Impact of DOTA Conjugation on Pharmacokinetics and Immunoreactivity of [177Lu]Lu-1C1m-Fc, an Anti TEM-1 Fusion Protein Antibody in a TEM-1 Positive Tumor Mouse Model.

1C1m-Fc, an anti-tumor endothelial marker 1 (TEM-1) scFv-Fc fusion protein antibody, was previously successfully radiolabeled with <sup>177</sup> Lu. TEM-1 specific tumor uptake was observed together with a non-saturation dependent liver uptake that could be related to the number of dodecane tetraacetic acid (DOTA) chelator per 1C1m-Fc. The objective of this study was to verify this hypothesis and to find the best DOTA per 1C1m-Fc ratio for theranostic applications. 1C1m-Fc was conjugated with six concentrations of DOTA. High-pressure liquid chromatography, mass spectrometry, immunoreactivity assessment, and biodistribution studies in mice bearing TEM-1 positive tumors were performed. A multi-compartment pharmacokinetic model was used to fit the data and a global pharmacokinetic model was developed to illustrate the effect of liver capture and immunoreactivity loss. Organ absorbed doses in mice were calculated from biodistribution results. A loss of immunoreactivity was observed with the highest DOTA per 1C1m-Fc ratio. Except for the spleen and bone, an increase of DOTA per 1C1m-Fc ratio resulted in an increase of liver uptake and absorbed dose and a decrease of uptake in tumor and other tissues. Pharmacokinetic models correlated these results. The number of DOTA per antibody played a determining role in tumor targeting. One DOTA per 1C1m-Fc gave the best pharmacokinetic behavior for a future translation of [ <sup>177</sup> Lu]Lu-1C1m-Fc in patients

A Pre-Landing Assessment of Regolith Properties at the InSight Landing Site

This article discusses relevant physical properties of the regolith at the Mars InSight landing site as understood prior to landing of the spacecraft. InSight will land in the northern lowland plains of Mars, close to the equator, where the regolith is estimated to be ≥3--5 m thick. These investigations of physical properties have relied on data collected from Mars orbital measurements, previously collected lander and rover data, results of studies of data and samples from Apollo lunar missions, laboratory measurements on regolith simulants, and theoretical studies. The investigations include changes in properties with depth and temperature. Mechanical properties investigated include density, grain-size distribution, cohesion, and angle of internal friction. Thermophysical properties include thermal inertia, surface emissivity and albedo, thermal conductivity and diffusivity, and specific heat. Regolith elastic properties not only include parameters that control seismic wave velocities in the immediate vicinity of the Insight lander but also coupling of the lander and other potential noise sources to the InSight broadband seismometer. The related properties include Poisson’s ratio, P- and S-wave velocities, Young’s modulus, and seismic attenuation. Finally, mass diffusivity was investigated to estimate gas movements in the regolith driven by atmospheric pressure changes. Physical properties presented here are all to some degree speculative. However, they form a basis for interpretation of the early data to be returned from the InSight mission.Additional co-authors: Nick Teanby and Sharon Keda

De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals where it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologs. Using RNA-sequencing, we show how 5’ splice site usage is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 bp region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide

From bench to bedside: 64Cu/177Lu 1C1m-Fc anti TEM-1: mice-to-human dosimetry extrapolations for future theranostic applications.

The development of diagnostic and therapeutic radiopharmaceuticals is an hot topic in nuclear medicine. Several radiolabeled antibodies are under development necessitating both biokinetic and dosimetry extrapolations for effective human translation. The validation of different animal-to-human dosimetry extrapolation methods still is an open issue. This study reports the mice-to-human dosimetry extrapolation of <sup>64</sup> Cu/ <sup>177</sup> Lu 1C1m-Fc anti-TEM-1 for theranostic application in soft-tissue sarcomas. We adopt four methods; direct mice-to-human extrapolation (M1); dosimetry extrapolation considering a relative mass scaling factor (M2), application of a metabolic scaling factor (M3) and combination of M2 and M3 (M4). Predicted in-human dosimetry for the [ <sup>64</sup> Cu]Cu-1C1m-Fc resulted in an effective dose of 0.05 mSv/MBq. Absorbed dose (AD) extrapolation for the [ <sup>177</sup> Lu]Lu-1C1m-Fc indicated that the AD of 2 Gy and 4 Gy to the red-marrow and total-body can be reached with 5-10 GBq and 25-30 GBq of therapeutic activity administration respectively depending on applied dosimetry method. Dosimetry extrapolation methods provided significantly different absorbed doses in organs. Dosimetry properties for the [ <sup>64</sup> Cu]Cu-1C1m-Fc are suitable for a diagnostic in-human use. The therapeutic application of [ <sup>177</sup> Lu]Lu-1C1m-Fc presents challenges and would benefit from further assessments in animals' models such as dogs before moving into the clinic

¹⁷⁷Lu radiolabeling and preclinical theranostic study of 1C1m-Fc: an anti-TEM-1 scFv-Fc fusion protein in soft tissue sarcoma.

TEM-1 (tumor endothelial marker-1) is a single-pass transmembrane cell surface glycoprotein expressed at high levels by tumor vasculature and malignant cells. We aimed to perform a preclinical investigation of a novel anti-TEM-1 scFv-Fc fusion antibody, 1C1m-Fc, which was radiolabeled with <sup>177</sup> Lu for use in soft tissue sarcomas models. 1C1m-Fc was first conjugated to p-SCN-Bn-DOTA using different excess molar ratios and labeled with <sup>177</sup> Lu. To determine radiolabeled antibody immunoreactivity, Lindmo assays were performed. The in vivo behavior of [177Lu]Lu-1C1m-Fc was characterized in mice bearing TEM-1 positive (SK-N-AS) and negative (HT-1080) tumors by biodistribution and single-photon emission SPECT/CT imaging studies. Estimated organ absorbed doses were obtained based on biodistribution results. The DOTA conjugation and the labeling with <sup>177</sup> Lu were successful with a radiochemical purity of up to 95%. Immunoreactivity after radiolabeling was 86% ± 4%. Biodistribution showed a specific uptake in TEM-1 positive tumor versus liver as critical non-specific healthy organ, and this specificity is correlated to the number of chelates per antibody. A 1.9-fold higher signal at 72 h was observed in SPECT/CT imaging in TEM-1 positive tumors versus control tumors. TEM-1 is a promising target that could allow a theranostic approach to soft-tissue sarcoma, and 1C1m-Fc appears to be a suitable targeting candidate. In this study, we observed the influence of the ratio DOTA/antibody on the biodistribution. The next step will be to investigate the best conjugation to achieve an optimal tumor-to-organ radioactivity ratio and to perform therapy in murine xenograft models as a prelude to future translation in patients

Copper-64-Labeled 1C1m-Fc, a New Tool for TEM-1 PET Imaging and Prediction of Lutetium-177-Labeled 1C1m-Fc Therapy Efficacy and Safety.

1C1m-Fc, a promising anti-TEM-1 DOTA conjugate, was labeled with <sup>64</sup> Cu to target cancer cells for PET imaging and predicting the efficacy and safety of a previously studied [ <sup>177</sup> Lu]Lu-1C1m-Fc companion therapy. DOTA-conjugated 1C1m-Fc was characterized by mass spectrometry, thin layer chromatography and immunoreactivity assessment. PET/CT and biodistribution studies were performed in human neuroblastoma xenografted mice. Absorbed doses were assessed from biodistribution results and extrapolated to <sup>177</sup> Lu based on the [ <sup>64</sup> Cu]Cu-1C1m-Fc data. The immunoreactivity was ≥ 70% after 48 h of incubation in serum, and the specificity of [ <sup>64</sup> Cu]Cu-1C1m-Fc for the target was validated. High-resolution PET/CT images were obtained, with the best tumor-to-organ ratios reached at 24 or 48 h and correlated with results of the biodistribution study. Healthy organs receiving the highest doses were the liver, the kidneys and the uterus. [ <sup>64</sup> Cu]Cu-1C1m-Fc could be of interest to give an indication of <sup>177</sup> Lu dosimetry for parenchymal organs. In the uterus and the tumor, characterized by specific TEM-1 expression, the <sup>177</sup> Lu-extrapolated absorbed doses are overestimated because of the lack of later measurement time points. Nevertheless, 1C1m-Fc radiolabeled with <sup>64</sup> Cu for imaging would appear as an interesting radionuclide companion for therapeutic application with [ <sup>177</sup> Lu]Lu-1C1m-Fc

Aspectos epidemiológicos da meningite tuberculosa em menores de 15 anos de idade, na Grande São Paulo, Brasil, 1982 -1983 Epidemiological aspects of tuberculous meningits in children under 15 years of age, Greater S. Paulo, Brazil, 1982 -1983

Foram estudadas algumas características epidemiológicas dos casos de meningite tuberculosa ocorridos em menores de 15 anos na Grande São Paulo (Brasil), nos anos de 1982 e 1983. O levantamento dos dados foi realizado em fontes oficiais de informação, complementado pela visitação domiciliária. Foram identificados 126 casos, analisados segundo distribuição etária, sexo, fonte de contágio, vacinação BCG, diagnóstico firmado, letalidade hospitalar, seqüelas e eventos ocorridos na seqüência do tratamento. Os resultados mostraram: demora no diagnóstico devido a prováveis falhas assistênciais, alta letalidade, identificação dos focos para a maioria dos casos. Houve dificuldade em avaliar a proteção conferida pela vacinação BCG e na ocorrência de seqüelas, o grupo de menores de 5 anos foi o mais comprometido (83,9%) enquanto que a maior letalidade ocorreu no grupo de 0 a 1 ano de idade (43,1%). Houve 38,9% de cura; 33,3% de óbito; 15,1% de abandono e em 12,7% dos casos alguns permaneceram sob controle; e o restante era desconhecido pelo sistema de controle de notificações.<br>Some epidemiological characteristics of cases of tuberculous meningitis which occurred in subjects under 15 years of age in Greater S. Paulo, S. Paulo State, Brazil, in 1982 and 1983, are studied. The cases were surveyed on the basis of official sources of information, complement by domiciliary visits. A hundred and twenty six (126) cases were identified and analysed by age, sex, source of contagion, BCG vaccination, confirmed diagnosis, hospital lethality, sequels and intercurrent events as part of the follow up. The results showed a delay in diagnosing the cases, possibly due to assistential failures; a high rate of lethality; identification of infectious focuses for the majority of the cases. There were difficulties in evaluating the protection provided by BCG vaccination and as regards sequels the most affected (83.9%) were the under-fives while the greatest lethality rate (43.1%) was found among the 0-1 year- olds. At the end of the study there were 38.9% of cases of cure; 33.3% of death; 15.1% of withdrawal, and the remaining 12.7% some were still under control and others unknown to the system of notification