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BackgroundNeuropilin-1 (NRP1) is a co-receptor that complexes with diverse ligands and their cognate receptors. As such, it plays a role in multiple different biological processes, including axon guidance and angiogenesis. NRP1 contains two CUB domains (a1 and a2) involved in binding the ligand Semaphorin3A (SEMA3A), two Factor V/VIII domains (b1 and b2) involved in VEGF ligand binding and one MAM domain (c domain). While functional antibodies with anti-tumor activity have been generated against the SEMA3A and VEGF binding domains, little attention has been paid to the c domain of NRP1, which has been implicated in the dimerization of NRP1, a prerequisite for functionality. We therefore hypothesized that c domain-binding antibodies would offer an opportunity to generate functional inhibitors of both SEMA3A and VEGF signaling and therefore improved anti-tumor activity.MethodsRecombinant human NRP1 comprising all subdomains was used to identify fully human anti-NRP1 antibodies. Specific antibodies were tested for their ability to block NRP1 interactions with recombinant SEMA3A and VEGF protein in vitro. Blocking antibodies were subsequently assessed for their functional effects, such as inhibition of SEMA3A-mediated growth cone collapse. Antibodies with diverse binding characteristics were then tested for in vivo anti-tumor activity in multiple cancer models of interest.ResultsRecombinant NRP1 containing the a1, a2, b1, b2 and c subdomains was used to successfully identify a series of specific monoclonal antibodies that cross-reacted with Cynomolgus monkey and mouse NRP1, but not human NRP2. Except for the a2 domain, epitope mapping showed an even distribution of mAbs for binding to each of the NRP1 subdomains, including the c domain that has been proposed to play a role in dimerization. Using biolayer interferometry, we identified antibody classes with direct SEMA3A and/or VEGF blocking properties. Further optimization of these antibodies yielded mAbs with subnanomolar affinities that showed significant tumor growth inhibition in multiple mouse models, including anti-PD1 non-responsive models.ConclusionsHere we demonstrate the identification of fully human monoclonal antibodies that specifically bind to the c domain of human NRP1. A subset of these c domain binders do not block either SEMA3A or VEGF binding to NRP1 but do show in vivo efficacy, suggesting a role for the c domain of NRP1 in the formation of functional (dimeric) complexes. Thus, c domain binding antibodies show remarkable inhibition of tumor growth in mouse cancer models and offer a novel means of therapeutic intervention in patients who are refractory to immune checkpoint inhibition.

BackgroundBispecific T Cell Engagers (BiTEs), consisting of an anti-CD3 scFv fused to an anti-tumor antigen scFv, are highly effective in the treatment of relapsed/refractory Acute Lymphoblastic Leukemia (ALL). However, the short half-life of BiTEs necessitates continuous intravenous administration at high doses for four-week increments. To overcome these pharmacokinetic shortcomings, we developed a method to engineer plasma cell precursors to continuously secrete transgenic biologics. Plasma cells were chosen for their high antibody production capacity (thousands of Ig molecules/cell/sec) and long-term survival (persisting for decades), making them a highly attractive cell-based platform for continuous biologic delivery.1 MethodsTo demonstrate proof-of-concept, we integrated a transgene coding for a bispecific non-Ig anti-CD3:CD19 scFv into the CCR5 safe-harbor locus of primary human B cells with CRISPR/Cas9, then initiated differentiation into plasma cells using a modified feeder-free culture system. Cells were characterized by flow cytometry, indel frequency, and droplet digital PCR. Edited cell supernatant was tested for therapeutic protein production with a timed ELISA and in vitro function with a cytolytic activity assay incorporating co-cultured T effector and CD19 expressing tumor cells. We also assessed in vivo anti-tumor activity of anti-CD3:CD19 scFv engineered B cell medicines (BeCMs) in NSG mice harboring a patient-derived xenograft (PDx). To avoid targeting of the BeCM by CD3+ cells, CD19 was knocked-out via a multiplexed CRISPR/Cas9 editing protocol. Mice were then inoculated with a luciferized B-ALL PDx line. Autologous T cells were delivered 24 hours and 72 hours following tumor transfer, and IVIS imaging was performed over the course of 17 days. A control GFP-engineered BeCM arm and PBS-dosed cohort were monitored in parallel, with n=6 mice per group.ResultsFlow cytometric analysis confirmed robust differentiation of BeCMs along the plasma cell lineage. We observed >85% cutting efficiency and 40–50% targeted integration, which translated to secretion rates (0.6–0.8 μg BiTE/106 cells/day) that were sufficient for in vitro functional assay tumor cytolytic activity. Significant reduction in tumor burden (bioluminescent flux, area under the curve) was observed in vivo in the anti-CD3:CD19 scFv cohort compared to the controls, which was in concordance with heightened in vivo T cell activation. The ~1000 pg/mL BiTE detected in mouse plasma demonstrates that BeCM-derived biologics can meet or even exceed the steady-state plasma concentrations achieved with clinically relevant doses.2 ConclusionsThese findings underscore the clinical potential of BeCMs as an emerging platform for sustained delivery of anti-tumor biologics.ReferencesHung KL, Meitlis I, Hale M, Chen C, Singh S, Jackson SW, Miao CH, Khan IF, Rawlings DJ, James RG. Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. Mol. Ther. 2018; 26:456–467.Franquiz MJ and Short NJ. Blinatumomab for the Treatment of Adult B-Cell Acute Lymphoblastic Leukemia: Toward a New Era of Targeted Immunotherapy. Biologics. 2020; 14: 23–34.Ethics ApprovalDeidentified human PBMCs were acquired under informed consent from the Fred Hutch Specimen Processing and Research Cell Bank (protocol #3942).

A combination of therapeutic vaccination with Ad26/MVA and stimulation of innate immune responses leads to improved virologic control and delayed rebound in SIV-infected macaques following discontinuation of antiretroviral therapy. Combining anti-HIV vaccination and immuostimulant These authors demonstrate that combining an experimental vaccine—consisting of an adenovirus serotype 26 vector vaccine and an MVA vector vaccine (Ad26/MVA)—with the innate immune stimulant Toll-like receptor 7 leads to improved virologic control and delayed rebound following discontinuation of stable antiretroviral therapy in SIV-infected macaques. The development of immunologic interventions that can target the viral reservoir in HIV-1-infected individuals is a major goal of HIV-1 research 1 , 2 . However, little evidence exists that the viral reservoir can be sufficiently targeted to improve virologic control following discontinuation of antiretroviral therapy. Here we show that therapeutic vaccination with Ad26/MVA (recombinant adenovirus serotype 26 (Ad26) prime, modified vaccinia Ankara (MVA) boost) 3 , 4 and stimulation of TLR7 (Toll-like receptor 7) improves virologic control and delays viral rebound following discontinuation of antiretroviral therapy in SIV-infected rhesus monkeys that began antiretroviral therapy during acute infection. Therapeutic vaccination with Ad26/MVA resulted in a marked increase in the magnitude and breadth of SIV-specific cellular immune responses in virologically suppressed, SIV-infected monkeys. TLR7 agonist administration led to innate immune stimulation and cellular immune activation. The combination of Ad26/MVA vaccination and TLR7 stimulation resulted in decreased levels of viral DNA in lymph nodes and peripheral blood, and improved virologic control and delayed viral rebound following discontinuation of antiretroviral therapy. The breadth of cellular immune responses correlated inversely with set point viral loads and correlated directly with time to viral rebound. These data demonstrate the potential of therapeutic vaccination combined with innate immune stimulation as a strategy aimed at a functional cure for HIV-1 infection.

Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causally associated with fetal microcephaly. The development of a safe and effective ZIKV vaccine is therefore an urgent global health priority. Here we demonstrate that three different vaccine platforms protect against ZIKV challenge in rhesus monkeys. A purified inactivated virus vaccine induced ZIKV-specific neutralizing antibodies and completely protected monkeys against ZIKV strains from both Brazil and Puerto Rico. Purified immunoglobulin from vaccinated monkeys also conferred passive protection in adoptive transfer studies. A plasmid DNA vaccine and a single-shot recombinant rhesus adenovirus serotype 52 vector vaccine, both expressing ZIKV premembrane and envelope, also elicited neutralizing antibodies and completely protected monkeys against ZIKV challenge. These data support the rapid clinical development of ZIKV vaccines for humans.

BackgroundBispecific T Cell Engagers (BiTEs), consisting of an anti-CD3 scFv fused to an anti-tumor antigen scFv, are highly effective in the treatment of relapsed/refractory Acute Lymphoblastic Leukemia (ALL). However, the short half-life of BiTEs necessitates continuous intravenous administration at high doses for four-week increments. To overcome these pharmacokinetic shortcomings, we developed a method to engineer plasma cell precursors to continuously secrete transgenic biologics. Plasma cells were chosen for their high antibody production capacity (thousands of Ig molecules/cell/sec) and long-term survival (persisting for decades), making them a highly attractive cell-based platform for continuous biologic delivery.1MethodsTo demonstrate proof-of-concept, we integrated a transgene coding for a bispecific non-Ig anti-CD3:CD19 scFv into the CCR5 safe-harbor locus of primary human B cells with CRISPR/Cas9, then initiated differentiation into plasma cells using a modified feeder-free culture system. Cells were characterized by flow cytometry, indel frequency, and droplet digital PCR. Edited cell supernatant was tested for therapeutic protein production with a timed ELISA and in vitro function with a cytolytic activity assay incorporating co-cultured T effector and CD19 expressing tumor cells. We also assessed in vivo anti-tumor activity of anti-CD3:CD19 scFv engineered B cell medicines (BeCMs) in NSG mice harboring a patient-derived xenograft (PDx). To avoid targeting of the BeCM by CD3+ cells, CD19 was knocked-out via a multiplexed CRISPR/Cas9 editing protocol. Mice were then inoculated with a luciferized B-ALL PDx line. Autologous T cells were delivered 24 hours and 72 hours following tumor transfer, and IVIS imaging was performed over the course of 17 days. A control GFP-engineered BeCM arm and PBS-dosed cohort were monitored in parallel, with n=6 mice per group.ResultsFlow cytometric analysis confirmed robust differentiation of BeCMs along the plasma cell lineage. We observed >85% cutting efficiency and 40–50% targeted integration, which translated to secretion rates (0.6–0.8 μg BiTE/106 cells/day) that were sufficient for in vitro functional assay tumor cytolytic activity. Significant reduction in tumor burden (bioluminescent flux, area under the curve) was observed in vivo in the anti-CD3:CD19 scFv cohort compared to the controls, which was in concordance with heightened in vivo T cell activation. The ~1000 pg/mL BiTE detected in mouse plasma demonstrates that BeCM-derived biologics can meet or even exceed the steady-state plasma concentrations achieved with clinically relevant doses.2ConclusionsThese findings underscore the clinical potential of BeCMs as an emerging platform for sustained delivery of anti-tumor biologics.ReferencesHung KL, Meitlis I, Hale M, Chen C, Singh S, Jackson SW, Miao CH, Khan IF, Rawlings DJ, James RG. Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. Mol. Ther. 2018; 26:456–467.Franquiz MJ and Short NJ. Blinatumomab for the Treatment of Adult B-Cell Acute Lymphoblastic Leukemia: Toward a New Era of Targeted Immunotherapy. Biologics. 2020; 14: 23–34.Ethics ApprovalDeidentified human PBMCs were acquired under informed consent from the Fred Hutch Specimen Processing and Research Cell Bank (protocol #3942).

Transcranial magnetic stimulation (TMS) can be used to probe for the location of cortical somatomotor representations in humans. These somatomotor representations are dynamic and are perturbed following motor training, systematic intervention, and in disease. Evidence suggests that these representations are maintained by the inhibitory neurotransmitter gamma-Aminobutyric acid (GABA). In the current study, we quantified the location, outline, and variability of the first dorsal interosseous (FDI) hand muscle somatomotor representation using a novel rapid-acquisition TMS method in 14 healthy young volunteers. In addition, resting motor thresholds were measured using established protocols. TMS was also used to examine short-interval intracortical inhibition (SICI), which is thought to measure transiently activated cortical gamma-Aminobutyric acid (GABA) interneurons. Using stepwise regression, our results showed that the level of intracortical inhibition was a significant predictor of the FDI somatomotor representation suggesting that greater excitability of the hand area representation is possibly governed by greater activation of transient GABA interneurons. Competing Interest Statement The authors have declared no competing interest.

A critical step in many biological studies is the estimation of evolutionary trees (phylogenies) from genomic data. Of particular interest is the species tree, which illustrates how a set of species evolved from a common ancestor. While species trees were previously estimated from a few regions of the genome (genes), it is now widely recognized that biological processes can cause the evolutionary histories of individual genes to differ from each other and from the species tree. This heterogeneity across the genome is phylogenetic signal that can be leveraged to estimate species evolution with greater accuracy. Hence, species tree estimation is expected to be greatly aided by current large-scale sequencing efforts, including the 5,000 Insect Genomes Project, the 10,000 Plant Genomes Project, the (~60,000) Vertebrate Genomes Project, and the Earth BioGenome Project, which aims to assemble genomes (or at least genome-scale data) for 1.5 million eukaryotic species in the next ten years. To analyze these forthcoming datasets, species tree estimation methods must scale to thousands of species and tens of thousands of genes; however, many of the current leading methods, which are heuristics for NP-hard optimization problems, can be prohibitively expensive on datasets of this size. In this dissertation, we argue that new methods are needed to enable scalable and statistically rigorous species tree estimation pipelines; we then seek to address this challenge through the introduction of three supertree-like methods: NJMerge, TreeMerge, and FastMulRFS. For these methods, we present theoretical results (worst-case running time analyses and proofs of statistical consistency) as well as empirical results on simulated datasets (and a fungal dataset for FastMulRFS). Overall, these methods enable statistically consistent species tree estimation pipelines that achieve comparable accuracy to the dominant optimization-based approaches while dramatically reducing running time.

The police department can find in the public library a place to connect one-on-one with citizens in a natural and comfortable setting, a ready-made audience for public relations and a helpful collaborating agency for promoting messages of safety and crime prevention. Marks-Molloy highlights police-library partnerships, a concept that has been successfully implemented in a number of US towns and cities. It will create positive relationships so that when something goes wrong, whether in the library or elsewhere, trust and cooperation will underlie emergency and investigative efforts.

Interprofessional education has served as a long-standing topic of interest in the health professions education community, mainly because of its reported capacity to develop the learner’s disposition for team-based practice. This chapter synthesises the findings from the health professional literature and focuses specifically on the impact of interprofessional education on students within clinical placements. The review sets the scene for the reporting of an empirical study conducted by the authors, examining medical students’ experiences of an interprofessional education placement. Medical students who rotated through the targeted interprofessional placement were interviewed via focus groups to elicit their experiences of the initiative. Their perspectives on how the context and the activities within the placement influenced their ability to learn ‘with, from and about’ other professionals were captured and analysed. Only a small percentage of participants reported that the workplace environment adequately supported opportunities for engagement with other professionals. The medical students, while able to voice the advantages of interprofessional practice “once they become” a practitioner, saw the agenda as relatively low on their priority list as busy students, subject to regular assessments of their ‘doctoring’ competencies. The results challenge the cheerfully optimistic literature on interprofessional education. It may be useful to acknowledge the resistance learners have in learning about other professions or disciplines when they have a fixed target of professional membership in sight. Undergraduate students seem to make judgements about what is important to learn, and who are the most legitimate models to learn from, very early in their curriculum. Students look to summative assessment as a key device to help differentiate the core from the peripheral within their program of study.

Background The identification and characterisation of differentially methylated regions (DMRs) between phenotypes in the human genome is of prime interest in epigenetics. We present a novel method, DMRcate , that fits replicated methylation measurements from the Illumina HM450K BeadChip (or 450K array) spatially across the genome using a Gaussian kernel. DMRcate identifies and ranks the most differentially methylated regions across the genome based on tunable kernel smoothing of the differential methylation (DM) signal. The method is agnostic to both genomic annotation and local change in the direction of the DM signal, removes the bias incurred from irregularly spaced methylation sites, and assigns significance to each DMR called via comparison to a null model. Results We show that, for both simulated and real data, the predictive performance of DMRcate is superior to those of Bumphunter and Probe Lasso , and commensurate with that of comb-p . For the real data, we validate all array-derived DMRs from the candidate methods on a suite of DMRs derived from whole-genome bisulfite sequencing called from the same DNA samples, using two separate phenotype comparisons. Conclusions The agglomeration of genomically localised individual methylation sites into discrete DMRs is currently best served by a combination of DM-signal smoothing and subsequent threshold specification. The findings also suggest the design of the 450K array shows preference for CpG sites that are more likely to be differentially methylated, but its overall coverage does not adequately reflect the depth and complexity of methylation signatures afforded by sequencing. For the convenience of the research community we have created a user-friendly R software package called DMRcate , downloadable from Bioconductor and compatible with existing preprocessing packages, which allows others to apply the same DMR-finding method on 450K array data.