The anti-CD40L monoclonal antibody AT-1501 promotes islet and kidney allograft survival and function in nonhuman primates.

Prior studies of anti-CD40 ligand (CD40L)-based immunosuppression demonstrated effective prevention of islet and kidney allograft rejection in nonhuman primate models; however, clinical development was halted because of thromboembolic complications. An anti-CD40L-specific monoclonal antibody, AT-1501 (Tegoprubart), was engineered to minimize risk of thromboembolic complications by reducing binding to Fcγ receptors expressed on platelets while preserving binding to CD40L. AT-1501 was tested in both a cynomolgus macaque model of intrahepatic islet allotransplantation and a rhesus macaque model of kidney allotransplantation. AT-1501 monotherapy led to long-term graft survival in both islet and kidney transplant models, confirming its immunosuppressive potential. Furthermore, AT-1501-based regimens after islet transplant resulted in higher C-peptide, greater appetite leading to weight gain, and reduced occurrence of cytomegalovirus reactivation compared with conventional immunosuppression. These data support AT-1501 as a safe and effective agent to promote both islet and kidney allograft survival and function in nonhuman primate models, warranting further testing in clinical trials.

Development of a classifier for [18F]fluorodeoxyglucose extravasation severity using semi-quantitative readings from topically applied detectors.

BACKGROUND: Radiotracer extravasations, caused largely by faulty tracer injections, can occur in up to 23% of 18F-fluorodeoxyglucose (FDG) PET/CT scans and negatively impact radiological review and tracer quantification. Conventional radiological assessment of extravasation severity on PET has limited performance (e.g., extravasations frequently resolve before scanning) and practical drawbacks. In this study, we develop a new topical detector-based FDG extravasation severity classifier, calibrated from semi-quantitative PET measurements, and assess its performance on human subjects. METHODS: A retrospective study examined patients whose FDG injections had been monitored as part of their standard workup for PET/CT imaging. Topical uncollimated gamma ray detectors were applied proximal to the injection site and on the same location on the opposing arm, and readings were acquired continuously during radiotracer uptake. Patients were imaged with their arms in the PET field of view and total extravasation activity quantified from static PET images through a volume of interest approach. The image-derived activities were considered ground truth and used to calibrate and assess quantification of topical detector readings extrapolated to the start of PET imaging. The classifier utilizes the calibrated detector readings to produce four extravasation severity classes: none, minor, moderate, and severe. In a blinded study, a radiologist qualitatively labeled PET images for extravasation severity using the same classifications. The radiologist's interpretations and topical detector classifications were compared to the ground truth PET results. RESULTS: Linear regression of log-transformed image-derived versus topical detector tracer extravasation activity estimates showed a strong correlation (R2 = 0.75). A total of 24 subject scans were cross-validated with the quantitatively based classifier through a leave-one-out methodology. For binary classification (none vs. extravasated), the topical detector classifier had the highest overall diagnostic performance for identifying extravasations. Specificity, sensitivity, accuracy, and positive predictive value were 100.0%, 80.0%, 95.8%, and 100.0%, respectively, for the topical detector classifier and 31.6%, 100.0%, 45.8%, and 27.8%, respectively, for the radiological analysis. The topical detector classifier, with an optimal detection threshold, produced a significantly higher Matthews correlation coefficient (MCC) than the radiological analysis (0.87 vs. 0.30). CONCLUSIONS: The topical detector binary classifier, calibrated using quantitative static PET measurements, significantly improves extravasation detection compared to qualitative image analysis.

A human-derived antibody targets misfolded SOD1 and ameliorates motor symptoms in mouse models of amyotrophic lateral sclerosis.

Mutations in the gene encoding superoxide dismutase 1 (SOD1) lead to misfolding and aggregation of SOD1 and cause familial amyotrophic lateral sclerosis (FALS). However, the implications of wild-type SOD1 misfolding in sporadic forms of ALS (SALS) remain unclear. By screening human memory B cells from a large cohort of healthy elderly subjects, we generated a recombinant human monoclonal antibody (α-miSOD1) that selectively bound to misfolded SOD1, but not to physiological SOD1 dimers. On postmortem spinal cord sections from 121 patients with ALS, α-miSOD1 antibody identified misfolded SOD1 in a majority of cases, regardless of their SOD1 genotype. In contrast, the α-miSOD1 antibody did not bind to its epitope in most of the 41 postmortem spinal cord sections from non-neurological control (NNC) patients. In transgenic mice overexpressing disease-causing human SOD1G37R or SOD1G93A mutations, treatment with the α-miSOD1 antibody delayed the onset of motor symptoms, extended survival by up to 2 months, and reduced aggregation of misfolded SOD1 and motor neuron degeneration. These effects were obtained whether α-miSOD1 antibody treatment was administered by direct brain infusion or peripheral administration. These results support the further development of α-miSOD1 antibody as a candidate treatment for ALS involving misfolding of SOD1.

C57BL/6J congenic Prp-TDP43A315T mice develop progressive neurodegeneration in the myenteric plexus of the colon without exhibiting key features of ALS.

ALS therapy development has been hindered by the lack of rodent animal models. The discovery of TDP-43, a transcription factor that accumulates in the cytoplasm of motor neurons (MNs) in most cases of ALS, prompted attempts to develop TDP-43-based models of the disease. The current study sought to examine, in extensive detail, the emerging disease phenotype of a transgenic mouse model that overexpresses a mutant human TDP-43 (hTDP-43) gene under mouse prion promoter control. Careful attention was given to ALS-like characteristics to determine the appropriateness of this model for testing therapies for ALS. In light of previous reports that gastrointestinal (GI) dysfunction is responsible for early death in these mice, gut immunohistochemistry (IHC) and longitudinal gut motility assays were used to identify the onset and the progression of these defects. IHC studies revealed that site-specific overexpression of the hTDP-43 transgene in colonic myenteric plexes resulted in progressive neurodegeneration in this region. This change was associated with progressively reduced GI motility, culminating in frank stasis that was primarily responsible for decreasing longevity in these mice. The disease phenotype was gender- and genetic background-dependent, with congenic C57BL/6J male mice exhibiting the most aggressive form of the disease. Spinal cord IHC revealed ubiquitin-positive inclusions, but not TDP-43 aggregates, in the cytoplasm of MNs. Neither gender exhibited compelling ALS-like neuromuscular deficits, irrespective of age. While this model may be useful for studying GI tract neurodegeneration, in its present state it does not display a phenotype suitable for testing ALS therapeutics.

The advantages of frontotemporal degeneration drug development (part 2 of frontotemporal degeneration: the next therapeutic frontier).

Frontotemporal degeneration (FTD) encompasses a spectrum of related neurodegenerative disorders with behavioral, language, and motor phenotypes for which there are currently no effective therapies. This is the second of two articles that summarize the presentations and discussions that occurred at two symposia in 2011 sponsored by the Frontotemporal Degeneration Treatment Study Group, a collaborative group of academic and industry researchers that is devoted to developing treatments for FTD. This article discusses the current status of FTD clinical research that is relevant to the conduct of clinical trials, and why FTD research may be an attractive pathway for developing therapies for neurodegenerative disorders. The clinical and molecular features of FTD, including rapid disease progression and relatively pure molecular pathology, suggest that there are advantages to developing drugs for FTD as compared with other dementias. FTD qualifies as orphan indication, providing additional advantages for drug development. Two recent sets of consensus diagnostic criteria will facilitate the identification of patients with FTD, and a variety of neuropsychological, functional, and behavioral scales have been shown to be sensitive to disease progression. Moreover, quantitative neuroimaging measurements demonstrate progressive brain atrophy in FTD at rates that may surpass Alzheimer's disease. Finally, the similarities between FTD and other neurodegenerative diseases with drug development efforts already underway suggest that FTD researchers will be able to draw on this experience to create a road map for FTD drug development. We conclude that FTD research has reached sufficient maturity to pursue clinical development of specific FTD therapies.

A call for transparent reporting to optimize the predictive value of preclinical research.

The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS.

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Targeting the lymphotoxin-beta receptor with agonist antibodies as a potential cancer therapy.

The lymphotoxin-beta receptor (LT beta R) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LT beta R monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LT beta R caused increased lymphocyte infiltration and necrosis of the tumor. A pattern of differential gene expression predictive of cellular and xenograft response to LT beta R activation was identified in a panel of colon carcinoma cell lines and when applied to a panel of clinical colorectal tumor samples indicated 35% likelihood a tumor response to CBE11. Consistent with this estimate, CBE11 decreased tumor size and/or improved long-term animal survival with two of six independent orthotopic xenografts prepared from surgical colorectal carcinoma samples. Targeting of LT beta R with agonistic mAbs offers a novel approach to the treatment of colorectal and potentially other types of cancers.

Identification of SFRP1 as a candidate mediator of stromal-to-epithelial signaling in prostate cancer.

Genetic changes in epithelial cells initiate the development of prostatic adenocarcinomas. As nascent tumors grow and undergo progression, epithelial tumor cells are intimately associated with stromal cells. Stromal cells within the tumor microenvironment acquire new properties, including the capacity to promote phenotypic and genetic progression in adjacent epithelial cells. Affymetrix microarrays were used to identify 119 genes differentially expressed between normal-derived and carcinoma-derived prostatic stromal cells. These included 31 genes encoding extracellular proteins that may act as stromal-to-epithelial paracrine signals. Further investigation of one of these genes, secreted frizzled related protein 1 (SFRP1), revealed that its expression parallels prostatic growth with high expression during prostatic development, low expression in the adult prostate, and elevated expression in prostatic tumor stroma. In addition, as prostatic epithelial cells progressed to a tumorigenic state under the influence of tumor stroma, SFRP1 became overexpressed in the progressed epithelial cells. To further understand the roles of SFRP1 in the prostate, we tested the affects of increased SFRP1 levels on prostatic tissues and cells. Treatment of developing prostates with SFRP1 in culture led to increased organ growth. Treatment of a human prostatic epithelial cell line with SFRP1 led to increased proliferation, decreased apoptosis, and decreased signaling through the Wnt/beta-catenin pathway in vitro and increased proliferation in vivo. These data suggest that overexpression of SFRP1 by prostatic tumor stroma may account for the previously reported capacity of prostatic tumor stroma to provide a pro-proliferative paracrine signal to adjacent epithelial cells.

LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex.

Axon regeneration in the adult CNS is prevented by inhibitors in myelin. These inhibitors seem to modulate RhoA activity by binding to a receptor complex comprising a ligand-binding subunit (the Nogo-66 receptor NgR1) and a signal transducing subunit (the neurotrophin receptor p75). However, in reconstituted non-neuronal systems, NgR1 and p75 together are unable to activate RhoA, suggesting that additional components of the receptor may exist. Here we describe LINGO-1, a nervous system-specific transmembrane protein that binds NgR1 and p75 and that is an additional functional component of the NgR1/p75 signaling complex. In non-neuronal cells, coexpression of human NgR1, p75 and LINGO-1 conferred responsiveness to oligodendrocyte myelin glycoprotein, as measured by RhoA activation. A dominant-negative human LINGO-1 construct attenuated myelin inhibition in transfected primary neuronal cultures. This effect on neurons was mimicked using an exogenously added human LINGO-1-Fc fusion protein. Together these observations suggest that LINGO-1 has an important role in CNS biology.