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CDH17 and TRAILR2 protein expression and their correlation with BI 905711 minPOC in CRC cell lines.

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Supplementary methods (S1-S4), Supplementary tables (1-4) and Supplementary Figures (1-5)

Anti-HER2/CD3, a T-cell–dependent bispecific antibody (TDB) construct, induces T-cell–mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell–targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ϵ expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell–rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell–containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776–85. ©2018 AACR.

Supplementary Material and Methods

Supplementary Data and Methods

S1: Evaluation of CDH17 and TRAILR2 IHC staining in hepatic metastases of colorectal cancer -S2:BI 905711 kinetic and steady-state measurements -S3: CDH17 and TRAILR2 mean fluorescence intensity, antibody binding capacity and BI 905711 / TAS266' minPOC in 24 CRC cell lines -S4: Intergroup comparison of gross pathology observations in Cynomolgus Monkey small intestine, large intestine and pancreas

Activation of TRAILR2 has emerged as an important therapeutic concept in cancer treatment. TRAILR2 agonistic molecules have only had limited clinical success, to date, due either to lack of efficacy or hepatotoxicity. BI 905711 is a novel tetravalent bispecific antibody targeting both TRAILR2 and CDH17 and represents a novel liver-sparing TRAILR2 agonist specifically designed to overcome the disadvantages of previous strategies. Here, we show that BI 905711 effectively triggered apoptosis in a broad panel of CDH17-positive colorectal cancer tumor cells in vitro. Efficient induction of apoptosis was dependent on the presence of CDH17, as exemplified by the greater than 1,000-fold drop in potency in CDH17-negative cells. BI 905711 demonstrated single-agent tumor regressions in CDH17-positive colorectal cancer xenografts, an effect that was further enhanced upon combination with irinotecan. Antitumor efficacy correlated with induction of caspase activation, as measured in both the tumor and plasma. Effective tumor growth inhibition was further demonstrated across a series of different colorectal cancer PDX models. BI 905711 induced apoptosis in both a cis (same cell) as well as trans (adjacent cell) fashion, translating into significant antitumor activity even in xenograft models with heterogeneous CDH17 expression. In summary, we demonstrate that BI 905711 has potent and selective antitumor activity in CDH17-positive colorectal cancer models both in vitro and in vivo. The high prevalence of over 95% CDH17-positive tumors in patients with colorectal cancer, the molecule preclinical efficacy together with its potential for a favorable safety profile, support the ongoing BI 905711 phase I trial in colorectal cancer and additional CDH17-positive cancer types (NCT04137289).

Supplemental Methods, followed by Supplemental Figures S1, S2, S3, S4 and S5. Supplementary Fig. S1 In vitro binding of TDBs to HER2 and CD3 expressing cell lines. Supplementary Fig. S2 HER2 expression in model cell lines in vitro and tumors in vivo. Supplementary Fig. S3 HER2, CD3� and MECA-32 staining and vascular characterization of HER2 positive and negative tumors. Supplementary Fig. S4 T cell infiltration and CD3 expression in murine CT26 and CT26-HER2 tumors. Supplementary Fig. S5 Pharmacokinetic data from whole blood and cell pellet fractions

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T-cell–dependent bispecific antibodies (TDB) have been a major advancement in the treatment of cancer, allowing for improved targeting and efficacy for large molecule therapeutics. TDBs are comprised of one arm targeting a surface antigen on a cancer cell and another targeting an engaging surface antigen on a cytotoxic T cell. To impart this function, the antibody must be in a bispecific format as opposed to the more conventional bivalent format. Through in vitro and in vivo studies, we sought to determine the impact of changing antibody valency on solid tumor distribution and catabolism. A bivalent anti-HER2 antibody exhibited higher catabolism than its full-length monovalent binding counterpart in vivo by both invasive tissue harvesting and noninvasive single photon emission computed tomography/X-ray computed tomography imaging despite similar systemic exposures for the two molecules. To determine what molecular factors drove in vivo distribution and uptake, we developed a mechanistic model for binding and catabolism of monovalent and bivalent HER2 antibodies in KPL4 cells. This model suggests that observed differences in cellular uptake of monovalent and bivalent antibodies are caused by the change in apparent affinity conferred by avidity as well as differences in internalization and degradation rates of receptor bound antibodies. To our knowledge, this is the first study to directly compare the targeting abilities of monovalent and bivalent full-length antibodies. These findings may inform diverse antibody therapeutic modalities, including T-cell–redirecting therapies and drug delivery strategies relying upon receptor internalization.

In response to genotoxic stress, multiple kinase signaling cascades are activated, many of them directed towards the tumor suppressor p53, which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10, and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation by modulating the p53-dependent transcriptional output. NEK10 directly phosphorylates p53 on Y327, revealing NEK10’s unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA-damaging agents. Further, a combinatorial score of NEK10 and TP53-target gene expression is an independent predictor of a favorable outcome in breast cancers.

The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted.Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells.We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion.Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases.This research was funded by Janssen, Pharmaceutical Companies of JohnsonJohnson.

TENB2, a transmembrane proteoglycan protein, is a promising target for antibody drug conjugate (ADC) therapy due to overexpression in human prostate tumors and rapid internalization. We previously characterized how predosing with parental anti-TENB2 monoclonal antibody (mAb) at 1 mg/kg in a patient-derived LuCap77 explant model with high (3+) TENB2 expression could (i) block target-mediated intestinal uptake of tracer (& 0.1 mg/kg) levels of radiolabeled anti-TENB2-monomethyl auristatin E ADC while preserving tumor uptake, and (ii) maintain efficacy relative to ADC alone. Here, we systematically revisit this strategy to evaluate the effects of predosing on tumor uptake and efficacy in LuCap96.1, a low TENB2-expressing (1+) patient-derived model that is more responsive to ADC therapy than LuCap77. Importantly, rather than using tracer (& 0.1 mg/kg) levels, radiolabeled ADC tumor uptake was assessed at 1 mg/kg – one of the doses evaluated in the tumor growth inhibition study – in an effort to bridge tissue distribution (PK) with efficacy (PD). Predosing with mAb up to 1 mg/kg had no effect on efficacy. These findings warrant further investigations to determine whether predosing prior to ADC therapy might improve therapeutic index by preventing ADC disposition and possible toxicological liabilities in antigen-expressing healthy tissues.

Background Covid-19 significantly affected healthcare delivery over the past year, with a shift in focus away from nonurgent care. Emerging data are showing that screening for breast and colon cancer has dramatically decreased. It is unknown whether the same trend has affected patients with melanoma. Methods This is a retrospective cohort study of melanoma patients at two large-volume cancer centers. Patients were compared for 8 months before and after the lockdown. Outcomes focused on delay in treatment and possible resultant upstaging of melanoma. Results A total of 375 patients were treated pre-lockdown and 313 patients were treated post-lockdown (17% decrease). Fewer patients presented with in situ disease post-lockdown (15.3% vs. 17.9%), and a higher proportion presented with stage III-IV melanoma (11.2% vs. 9.9%). Comparing patients presenting 2 months before versus 2 months after the lockdown, there was an even more significant increase in Stage III-IV melanoma from 7.1% to 27.5% (p < 0.0001). Finally, in Stage IIIB-IIID patients, there was a decrease in patients receiving adjuvant therapy in the post lockdown period (20.0% vs. 15.2%). Conclusions As a result of the recent pandemic, it appears there has been a shift away from melanoma in situ and toward more advanced disease, which may have significant downstream effects on prognosis and could be due to a delay in screening. Significantly patients have presented after the lockdown, and fewer patients are undergoing the recommended adjuvant therapies. Patient outreach efforts are essential to ensure that patients continue to receive preventative medical care and screening as the pandemic continues.

T-cell–dependent bispecific antibodies (TDB) have been a major advancement in the treatment of cancer, allowing for improved targeting and efficacy for large molecule therapeutics. TDBs are comprised of one arm targeting a surface antigen on a cancer cell and another targeting an engaging surface antigen on a cytotoxic T cell. To impart this function, the antibody must be in a bispecific format as opposed to the more conventional bivalent format. Through in vitro and in vivo studies, we sought to determine the impact of changing antibody valency on solid tumor distribution and catabolism. A bivalent anti-HER2 antibody exhibited higher catabolism than its full-length monovalent binding counterpart in vivo by both invasive tissue harvesting and noninvasive single photon emission computed tomography/X-ray computed tomography imaging despite similar systemic exposures for the two molecules. To determine what molecular factors drove in vivo distribution and uptake, we developed a mechanistic model for binding and catabolism of monovalent and bivalent HER2 antibodies in KPL4 cells. This model suggests that observed differences in cellular uptake of monovalent and bivalent antibodies are caused by the change in apparent affinity conferred by avidity as well as differences in internalization and degradation rates of receptor bound antibodies. To our knowledge, this is the first study to directly compare the targeting abilities of monovalent and bivalent full-length antibodies. These findings may inform diverse antibody therapeutic modalities, including T-cell–redirecting therapies and drug delivery strategies relying upon receptor internalization.

The tumor suppressor PTEN is disrupted in a large proportion of cancers, including in HER2-positive breast cancer, where its loss is associated with resistance to therapy. Upon genotoxic stress, ataxia telangiectasia mutated (ATM) is activated and phosphorylates PTEN on residue 398. To elucidate the physiological role of this molecular event, we generated and analyzed knock-in mice expressing a mutant form of PTEN that cannot be phosphorylated by ATM (PTEN-398A). This mutation accelerated tumorigenesis in a model of HER2-positive breast cancer. Mammary tumors in bi-transgenic mice carrying MMTV-neu and Pten398A were characterized by DNA damage accumulation but reduced apoptosis. Mechanistically, phosphorylation of PTEN at position 398 is essential for the proper activation of the S phase checkpoint controlled by the PI3K–p27Kip1–CDK2 axis. Moreover, we linked these defects to the impaired ability of the PTEN-398A protein to relocalize to the plasma membrane in response to genotoxic stress. Altogether, our results uncover a novel role for ATM-dependent PTEN phosphorylation in the control of genomic stability, cell cycle progression, and tumorigenesis.

IL-2 is a powerful cytokine that enhances the activity of and induces the expansion of tumoricidal effector cells including T and NK cells. Despite promising signs of activity, the clinical utility of IL-2 is limited due to severe treatment-associated toxicities including vascular leak syndrome and the concurrent expansion of immunosuppressive regulatory T cells, which blunts efficacy. We developed a molecular targeted cytokine (MTC) platform that combines an engineered IL-2 variant with reduced affinity for the IL-2 receptor and high affinity single-domain antibodies (sdAb) to restrict IL-2 receptor signaling to cells expressing the target antigen (termed cis-signaling). INBRX-121 is an MTC designed to deliver IL-2 to NK cells using an NKp46-specific sdAb as the targeting moiety. In vitro, INBRX-121 induces dose-dependent STAT5 phosphorylation and proliferation in NK cells with comparable potency to wild-type IL-2, while showing no NKp46-independent activity at concentrations of more than three magnitudes higher than that required for NK cell activity. INBRX-121 enhances NK cell activation and cytotoxic capacity, thereby reducing the NK-to-target cell ratio as well as the target receptor density needed for optimal tumor cell killing. Furthermore, INBRX-121 lowers the threshold for antibody-dependent cellular cytotoxicity (ADCC) and increases maximal target cell killing in combination with ADCC-competent antibodies to an extent that may lessen the need for Fc engineering to enhance binding to the low affinity CD16A variant. In vivo, INBRX-121 demonstrates potent anti-tumor activity with increased intra-tumoral NK cell accumulation and upregulation of NK cell activation markers. INBRX-121 monotherapy reduces primary tumor growth and the formation of metastasis, while combination with ADCC-competent antibodies further increases NK cell activity to convert tumor growth suppression into complete tumor elimination. INBRX-121 is well tolerated in rodents and non-human primates and no overt signs of toxicity have been observed in cynomolgus monkeys up to the highest administered dose of 30 mg/kg. Our MTC platform provides a novel path to overcoming the limitations of IL-2 therapy through the pinpointed delivery of IL-2 activity to target cells of interest. In the case of INBRX-121 this ensures potent enhancement of the naturally broad anti-tumor activity of NK cells, which have distinct advantages over T cell therapeutics. Unlike T cells, tumor recognition by NK cells is independent of MHC-presented antigens and can elicit immediate cytotoxic activity. Further, many of the toxicities associated with T cell therapeutics have not been observed with NK-centric treatments. INBRX-121 therefore provides a promising safe and efficacious treatment option for a broad array of cancer indications both as a monotherapy and in combination with ADCC-enabled antibodies. Citation Format: Heather Kinkead, Florian J. Sulzmaier, Anya Polovina, Nadja Kern, Angelica Sanabria, Jason Ho, Chelsie Macedo, Ryan Henderson, Abrahim Hussain, Rajay Pandit, William Crago, Emily Rowell, Brendan P. Eckelman. INBRX-121, a safe and efficacious molecular targeted cytokine that enhances NK cell-mediated tumor killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3513.

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases associated with diverse cellular activities (AAA+) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released into the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe, generating a list of many previously unknown potential Cdc48-binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes, Cdc48-Ufd1 and Cdc48-Rbd2, are required for SREBP activation and low-oxygen adaptation in S. pombe.

Linear shaped charges (LSC) are typically manufactured in continuous lengths and formed into an inverted “V”. They use explosive force to cut through a target with a straight blade, usually in the demolition industry, but there is also significant interest in cutting a circle with the LSC formation for military and breaching applications. While some curved LSCs do exist, there are limitations for the curve due to the manufacturing process; additionally depth of penetration is reduced as the blade is formed at an angle due to varying inside and outside dimensions of the LSC. The run-up/run-down effect that is prevalent with the use of LSCs also poses as an obstacle towards cutting a full circle, as the optimal penetration depth is not reached in the run-up/run-down areas. Additive manufacturing allows for geometric complexity not possible in other manufacturing techniques. In this work, selective laser melting with a Renishaw 250 system was utilized. Using additive manufacturing, two separate design challenges were addressed; reducing the amount of run-up that occurs, and producing curved penetration with the LSC blade. LSC performance was evaluated by the depth of penetration, reduction of the amount of run-up that occurs, and curvature in the cut compared to traditional liners. The aim of this work is to show the potential for reducing the amount of run-up and curving the blade of a LSC through additive manufacturing of LSC liners.

Full-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted trans-cyclooctene (TCO)-modified antibodies. Noninvasive SPECT imaging of mice bearing HER2-expressing xenografts was performed both directly (111In-labeled antibody) and indirectly (pretargeted TCO-modified antibody followed by 111In-labeled tetrazine). Pharmacokinetic modulation of antibodies was achieved by two distinct methods: Fc engineering to reduce binding affinity to FcRn, and delayed administration of an antibody that competes with binding to FcRn. Tumor imaging with directly labeled antibodies was feasible in the absence of FcRn binding, rapidly attaining high tumor-to-blood ratios, but accompanied by moderate liver and spleen uptake. Pretargeted imaging of tumors with non-FcRn-binding antibody was also feasible, but systemic click reaction still occurred, albeit at lower levels than with parental antibody. Our findings demonstrate that FcRn binding impairment of full-length IgG antibodies moderately lowers tumor accumulation of radioactivity, and shifts background activity from blood pool to liver and spleen. Furthermore, reduction of FcRn binding did not eliminate systemic click reaction, but yielded greater improvements in tumor-to-blood ratio when imaging with directly labeled antibodies than with pretargeting.

In response to genotoxic stress, multiple kinase signaling cascades are activated, many of them directed towards the tumor suppressor p53, which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10, and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation by modulating the p53-dependent transcriptional output. NEK10 directly phosphorylates p53 on Y327, revealing NEK10's unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA-damaging agents. Further, a combinatorial score of NEK10 and TP53-target gene expression is an independent predictor of a favorable outcome in breast cancers.

BACKGROUND/AIMS: Poor visual outcomes have recently been reported in black patients undergoing vitrectomy and delamination surgery for complications of proliferative diabetic retinopathy. We therefore investigated the outcome of surgery on a similar cohort of black patients and examined for potential predictors of visual success. METHODS: A single-centre retrospective case review of consecutive black patients who underwent vitrectomy and delamination surgery for complications of PDR between July 2010 and September 2017. The primary outcome measure was change in visual acuity (VA) at 6 months post-operatively. Multiple linear regression analysis was performed to evaluate determinants of change in VA. RESULTS: A total of 44 eyes of 44 patients were included. Mean age was 53.7 (range: 24.3–75.8) years. In all, 43% were male. A total of 52% had adjunctive pre-operative anti-VEGF therapy. Mean pre-operative VA was 1.49 logMAR ± 0.73 (range 0.18 to 2.6 logMAR). Mean change in VA at 6 months was a gain of 0.59 ± 0.94 logMAR (range 1.9 logMAR acuity loss to 2.5 logMAR acuity gain). Four cases required further surgery to treat rhegmatogenous detachment. The acuity of 68% improved by 0.3 or more logMAR. Silicone oil was used as primary tamponade in 7%. Pre-operative VA and use of silicone oil significantly predicted VA decline at 6 months (p = 0.001 and p = 0.007). CONCLUSIONS: The majority of our cohort derived visual benefit from vitrectomy and delamination for PDR-related complications. Improvement in VA was comparable to outcomes from the UK National Ophthalmic Database report. Silicone oil should be avoided these patients if possible.

BACKGROUND: Ophthalmic emergencies comprise a significant portion of junior doctors’ workload in accident and emergency (A&E). However, previous studies have demonstrated a lack of training and confidence in the management of such emergencies. This study assessed changes in basic ophthalmic training that A&E junior doctors received in dealing with eye emergencies, their perceived level of confidence and the availability of appropriate ophthalmic equipment in A&E over the last 15 years. METHODS: A prospective, national, combined online and telephone survey using a previously published questionnaire was performed. Foundation year two doctors (FY2s) from each A&E department in the UK listed on the official NHS directory were contacted for participation. RESULTS: Two hundred and ten A&E departments were contacted and 202 responded (response rate of 96.2%). There was no significant change in the number of A&E departments equipped with slit lamps (82.5% in 2003 vs 79.7% in 2018; p = 0.26). However, the prevalence of training in its use has decreased significantly (68.4% in 2003 vs 52% in 2018; p = 0.005). There was also a significant reduction in the prevalence of training in the management of eye emergencies (77.4% in 2003 vs 45.5% in 2018; p

AIMS: To report anatomical and functional outcomes in patients with proliferative sickle retinopathy (PSR) who underwent 23-gauge (23G) and 20-gauge (20G) vitrectomy. METHODS: Retrospective consecutive case series of patients who underwent vitreoretinal intervention for complications of PSR between April 2009 and February 2015. Operations were performed at a tertiary referral centre, Moorfields Eye Hospital. Visual acuity and anatomical success rates were evaluated for PSR complicated by retinal detachment, tractional vitreous haemorrhage and macular hole. Proliferative diabetic retinopathy cases were excluded. RESULTS: A total of 71 eyes (63 patients) underwent vitreoretinal surgery for PSR complications with 26 months mean follow-up. Primary indications were: tractional retinal detachment (TRD, n = 17), TRD with rhegmatogenous retinal detachment (n = 16), rhegmatogenous retinal detachment (n = 5, macula-on: 1, macula-off: 4), vitreous haemorrhage (n = 19), epiretinal membrane (n = 6), and full thickness macula hole (n = 8). Thirty-nine cases underwent 20G vitrectomy, and 23G surgery was performed in 32 eyes. Mean best corrected visual acuity (BCVA) improved from pre-operative 1.30 LogMAR to final BCVA of 0.74 LogMAR (p

PURPOSE To evaluate the real world clinical outcomes of intravitreal ocriplasmin in patients with vitreomacular traction (VMT) with and without full thickness macular holes (FTMH) treated according to NICE guidance. METHODS Retrospective observational case series of 25 patients treated with a single intravitreal ocriplasmin injection between December 2013 and December 2015. Best corrected visual acuity and optical coherence tomography exams were performed to determine visual outcomes and anatomical VMT release and FTMH closure over time. Two patient groups were identified: ocular macular co-morbidity (OCM) and no OCM (nOCM), with follow-up at 4, 12, and 24 weeks. RESULTS Twenty-five patients were identified that included 19 patients with VMT, and 6 patients with VMT plus FTMH. In the nOCM group of 22 patients, the release rate of VMT was 44%, 63%, and 69% at 4, 12 and 24 weeks respectively. In the "real-world" OCM group of 25 patients, the VMT release rate was 37%, 53%, and 58% at the same time-points. In both groups, the FTMH closure rate was 33%, 50%, and 67% at 4, 12, and 24 weeks. At mean follow-up of 30 weeks in the VMT group with nOCM, the mean LogMAR VA improved significantly from 0.44 to 0.28 (p = 0.0068, paired t-test). Three were no serious adverse events. CONCLUSIONS This study reports improved efficacy of intravitreal ocriplasmin for both VMT and FTMH, and is more favourable in patients with no ocular co-morbidity. We highlight the importance of careful patient selection and structured standard of care pathways to identify patients who will benefit from the positive visual and anatomical effects of intravitreal ocriplasmin.

How do household members influence one another9s television-viewing behaviors, and how can these behaviors affect new programming? The current study offers a method to separate two different sources of interpersonal influence among television viewers in the same household: what the authors call “social co-viewing” and the intrinsic preferences of another viewer independent of co-viewing. Applying the method to people-meter data from Mexico, the researchers found that, ultimately, wives were more influential than husbands in building audiences. The authors believe their method can be applied to any people-meter data, providing insight for programmers promoting new shows and for advertisers choosing programs to sponsor in the upfront market.

Anti-HER2/CD3, a T-cell–dependent bispecific antibody (TDB) construct, induces T-cell–mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell–targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ϵ expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell–rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell–containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776–85. ©2018 AACR.

For years, clinical and basic researchers have been aware of the presence of PTEN in the nucleus in cell culture, animal models, and both healthy and diseased human tissues. Despite the early recognition of nuclear PTEN, the understanding of the mechanisms of its nuclear localization, function in the nucleus, and importance in biology and human disease has been lacking. Over the last decade, emerging concepts for the complex involvement of nuclear PTEN in a variety of processes, including genome maintenance and DNA repair, cell-cycle control, gene expression, and DNA replication, are illuminating what could prove to be the key path toward a full understanding of PTEN function in health and disease. Dysregulation of nuclear PTEN is now considered an important aspect of the etiology of many pathologic conditions, prompting reconsideration of the therapeutic approaches aimed at countering the consequences of PTEN deficiency. This new knowledge is fueling the development of innovative therapeutic modalities for a broad spectrum of human conditions, from cancer and metabolic diseases, to neurological disorders and autism.

PTEN Variations The product of the tumor suppressor gene phosphate and tensin homolog on chromosome ten ( PTEN) is a lipid and protein phosphatase that regulates important cellular processes, including growth, survival, and metabolism (see the Perspective by Leslie and Brunton ). Though PTEN is best known for effects on the phosphatidylnositol 3-kinase (PI3K) signaling pathway, the PTEN protein is also found in the nucleus. Bassi et al. (p. 395 ) found that PTEN's presence in the nucleus was regulated in response to covalent modification of the protein by SUMOylation and phosphorylation. Cells lacking nuclear PTEN showed increased sensitivity to DNA damage and underwent cell death if the PI3K pathway was also inhibited. Hopkins et al. (p. 399 , published online 6 June) discovered an alternative translation start site in human PTEN messenger RNA that allowed expression of a protein, PTEN-Long, with about 170 extra amino acids. The unusual enzyme was released from cells and then taken up into other cells. In a mouse tumor model, uptake of the enzyme inhibited the PI3K pathway and inhibited tumor growth.

Terminal differentiation of B cells and hypergammaglobulinemia are hallmarks of B-cell hyperactivity in HIV disease. Plasmablasts are terminally differentiating B cells that circulate transiently in the blood following infection or vaccination; however, in HIV infection, they arise early and are maintained at abnormally high levels in viremic individuals. Here we show that only a small fraction of plasmablasts in the blood of viremic individuals is HIV specific. Assessment of plasmablast immunoglobulin isotype distribution revealed increased IgG + plasmablasts in early and most prominently during chronic HIV viremia, contrasting with a predominantly IgA + plasmablast profile in HIV-negative individuals or in aviremic HIV-infected individuals on treatment. Of note, IgG is the predominant immunoglobulin isotype of plasmablasts that arise transiently in the blood following parenteral immunization. Serum immunoglobulin levels were also elevated in HIV-infected viremic individuals, especially IgG, and correlated with levels of IgG + plasmablasts. Several soluble factors associated with immune activation were also increased in the sera of HIV-infected individuals, especially in viremic individuals, and correlated with serum immunoglobulin levels, particularly IgG. Thus, our data suggest that while plasmablasts in the blood may contribute to the HIV-specific immune response, the majority of these cells are not HIV specific and arise early, likely from indirect immune-activating effects of HIV replication, and reflect over time the effects of chronic antigenic stimulation. Such B-cell dysregulation may help explain why the antibody response is inadequate in HIV-infected individuals, even during early infection.

Cancer sequencing predicts driver genes using recurrent protein-altering mutations, but detecting recurrence for non-coding mutations remains unsolved. Here, we present a convergence framework for recurrence analysis of non-coding mutations using three-dimensional co-localization of epigenomically-identified regions. We define the regulatory plexus of each gene as its cell-type-specific three-dimensional gene-regulatory neighborhood, inferred using Hi-C chromosomal interactions and chromatin state annotations. Using 16 matched tumor-normal prostate transcriptomes, we predict tumor-upregulated genes, and find enriched plexus mutations in distal regulatory regions normally repressed in prostate, suggesting out-of-context de-repression. Using 55 matched tumor-normal prostate genomes, we predict 15 driver genes by convergence of dispersed, low-frequency mutations into high-frequency dysregulation events along prostate-specific plexi, while controlling for mutational heterogeneity across regions, chromatin states, and patients. These putative drivers play roles in growth signaling, immune evasion, mitochondrial function, and vascularization, suggesting higher-order pathway-level convergence. We experimentally validate the PLCB4 plexus and its ability to affect the canonical PI3K cancer pathway.

Central and split systems are the two most common air conditioning (AC) systems used in residential applications. Central system employs one large unit to produce and distribute conditioned air through a system of ductwork. On the other hand, the split system, employs several small ACs. Each AC consisted of outdoor and indoor units to produce conditioned air directly to the designed area. Each system has distinct strengths and weaknesses. Depending on the structure of cooling area and operating schedule, the performance of each system will be different. The aim of this paper is to examine the impact of various parameters such as operating schedule and building characteristics to the performance of central and split AC systems over the 25 years of their operation. The life cycle analysis (LCA) considered essential factors which have significant impact on the energy consumption and both initial and operating costs of the two systems. All required sections of life cycle analysis are included according to the relevant Australian Standards. The results indicated that under standard operating conditions, central system is more economical and energy efficient than split system. However, when the flexibility in operation of split system is considered, there was a significant reduction in its operating cost, which was below that of central system. Overall, total life cycle cost of split system was slightly lower than central system. Also, considering the usage flexibility and the comfort of users, it appears that the split system is more suitable than the central AC system for residential buildings.

Head pose implies a person's visual attention and interest. It plays an important role in many applications. Existing head pose estimation methods work in the original head pose space. However, the large number of head pose candidates in the space makes the estimation task quite challenging. In this paper, we propose a coarse-to-fine head pose estimation method by decomposing the original pose space into a hierarchical structure. The estimation begins by detecting the region of interest (ROI) within a face image via measuring the importance scores of key image points. After that, a coarse head pose estimation step is applied to identify a subset of head pose candidates, based on Gabor filter and random forest. A fine estimation is then employed within the subset, using histogram of oriented gradient (HOG) and support vector machine (SVM). Finally, we apply the proposed method to TV viewers' behavior analysis by determining whether a viewer is focused or unfocused, which can be useful for marketing research.

Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumab was administered alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography–X-ray computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastuzumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-VEGF treatment, with no significant differences in the other physiologic parameters measured despite immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to achieve improved clinical outcomes. Mol Cancer Ther; 11(3); 752–62. ©2012 AACR.

Television co-viewing is a frequent behavior with important social and economic implications. This study proposes a measure of co-viewing on people meter panel data, tests it on a data set in Mexic...