Targeting breast and pancreatic cancer metastasis using a dual-cadherin antibody.

The successful application of antibody-based therapeutics in either primary or metastatic cancer depends upon the selection of rare cell surface epitopes that distinguish cancer cells from surrounding normal epithelial cells. By contrast, as circulating tumor cells (CTCs) transit through the bloodstream, they are surrounded by hematopoietic cells with dramatically distinct cell surface proteins, greatly expanding the number of targetable epitopes. Here, we show that an antibody (23C6) against cadherin proteins effectively suppresses blood-borne metastasis in mouse isogenic and xenograft models of triple negative breast and pancreatic cancers. The 23C6 antibody is remarkable in that it recognizes both the epithelial E-cadherin (CDH1) and mesenchymal OB-cadherin (CDH11), thus overcoming considerable heterogeneity across tumor cells. Despite its efficacy against single cells in circulation, the antibody does not suppress primary tumor formation, nor does it elicit detectable toxicity in normal epithelial organs, where cadherins may be engaged within intercellular junctions and hence inaccessible for antibody binding. Antibody-mediated suppression of metastasis is comparable in matched immunocompetent and immunodeficient mouse models. Together, these studies raise the possibility of antibody targeting CTCs within the vasculature, thereby suppressing blood-borne metastasis.

Rapid evolution of promoters from germline-specifically expressed genes including transposon silencing factors.

BACKGROUND: The piRNA pathway in animal gonads functions as an 'RNA-based immune system', serving to silence transposable elements and prevent inheritance of novel invaders. In Drosophila, this pathway relies on three gonad-specific Argonaute proteins (Argonaute-3, Aubergine and Piwi) that associate with 23-28 nucleotide piRNAs, directing the silencing of transposon-derived transcripts. Transposons constitute a primary driver of genome evolution, yet the evolution of piRNA pathway factors has not received in-depth exploration. Specifically, channel nuclear pore proteins, which impact piRNA processing, exhibit regions of rapid evolution in their promoters. Consequently, the question arises whether such a mode of evolution is a general feature of transposon silencing pathways. RESULTS: By employing genomic analysis of coding and promoter regions within genes that function in transposon silencing in Drosophila, we demonstrate that the promoters of germ cell-specific piRNA factors are undergoing rapid evolution. Our findings indicate that rapid promoter evolution is a common trait among piRNA factors engaged in germline silencing across insect species, potentially contributing to gene expression divergence in closely related taxa. Furthermore, we observe that the promoters of genes exclusively expressed in germ cells generally exhibit rapid evolution, with some divergence in gene expression. CONCLUSION: Our results suggest that increased germline promoter evolution, in partnership with other factors, could contribute to transposon silencing and evolution of species through differential expression of genes driven by invading transposons.

Evaluation of serological lateral flow assays for severe acute respiratory syndrome coronavirus-2.

BACKGROUND: COVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed. METHODS: We evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays' performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence. RESULTS: Combined IgG + IgM sensitivities ranged from 33.9 to 94.6%, while combined specificities ranged from 92.6 to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG + IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG + IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 µg/mL), followed by a similar LOD of 1.5 µg/mL for CareHealth, Cellex, KHB, and Vivachek. CONCLUSION: We provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model.

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara-C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara-C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara-C were elucidated by whole-exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara-C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.

Pediatric Blunt Neck Trauma Causing Esophageal and Complete Tracheal Transection.

BACKGROUND: Blunt injuries to the cervical trachea remain rare but present unique and challenging clinical scenarios for prehospital providers. These injuries depend on prehospital providers either definitively securing the injured airway or bridging the patient to a treatment facility that can mobilize the necessary resources. CASE SUMMARY: The case presented here involves a clothesline injury to a pediatric patient that resulted in complete tracheal transection and partial esophageal transection. Ground and air prehospital providers utilized a stepwise approach to this airway injury and achieved a favorable outcome. The patient was serendipitously intubated through a blind nasal approach that entered the proximal esophagus, exited through the tear and entered the distal trachea. DISCUSSION: There is a paucity of literature describing the successful management of these devastating injuries. While some authors have advocated for early flexible fiberoptic intubation or proceeding directly to tracheostomy, these techniques are not available in the prehospital environment. This case also highlights the inherent issues with proceeding to cricothyroidotomy in patients with tracheal trauma and should give all providers pause before considering this management technique. CONCLUSION: Ultimately, a systematic approach to all airways will ensure that prehospital providers are best prepared for even the most challenging scenarios.

Helicopter Transport in Regionalized Burn Care: One Program's Perspective.

BACKGROUND: The decision to use helicopter EMS (HEMS) for the transport of burn patients is a complex decision. This analysis sought to evaluate burn patients flown to burn centers who met predetermined criteria for patients who likely benefit from HEMS care. METHODS: A retrospective transport chart review of all burn transports covering the preceding nine and a half years was conducted to evaluate for HEMS appropriate criteria defined as patients requiring advanced airway management, ventilator support, facial burns, inhalation injury, circumferential burns, electrical or chemical burn, or major burns. All ages were included. RESULTS: A total of 171 cases were identified. Thirty-one (18.1%) were pediatric. Facial burns constituted the most frequent criteria met with 112 (65.5%) patients identified. Sixty-nine (40.4%) had suspected inhalation injuries. Fifty-five (32.2%) patients were intubated. Forty (28.6%) adults and twelve (38.7%) children had major burns. CONCLUSION: Of the 171 burn patient transported, twenty-one (12.3%) patients did not meet any HEMS criteria. Excluding those who did not meet any criteria, 98 (57.3%) patients were flown with non-major burns. Efforts are needed to determine the risks burn patients face if slower, non-critical care transport is utilized and which patients are appropriate for HEMS.

Effect of ranibizumab on high-speed indocyanine green angiography and minimum intensity projection optical coherence tomography findings in neovascular age-related macular degeneration.

PURPOSE: The purpose of this 1-year prospective study was to investigate how induction/pro re nata ranibizumab intravitreal treatment of eyes with neovascular age-related macular degeneration affects the anatomy of choroidal neovascularization (CNV) and the overlying outer retinal tissue. METHODS: High-speed indocyanine green (HS-ICG) angiography measurements provided quantification of the CNV size in 60 patients followed for 1 year. Minimum intensity projection optical coherence tomography (MinIP OCT), a novel algorithm assessing minimum optical intensity between the internal limiting membrane and retinal pigment epithelium, measured the area of outer retinal disruption overlying the CNV. Fluorescein angiography was also assessed to evaluate late retinal leakage. RESULTS: After 1 year, the mean area of CNV measured with indocyanine green angiography decreased by 5.8%. The mean area of MinIP OCT of outer retinal disruption overlying the CNV decreased by 4.2%. Mean area of fluorescein angiography leakage decreased by 6.3%. Both the area of outer retinal disruption measured with MinIP OCT and the area of leakage on fluorescein angiography typically exceeded the area of CNV on indocyanine green angiography at baseline and 1 year. CONCLUSION: Choroidal neovascularization treated with induction/pro re nata intravitreal ranibizumab for 1 year essentially remained static. Minimum intensity projection optical coherence tomography suggests that the area of outer retinal disruption overlying the CNV may be greater than the CNV itself and often correlates with the leakage area on fluorescein angiography. Additionally, there was minimal change in the area of outer retinal disruption on MinIP OCT even when fluid resolved. Measurements of the extent of CNV lesions based on indocyanine green angiography and MinIP OCT may provide useful outcome variables to help assess the CNV complex longitudinally and warrant further validation.

Epidemiology of epiretinal membrane in a large cohort of patients with uveitis.

OBJECTIVE: To identify clinical characteristics associated with the presence of epiretinal membrane (ERM) in patients with uveitis. DESIGN: Case-control study. PARTICIPANTS AND CONTROLS: Five hundred ninety-eight subjects seen in a single tertiary referral clinic between January 1, 2008, and December 31, 2011, who were diagnosed with uveitis. METHODS: Spectral-domain optical coherence tomography (SD OCT) images of all subjects were reviewed to assess for ERM. A multivariate logistic regression analysis was performed to compare characteristics of subjects with ERM (cases) with characteristics of subjects without ERM (controls). A second multivariate analysis assessed the relationship between ERM and visual acuity. Fundus photographs were reviewed to compare SD OCT ascertainment of ERM with photographic ascertainment. MAIN OUTCOME MEASURES: Presence or absence of ERM on OCT imaging. RESULTS: Of 598 uveitic participants, 246 (41%) were found to have ERM in at least 1 eye on SD OCT imaging. The prevalence of ERM by Standardization of Uveitis Nomenclature anatomic subtype was 28.1% for anterior uveitis, 57.0% for intermediate uveitis, and 43.4% for posterior uveitis and panuveitis. Multivariate analysis showed that the following clinical factors were associated significantly with ERM: older age (3% increased risk per year of age; 95% confidence interval [CI], 1.02-1.05), intermediate uveitis (odds ratio [OR], 3.41; 95% CI, 1.67-6.96), posterior uveitis and panuveitis (OR, 1.81; 95% CI, 1.09-3.01), male sex (OR, 1.59; 95% CI, 1.05-2.42), and history of cataract surgery (OR, 1.78; 95% CI, 1.13-2.79). When adjusted for covariates, eyes with ERM had a mean logarithm of the minimum angle of resolution visual acuity of 0.58 (20/76) versus 0.48 (20/60) in non-ERM eyes (P = 0.039). Of OCT-defined ERMs in this cohort, 38% were not detectable on fundus photographs. CONCLUSIONS: Epiretinal membrane is a common complication of uveitis that is associated with patient age, intermediate uveitis, posterior uveitis, panuveitis, male sex, and previous cataract surgery. It can contribute independently to vision loss in uveitic eyes. In uveitis, OCT is more sensitive than fundus photography for identification of ERM.

Visual acuity after cataract surgery in patients with age-related macular degeneration: age-related eye disease study 2 report number 5.

OBJECTIVE: To evaluate visual acuity outcomes after cataract surgery in persons with varying degrees of severity of age-related macular degeneration (AMD). DESIGN: Cohort study. PARTICIPANTS: A total of 1232 eyes of 793 participants who underwent cataract surgery during the Age-Related Eye Disease Study 2, a prospective, multicenter, randomized controlled trial of nutritional supplements for treatment of AMD. METHODS: Preoperative and postoperative characteristics of participants who underwent cataract extraction during the 5-year trial were analyzed. Both clinical data and standardized red-reflex lens and fundus photographs were obtained at baseline and annually. Photographs were graded by a centralized reading center for cortical and posterior subcapsular lens opacities and for AMD severity. Cataract surgery was documented at annual study visits or by history during the 6-month telephone calls. Analyses were conducted using multivariate repeated-measures regression. MAIN OUTCOME MEASURES: Change in best-corrected visual acuity (BCVA) after cataract surgery compared with preoperative BCVA. RESULTS: Adjusting for age at time of surgery, gender, interval between preoperative and postoperative visits, and type and severity of cataract, the mean changes in visual acuity were as follows: eyes with mild AMD (n = 30) gained 11.2 letters (95% confidence interval [CI], 6.9-15.5), eyes with moderate AMD (n = 346) gained 11.1 letters (95% CI, 9.1-13.2), eyes with severe AMD (n = 462) gained 8.7 letters (95% CI, 6.7-10.7), eyes with noncentral geographic atrophy (n = 70) gained 8.9 letters (95% CI, 5.8-12.1), and eyes with advanced AMD (central geographic atrophy, neovascular disease, or both; n = 324) gained 6.8 letters (95% CI, 4.9-8.8). The visual acuity gain across all AMD severity groups was statistically significant from preoperative values (P < 0.0001). CONCLUSIONS: Mean visual acuities improved significantly after cataract surgery across varying degrees of AMD severity.

Global Emergency Medicine: A Scoping Review of the Literature from 2022.

OBJECTIVE: The objective was to identify the highest quality global emergency medicine (GEM) research published in 2022. The top articles are compiled in a comprehensive list of all the year's GEM articles and narrative summaries are performed on those included. METHODS: A systematic PubMed search was conducted to identify all GEM articles published in 2022 and included a manual supplemental screen of 11 organizational websites for gray literature (GRAY). A team of trained reviewers and editors screened all identified titles and abstracts, based on three case definition categories: disaster and humanitarian response (DHR), emergency care in resource-limited settings (ECRLS), and emergency medicine development (EMD). Articles meeting these definitions were independently scored by two reviewers using rubrics for original research (OR), review (RE) articles, and GRAY. Articles that scored in the top 5% from each category as well as the overall top 5% of articles were included for narrative summary. RESULTS: The 2022 search identified 58,510 articles in the main review, of which 524 articles screened in for scoring, respectively, 30% and 18% increases from last year. After duplicates were removed, 36 articles were included for narrative summary. The GRAY search identified 7755 articles, of which 33 were scored and one was included for narrative summary. ECRLS remained the largest category (27; 73%), followed by DHR (7; 19%) and EMD (3; 8%). OR articles remained more common than RE articles (64% vs. 36%). CONCLUSIONS: The waning of the COVID-19 pandemic has not affected the continued growth in GEM literature. Articles related to prehospital care, mental health and resilience among patients and health care workers, streamlining pediatric infectious disease care, and disaster preparedness were featured in this year's review. The continued lack of EMD studies despite the global growth of GEM highlights a need for more scholarly dissemination of best practices.

XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies.

IL12 is a proinflammatory cytokine, that has shown promising antitumor activity in humans by promoting the recruitment and activation of immune cells in tumors. However, the systemic administration of IL12 has been accompanied by considerable toxicity, prompting interest in researching alternatives to drive preferential IL12 bioactivity in the tumor. Here, we have generated XTX301, a tumor-activated IL12 linked to the human Fc protein via a protease cleavable linker that is pharmacologically inactivated by an IL12 receptor subunit beta 2 masking domain. In vitro characterization demonstrates multiple matrix metalloproteases, as well as human primary tumors cultured as cell suspensions, can effectively activate XTX301. Intravenous administration of a mouse surrogate mXTX301 demonstrated significant tumor growth inhibition (TGI) in inflamed and non-inflamed mouse models without causing systemic toxicities. The superiority of mXTX301 in mediating TGI compared with non-activatable control molecules and the greater percentage of active mXTX301 in tumors versus other organs further confirms activation by the tumor microenvironment-associated proteases in vivo. Pharmacodynamic characterization shows tumor selective increases in inflammation and upregulation of immune-related genes involved in IFNγ cell signaling, antigen processing, presentation, and adaptive immune response. XTX301 was tolerated following four repeat doses up to 2.0 mg/kg in a nonhuman primate study; XTX301 exposures were substantially higher than those at the minimally efficacious dose in mice. Thus, XTX301 has the potential to achieve potent antitumor activity while widening the therapeutic index of IL12 treatment and is currently being evaluated in a phase I clinical trial.

Ubiquitin-based anticancer therapy: carpet bombing with proteasome inhibitors vs surgical strikes with E1, E2, E3, or DUB inhibitors.

The proteasome inhibitor bortezomib remains the only ubiquitin pathway effector to become a drug (VELCADE®) and has become a successful treatment for hematological malignancies. While producing a global cellular effect, proteasome inhibitors have not triggered the catastrophe articulated initially in terms such as "buildup of cellular garbage". Proteasome inhibitors, in fact, do have a therapeutic window, although in the case of the prototype bortezomib it is small owing to peripheral neuropathy, myelosuppression and, as recently reported, cardiotoxicity [1]. Currently, several second-generation molecules are undergoing clinical evaluation to increase this window. An alternative strategy is to target ubiquitin pathway enzymes acting at non-proteasomal sites-E1, E2, and E3, associated with ubiquitin conjugation, and deubiquitylating enzymes ("DUBs")-that act locally on selected targets rather than on the whole cell. Inhibitors (or activators, in some cases) of these enzymes should be developable as selective antitumor agents with toxicity profiles superior to that of bortezomib. Various therapeutic hypotheses follow from known cellular mechanisms of these target enzymes; most hypotheses relate to cancer, reminiscent of the FDA-approved protein kinase inhibitors now marketed. Since ubiquitin tagging controls the cellular content, activity, or compartmentation of proteins associated with disease, inhibitors or activators of ubiquitin conjugation or deconjugation are predicted to have an impact on disease. For practical and empirical reasons, inhibitors of ubiquitin pathway enzymes have been the favored therapeutic avenue. In approximately the time that has elapsed since the approval of bortezomib in 2003, there has been some progress in developing potential anticancer drugs that target various ubiquitin pathway enzymes. An E1 inhibitor and inhibitors of E3 are now in clinical trial, with some objective responses reported. Appropriate assays and/or rational design may uncover improved inhibitors of these enzymes, as well as E2 and DUBs, for further development. Presently, it should become clear whether one or both of the two general strategies for ubiquitin-based drug discovery will lead to truly superior new medicines for cancer and other diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Detection of ubiquitin-proteasome enzymatic activities in cells: application of activity-based probes to inhibitor development.

BACKGROUND: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin-proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. SCOPE OF REVIEW: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. MAJOR CONCLUSIONS: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. GENERAL SIGNIFICANCE: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Spongiacidin C, a pyrrole alkaloid from the marine sponge Stylissa massa, functions as a USP7 inhibitor.

USP7, a deubiquitylating enzyme hydrolyzing the isopeptide bond at the C-terminus of ubiquitin, is an emerging cancer target. We isolated spongiacidin C from the marine sponge Stylissa massa as the first USP7 inhibitor from a natural source. This compound inhibited USP7 most strongly with an IC50 of 3.8 µM among several USP family members tested.

A novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from Bortezomib.

Bortezomib therapy has proven successful for the treatment of relapsed and/or refractory multiple myeloma (MM); however, prolonged treatment is associated with toxicity and development of drug resistance. Here, we show that the novel proteasome inhibitor NPI-0052 induces apoptosis in MM cells resistant to conventional and Bortezomib therapies. NPI-0052 is distinct from Bortezomib in its chemical structure, effects on proteasome activities, mechanisms of action, and toxicity profile against normal cells. Moreover, NPI-0052 is orally bioactive. In animal tumor model studies, NPI-0052 is well tolerated and prolongs survival, with significantly reduced tumor recurrence. Combining NPI-0052 and Bortezomib induces synergistic anti-MM activity. Our study therefore provides the rationale for clinical protocols evaluating NPI-0052, alone and together with Bortezomib, to improve patient outcome in MM.

Subconjunctival Palomid 529 in the treatment of neovascular age-related macular degeneration.

BACKGROUND: Recent evidence suggests that neovascular age-related macular degeneration (AMD) may have an immune mediated component. Palomid 529, an investigational medication involving the immune Akt/mTOR pathway, is unique in dissociating both targets of rapamycin complexes TORC1 and TORC2. This small short-term pilot study assesses the safety of subconjunctival Palomid 529 in the treatment of neovascular AMD, with some limited efficacy information. METHODS: In this 12-week phase I open-label prospective pilot study, five participants with neovascular age-related macular degeneration that were refractory to intravitreal anti-vascular endothelial growth factor (VEGF) received three serial monthly subconjunctival doses of 1.9 mg Palomid 529. All participants were also offered concomitant monthly intravitreal anti-VEGF injections. Safety was monitored via adverse events recording. Additional outcome measures included visual acuity, optical coherence tomography, fluorescein angiography, indocyanine green angiography and fundus photography. RESULTS: The study drug was well-tolerated by all participants. There were no drug-related adverse events and no serious adverse events. A depot formed at the injection site, which persisted at the end of the study. In these anti-VEGF refractory patients, no clinically important changes in best-corrected visual acuity, fluorescein leakage pattern, choroidal neovascularization size on indocyanine green angiography, or autofluorescence pattern on fundus autofluorescence were observed compared to baseline. The fluid status, assessed with optical coherence tomography showed that central retinal thickness and macular volume remained stable in three participants, while the other two participants clinically progressed. CONCLUSIONS: Serial subconjunctival injections of Palomid 529 were well-tolerated and resulted in depot formation. There were no concerns for any ocular or systemic toxicity during this small short-term study. Larger randomized studies are required to determine efficacy.

Recurrent activity in neuronal avalanches.

A new statistical analysis of large neuronal avalanches observed in mouse and rat brain tissues reveals a substantial degree of recurrent activity and cyclic patterns of activation not seen in smaller avalanches. To explain these observations, we adapted a model of structural weakening in materials. In this model, dynamical weakening of neuron firing thresholds closely replicates experimental avalanche size distributions, firing number distributions, and patterns of cyclic activity. This agreement between model and data suggests that a mechanism like dynamical weakening plays a key role in recurrent activity found in large neuronal avalanches. We expect these results to illuminate the causes and dynamics of large avalanches, like those seen in seizures.

Unistrand piRNA clusters are an evolutionarily conserved mechanism to suppress endogenous retroviruses across the Drosophila genus.

The PIWI-interacting RNA (piRNA) pathway prevents endogenous genomic parasites, i.e. transposable elements, from damaging the genetic material of animal gonadal cells. Specific regions in the genome, called piRNA clusters, are thought to define each species' piRNA repertoire and therefore its capacity to recognize and silence specific transposon families. The unistrand cluster flamenco (flam) is essential in the somatic compartment of the Drosophila ovary to restrict Gypsy-family transposons from infecting the neighbouring germ cells. Disruption of flam results in transposon de-repression and sterility, yet it remains unknown whether this silencing mechanism is present more widely. Here, we systematically characterise 119 Drosophila species and identify five additional flam-like clusters separated by up to 45 million years of evolution. Small RNA-sequencing validated these as bona-fide unistrand piRNA clusters expressed in somatic cells of the ovary, where they selectively target transposons of the Gypsy family. Together, our study provides compelling evidence of a widely conserved transposon silencing mechanism that co-evolved with virus-like Gypsy-family transposons.

XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 monoclonal antibody, demonstrates tumor-growth inhibition and tumor-selective pharmacodynamics in mouse models of cancer.

INTRODUCTION: The clinical benefit of the anti-CTLA-4 monoclonal antibody (mAb) ipilimumab has been well established but limited by immune-related adverse events, especially when ipilimumab is used in combination with anti-PD-(L)1 mAb therapy. To overcome these limitations, we have developed XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 mAb. METHODS: XTX101 consists of an anti-human CTLA-4 mAb covalently linked to masking peptides that block the complementarity-determining regions, thereby minimizing the mAb binding to CTLA-4. The masking peptides are designed to be released by proteases that are typically dysregulated within the tumor microenvironment (TME), resulting in activation of XTX101 intratumorally. Mutations within the Fc region of XTX101 were included to enhance affinity for FcγRIII, which is expected to enhance potency through antibody-dependent cellular cytotoxicity. RESULTS: Biophysical, biochemical, and cell-based assays demonstrate that the function of XTX101 depends on proteolytic activation. In human CTLA-4 transgenic mice, XTX101 monotherapy demonstrated significant tumor growth inhibition (TGI) including complete responses, increased intratumoral CD8+T cells, and regulatory T cell depletion within the TME while maintaining minimal pharmacodynamic effects in the periphery. XTX101 in combination with anti-PD-1 mAb treatment resulted in significant TGI and was well tolerated in mice. XTX101 was activated in primary human tumors across a range of tumor types including melanoma, renal cell carcinoma, colon cancer and lung cancer in an ex vivo assay system. CONCLUSIONS: These data demonstrate that XTX101 retains the full potency of an Fc-enhanced CTLA-4 antagonist within the TME while minimizing the activity in non-tumor tissue, supporting the further evaluation of XTX101 in clinical studies.