domain phage libraries. Target-specific knob domains can be enriched by panning the libraries against an antigen of interest. Phage display of knob domains exploits the link between phage genotype and phenotype and could prove to be a high throughput method to discover target-specific knob domains, helping to explore the pharmacological properties of this unique antibody fragment.

Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery.

Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα. The receptors are shared with IL-4, but IL-4 initially binds IL-4Rα. Here we report the identification and characterisation of a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 (KD 40 nM-5.5 µM) and inhibit downstream IL-13 signalling (IC50 0.2-53.8 µM). NMR mapping showed that the VHHs recognise a number of epitopes on IL-13, including previously unknown allosteric sites. Further NMR investigation of VHH204 bound to IL-13 revealed a novel allosteric mechanism of inhibition, with the antibody stabilising IL-13 in a conformation incompatible with receptor binding. This also led to the identification of a conformational equilibrium for free IL-13, providing insights into differing receptor signalling complex assembly seen for IL-13 compared to IL-4, with formation of the IL-13:IL-13Rα1 complex required to stabilise IL-13 in a conformation with high affinity for IL-4Rα. These findings highlight new opportunities for therapeutic targeting of IL-13 and we report a successful 19F fragment screen of the IL-13:VHH204 complex, including binding sites identified for several hits. To our knowledge, these 19F containing fragments represent the first small-molecules shown to bind to IL-13 and could provide starting points for a small-molecule drug discovery programme.

Serum albumin binding knob domains engineered within a VH framework III bispecific antibody format and as chimeric peptides.

Background: Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering. Methods: Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion. Results: By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity. Conclusions: This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.

Evaluating implementation of Diabetes Self-Management Education in Maryland County, Liberia: protocol for a pilot prospective cohort study.

INTRODUCTION: Achieving glycaemic targets for people living with diabetes (PLWD) is challenging, especially in settings with limited resources. Programmes need to address gaps in knowledge, skills and self-management. Diabetes Self-Management Education (DSME) is an evidence-based intervention to educate and empower PLWD to improve self-management activities. This protocol describes a pilot study assessing the feasibility, acceptability and effect on clinical outcomes of implementing DSME in clinics caring for people living with insulin-dependent diabetes in Liberia. METHODS AND ANALYSIS: Our protocol is a three-phased, mixed-methods, quasi-experimental prospective cohort study. Phase 1 focuses on (a) establishing a Patient Advisory Board and (b) training providers in DSME who provide care for PLWD. In phase 2, clinicians will implement DSME. In phase 3, we will train additional providers who interact with PLWD.We will assess whether this DSME programme can lead to increased provider knowledge of DSME, improvements in diabetes self-management behaviours, glycaemic control, diabetes knowledge and psychosocial well-being, and a reduction in severe adverse events. Primary outcomes of interest are implementation outcomes and change in frequency of self-management behaviours by patients. Secondary outcomes include change in haemoglobin A1c, psychosocial well-being, severe adverse events and change in provider knowledge of DSME. ETHICS AND DISSEMINATION: Ethical approval was obtained from the University of Liberia Institutional Review Board (IRB) and the Brigham and Women's Hospital IRB. Findings from the study will be shared with local and national clinical and programmatic stakeholders and published in an open-access, peer-reviewed journal.

Combating the Current Pandemic and Preparing for the Next: Lessons Learned From the COVID-19 Pandemic From the Perspective of Deployed Special Operations Forces.

The coronavirus 2019 (COVID-19) pandemic continues to be a threat to global health, including the health of deployed armed forces. Servicemembers had to adjust to the "new normal" while maintaining the interests of the nation's security as well as that of our host nation partners. This commentary examines how Special Operations Forces operating within four different regions worldwide leveraged the challenges presented by the onset of this pandemic in maintaining stability, sustaining a ready force, and operating forward deployed. Deployed forces face constant difficulties with logistical support, varied medical resources access and a medical system predominantly focused on trauma care. At the onset of the COVID-19 pandemic there was little guidance specific to these circumstances which required an improvised adaptation of the recommendations set by national and Department of Defense medical authorities. Plans were constantly revised to match the ever changing medical and operational environment. Strategies such as the "Bubble Philosophy" and tiered force protection measures helped our units to maintain a rigorous training cycle. New methods of communication and training with our host nation partners such as the use of Unmanned Aerial Systems (UAS) platforms to survey host nation training became standard. Through these measures all of our forces were able to maintain operational capacity, protect the force, and maintain rapport with the host nations. We hope these experiences will provide a rough framework for future forces faced with a similar struggle. We also want to stress that challenges vary depending on the area of operations and the pathogen responsible for the pandemic. Any feedback and collaboration that may come from this work is appreciated and encouraged.

Drug-like antibodies with high affinity, diversity and developability directly from next-generation antibody libraries.

Therapeutic antibodies must have "drug-like" properties. These include high affinity and specificity for the intended target, biological activity, and additional characteristics now known as "developability properties": long-term stability and resistance to aggregation when in solution, thermodynamic stability to prevent unfolding, high expression yields to facilitate manufacturing, low self-interaction, among others. Sequence-based liabilities may affect one or more of these characteristics. Improving the stability and developability of a lead antibody is typically achieved by modifying its sequence, a time-consuming process that often results in reduced affinity. Here we present a new antibody library format that yields high-affinity binders with drug-like developability properties directly from initial selections, reducing the need for further engineering or affinity maturation. The innovative semi-synthetic design involves grafting natural complementarity-determining regions (CDRs) from human antibodies into scaffolds based on well-behaved clinical antibodies. HCDR3s were amplified directly from B cells, while the remaining CDRs, from which all sequence liabilities had been purged, were replicated from a large next-generation sequencing dataset. By combining two in vitro display techniques, phage and yeast display, we were able to routinely recover a large number of unique, highly developable antibodies against clinically relevant targets with affinities in the subnanomolar to low nanomolar range. We anticipate that the designs and approaches presented here will accelerate the drug development process by reducing the failure rate of leads due to poor antibody affinities and developability.Abbreviations: AC-SINS: affinity-capture self-interaction nanoparticle spectroscopy; CDR: complementarity-determining region; CQA: critical quality attribute; ELISA: enzyme-linked immunoassay; FACS: fluorescence-activated cell sorting; Fv: fragment variable; GM-CSF: granulocyte-macrophage colony-stimulating factor; HCDR3: heavy chain CDR3; IFN2a: interferon α-2; IL6: interleukin-6; MACS: magnetic-activated cell sorting; NGS: next generation sequencing; PCR: polymerase chain reaction; SEC: size-exclusion chromatography; SPR: surface plasmon resonance; TGFß-R2: transforming growth factor ß-R2; VH: variable heavy; VK: variable kappa; VL: variable light; Vl: variable lambda.

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.

Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies.

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.

Exploiting Single Domain Antibodies as Regulatory Parts to Modulate Monoterpenoid Production in E. coli.

Synthetic biology and metabolic engineering offer potentially green and attractive routes to the production of high value compounds. The provision of high-quality parts and pathways is crucial in enabling the biosynthesis of chemicals using synthetic biology. While a number of regulatory parts that provide control at the transcriptional and translational level have been developed, relatively few exist at the protein level. Single domain antibodies (sdAb) such as camelid heavy chain variable fragments (VHH) possess binding characteristics which could be exploited for their development and use as novel parts for regulating metabolic pathways at the protein level in microbial cell factories. Here, a platform for the use of VHH as tools in Escherichia coli is developed and subsequently used to modulate linalool production in E. coli. The coproduction of a Design of Experiments (DoE) optimized pBbE8k His6-VHHCyDisCo system alongside a heterologous linalool production pathway facilitated the identification of anti-bLinS VHH that functioned as modulators of bLinS. This resulted in altered product profiles and significant variation in the titers of linalool, geraniol, nerolidol, and indole obtained. The ability to alter the production levels of high value terpenoids, such as linalool, in a tunable manner at the protein level could represent a significant step forward for the development of improved microbial cell factories. This study serves as a proof of principle indicating that VHH can be used to modulate enzyme activity in engineered pathways within E. coli. Given their almost limitless binding potential, we posit that single domain antibodies could emerge as powerful regulatory parts in synthetic biology applications.

Structurally Mapping Antibody Repertoires.

Every human possesses millions of distinct antibodies. It is now possible to analyze this diversity via next-generation sequencing of immunoglobulin genes (Ig-seq). This technique produces large volume sequence snapshots of B-cell receptors that are indicative of the antibody repertoire. In this paper, we enrich these large-scale sequence datasets with structural information. Enriching a sequence with its structural data allows better approximation of many vital features, such as its binding site and specificity. Here, we describe the structural annotation of antibodies pipeline that maps the outputs of large Ig-seq experiments to known antibody structures. We demonstrate the viability of our protocol on five separate Ig-seq datasets covering ca. 35 m unique amino acid sequences from ca. 600 individuals. Despite the great theoretical diversity of antibodies, we find that the majority of sequences coming from such studies can be reliably mapped to an existing structure.

Rapid intervention to reduce Ebola transmission in a remote village - Gbarpolu County, Liberia, 2014.

As late as September 14, 2014, Liberia's Gbarpolu County had reported zero cases of Ebola virus disease (Ebola). On October 25, the Bong County Health Team, a local health department in the Liberian Ministry of Health and Social Welfare (MOHSW), received confirmation of Ebola in a man who had recently left Geleyansiesu, a remote village of approximately 800 residents, after his wife and daughter had died of illnesses consistent with Ebola. MOHSW requested assistance from CDC, the World Health Organization, and other international partners to investigate and confirm the outbreak in Geleyansiesu and begin interventions to interrupt transmission. A total of 22 cases were identified, of which 18 (82%) were laboratory confirmed by real-time polymerase chain reaction. There were 16 deaths (case-fatality rate = 73%). Without road access to or direct telecommunications with the village, interventions had to be tailored to the local context. Public health interventions included 1) education of the community about Ebola, transmission of the virus, signs and symptoms, the importance of isolating ill patients from family members, and the potential benefits of early diagnosis and treatment; 2) establishment of mechanisms to alert health authorities of possibly infected persons leaving the village to facilitate safe transport to the closest Ebola treatment unit (ETU); 3) case investigation, contact tracing, and monitoring of contacts; 4) training in hygienic burial of dead bodies; 5) active case finding and diagnosis; and 6) isolation and limited no-touch treatment in the village of patients unwilling or unable to seek care at an ETU. The findings of this investigation could inform interventions aimed at controlling focal outbreaks in difficult-to-reach communities, which has been identified as an important component of the effort to eliminate Ebola from Liberia.

Rapid response to Ebola outbreaks in remote areas - Liberia, July-November 2014.

West Africa is experiencing its first epidemic of Ebola virus disease (Ebola). As of February 9, Liberia has reported 8,864 Ebola cases, of which 3,147 were laboratory-confirmed. Beginning in August 2014, the Liberia Ministry of Health and Social Welfare (MOHSW), supported by CDC, the World Health Organization (WHO), and others, began systematically investigating and responding to Ebola outbreaks in remote areas. Because many of these areas lacked mobile telephone service, easy road access, and basic infrastructure, flexible and targeted interventions often were required. Development of a national strategy for the Rapid Isolation and Treatment of Ebola (RITE) began in early October. The strategy focuses on enhancing capacity of county health teams (CHT) to investigate outbreaks in remote areas and lead tailored responses through effective and efficient coordination of technical and operational assistance from the MOHSW central level and international partners. To measure improvements in response indicators and outcomes over time, data from investigations of 12 of 15 outbreaks in remote areas with illness onset dates of index cases during July 16-November 20, 2014, were analyzed. The times to initial outbreak alerts and durations of the outbreaks declined over that period while the proportions of patients who were isolated and treated increased. At the same time, the case-fatality rate in each outbreak declined. Implementation of strategies, such as RITE, to rapidly respond to rural outbreaks of Ebola through coordinated and tailored responses can successfully reduce transmission and improve outcomes.

A randomized comparison of the efficacy of 2 techniques for piriformis muscle injection: ultrasound-guided versus nerve stimulator with fluoroscopic guidance.

BACKGROUND: Piriformis muscle injections are most often performed using fluoroscopic guidance; however, ultrasound (US) guidance has recently been described extensively in the literature. No direct comparisons between the 2 techniques have been performed. Our objective was to compare the efficacy and efficiency of fluoroscopic- and US-guided techniques. METHODS: A randomized, comparative trial was carried out to compare the 2 techniques. Twenty-eight patients with a diagnosis of piriformis syndrome, based on history and physical examination, who had failed conservative treatment were enrolled in the study. Patients were randomized to receive the injection either via US or fluoroscopy. Injections consisted of 10 mL of 1% lidocaine with 80 mg of triamcinalone. The primary outcome measure was numeric pain score, and secondary outcome measures included functional status as measured by the Multidimensional Pain Inventory, patient satisfaction as measured by the Patient Global Impression of Change scale, and procedure timing characteristics. Outcome data were measured preprocedure, immediately postprocedure, and 1 to 2 weeks and 3 months postprocedure. RESULTS: We found no statistically significant differences in numeric pain scores, patient satisfaction, procedure timing characteristics, or most functional outcomes when comparing the 2 techniques. Statistically significant differences between the 2 techniques were found with respect to the outcome measures of household chores and outdoor work. CONCLUSIONS: Ultrasound-guided piriformis injections provide similar outcomes to fluoroscopically guided injections without differences in imaging, needling, or overall procedural times.

Treatment of meralgia paresthetica with ultrasound-guided pulsed radiofrequency ablation of the lateral femoral cutaneous nerve.

A 23-year-old female with an 18-month history of left anterolateral thigh paresthesias and burning pain consistent with meralgia paresthetica was referred to our clinic after failing trials of physical therapy, nonsteroidal anti-inflammatories, gabapentin, and amitriptyline. We performed 3 lateral femoral cutaneous nerve blocks with corticosteroid over a 4-month period; however, each block provided only temporary relief. As this pain was limiting the patient's ability to perform her functions as an active duty service member, we elected to perform a pulsed radiofrequency treatment of the lateral femoral cutaneous nerve with ultrasound guidance and nerve stimulation. After locating the lateral femoral cutaneous nerve with ultrasound and reproducing the patient's dysthesia with stimulation, pulsed radiofrequency treatment was performed at 42°C for 120 seconds. The needle was then rotated 180° and an additional cycle of pulsed radiofrequency treatment was performed followed by injection of 0.25% ropivacaine with 4 mg of dexamethasone. At 1.5 and 3 month follow-up visits, the patient reported excellent pain relief with activity and improved ability to perform her duties as an active duty service member. ▪

The unusual extended signal peptide region is not required for secretion and function of an Escherichia coli autotransporter.

The plasmid-encoded toxin, Pet, a prototypical member of the serine protease autotransporters of the Enterobacteriaceae, possesses an unusually long signal peptide, which can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions correspond to a conserved N-terminal extension previously designated the extended signal peptide region (ESPR), while the N2, H2 and C regions resemble typical Sec-dependent signal sequences and exhibit considerable sequence variability. We have shown previously that the ESPR directs Sec-dependent, post-translational translocation of Pet across the bacterial inner membrane. In this study, we demonstrate that the ESPR is not essential for the secretion or the function of Pet.