RESUMEN
Rare coding variants that substantially affect function provide insights into the biology of a gene1-3. However, ascertaining the frequency of such variants requires large sample sizes4-8. Here we present a catalogue of human protein-coding variation, derived from exome sequencing of 983,578 individuals across diverse populations. In total, 23% of the Regeneron Genetics Center Million Exome (RGC-ME) data come from individuals of African, East Asian, Indigenous American, Middle Eastern and South Asian ancestry. The catalogue includes more than 10.4 million missense and 1.1 million predicted loss-of-function (pLOF) variants. We identify individuals with rare biallelic pLOF variants in 4,848 genes, 1,751 of which have not been previously reported. From precise quantitative estimates of selection against heterozygous loss of function (LOF), we identify 3,988 LOF-intolerant genes, including 86 that were previously assessed as tolerant and 1,153 that lack established disease annotation. We also define regions of missense depletion at high resolution. Notably, 1,482 genes have regions that are depleted of missense variants despite being tolerant of pLOF variants. Finally, we estimate that 3% of individuals have a clinically actionable genetic variant, and that 11,773 variants reported in ClinVar with unknown significance are likely to be deleterious cryptic splice sites. To facilitate variant interpretation and genetics-informed precision medicine, we make this resource of coding variation from the RGC-ME dataset publicly accessible through a variant allele frequency browser.
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Exoma , Variación Genética , Proteínas , Humanos , Alelos , Exoma/genética , Secuenciación del Exoma , Frecuencia de los Genes , Variación Genética/genética , Heterocigoto , Mutación con Pérdida de Función/genética , Mutación Missense/genética , Sistemas de Lectura Abierta/genética , Proteínas/genética , Sitios de Empalme de ARN/genética , Medicina de PrecisiónRESUMEN
Fc fusion proteins are a new emerging class of molecules for immune-targeted delivery of therapeutic proteins. Biophysical and bioanalytical characterization is critical for clinical development and delivery of therapeutic proteins. Here we report molecular and functional characterization of a recombinant human fusion protein Mutant IL-15/Fc. MutIL-15/Fc has a molecular weight of â¼95 kDa as determined by multiangle laser light scattering with online size exclusion chromatography and migrated at a faster rate (lower retention time) in gel filtration column. The kinetics of binding of MutIL-15/Fc to Fcγ receptor is best fitted in a bivalent modal with K(D1) 5 µM and K(D2) 9 µM determined by surface plasmon resonance (BIAcore). N-Glycoprofiling analysis revealed extensive glycosylation of MutIL-15/Fc. The Fc and IL-15 components in the MutIL-15/Fc are detected using the dual mode ELISA. The HT-2 cell proliferation inhibition assay is qualified as a quantitative in vitro marker functional assay. Molecular state changes associated with forced stress analyzed by SEC-MALS resulted in changes in bioactivity and Fc:Fcγ receptor interaction affinity. These data provide a systematic approach to molecular and functional characterization of the MutIL-15/Fc to establish product consistency and stability monitoring during storage and under drug delivery conditions.
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Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Interleucina-15/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proliferación Celular , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Cinética , Unión Proteica , Receptores de IgG/metabolismoRESUMEN
BACKGROUND: In December 2019 the World Health Organization announced that the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection had become a global pandemic. The most affected organ by the novel virus is the lung, and imaging exploration of the thorax using computer tomography (CT) scanning and X-ray has had an important impact. MATERIALS AND METHODS: We assessed the prevalence of lung lesions in vaccinated versus unvaccinated SARS-CoV-2 patients using an artificial intelligence (AI) platform provided by Medicai. The software analyzes the CT scans, performing the lung and lesion segmentation using a variant of the U-net convolutional network. RESULTS: We conducted a cohort study at a tertiary lung hospital in which we included 186 patients: 107 (57.52%) male and 59 (42.47%) females, of which 157 (84.40%) were not vaccinated for SARS-CoV-2. Over five times more unvaccinated patients than vaccinated ones are admitted to the hospital and require imaging investigations. More than twice as many unvaccinated patients have more than 75% of the lungs affected. Patients in the age group 30-39 have had the most lung lesions at almost 69% of both lungs affected. Compared to vaccinated patients with comorbidities, unvaccinated patients with comorbidities had developed increased lung lesions by 5%. CONCLUSION: The study revealed a higher percentage of lung lesions among unvaccinated SARS-CoV-2-positive patients admitted to The National Institute of Pulmonology "Marius Nasta" in Bucharest, Romania, underlining the importance of vaccination and also the usefulness of artificial intelligence in CT interpretation.
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Coding variants that have significant impact on function can provide insights into the biology of a gene but are typically rare in the population. Identifying and ascertaining the frequency of such rare variants requires very large sample sizes. Here, we present the largest catalog of human protein-coding variation to date, derived from exome sequencing of 985,830 individuals of diverse ancestry to serve as a rich resource for studying rare coding variants. Individuals of African, Admixed American, East Asian, Middle Eastern, and South Asian ancestry account for 20% of this Exome dataset. Our catalog of variants includes approximately 10.5 million missense (54% novel) and 1.1 million predicted loss-of-function (pLOF) variants (65% novel, 53% observed only once). We identified individuals with rare homozygous pLOF variants in 4,874 genes, and for 1,838 of these this work is the first to document at least one pLOF homozygote. Additional insights from the RGC-ME dataset include 1) improved estimates of selection against heterozygous loss-of-function and identification of 3,459 genes intolerant to loss-of-function, 83 of which were previously assessed as tolerant to loss-of-function and 1,241 that lack disease annotations; 2) identification of regions depleted of missense variation in 457 genes that are tolerant to loss-of-function; 3) functional interpretation for 10,708 variants of unknown or conflicting significance reported in ClinVar as cryptic splice sites using splicing score thresholds based on empirical variant deleteriousness scores derived from RGC-ME; and 4) an observation that approximately 3% of sequenced individuals carry a clinically actionable genetic variant in the ACMG SF 3.1 list of genes. We make this important resource of coding variation available to the public through a variant allele frequency browser. We anticipate that this report and the RGC-ME dataset will serve as a valuable reference for understanding rare coding variation and help advance precision medicine efforts.
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PURPOSE: The use of recombinant human interleukin (rhIL)-15 as a potential therapeutic immune modulator and anticancer agent requires pure, stable preparations. However, purified rhIL-15 preparations readily accumulated heterogeneities. We sought to improve rhIL-15 stability through process, formulation, and targeted amino acid changes. METHODS: The solution state of rhIL-15 versus buffer composition and temperature was studied using SEC and IEX methods. rhIL-15 deamidation was confirmed using RP-HPLC/ESI-MS, enzymatic labeling, and peptide mapping. Deamidation kinetics were measured versus buffer composition and pH using RP-HPLC. Deamidation-resistant rhIL-15 variants (N77A, N77S, N77Q, G78A, and [N71S/N72A/N77A]) were produced in E. coli, then assayed for T-cell culture expansion potency and deamidation resistance. RESULTS: Adding 20% ethanol to buffers or heating at ≥32°C dispersed rhIL-15 transient pairs, improving purification efficiencies. Asparagine 77 deamidated rapidly at pH 7.4 with activation energy of 22.9 kcal per mol. Deamidation in citrate buffer was 17-fold slower at pH 5.9 than at pH 7.4. Amino acid substitutions at N77 or G78 slowed deamidation ≥23-fold. rhIL-15 variants N77A and (N71S/N72A/N77A) were active in a CTLL-2 proliferation assay equivalent to unsubstituted rhIL-15. CONCLUSIONS: The N77A and (N71S/N72A/N77A) rhIL-15 variants are resistant to deamidation and remain potent, thus providing enhanced drug substances for clinical evaluation.
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Sustitución de Aminoácidos , Asparagina/química , Interleucina-15/química , Interleucina-15/genética , Secuencia de Aminoácidos , Animales , Asparagina/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Interleucina-15/farmacología , Ratones , Datos de Secuencia Molecular , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacosRESUMEN
Recombinant adeno-associated viral (rAAV) vector-based gene therapy has been adapted for use in more than 100 clinical trials. This is mainly because of its excellent safety profile, ability to target a wide range of tissues, stable transgene expression, and significant clinical benefit. However, the major challenge is to produce a high-titer, high-potency vector to achieve a better therapeutic effect. Even though the three plasmid-based transient transfection method is currently being used for AAV production in many clinical trials, there are complications associated with scalability and it is not cost-effective. Other methods require either large-scale production of two herpes simplex viruses, rHSV-RepCap and rHSV-GOI (gene of interest), with high titers, or a stable cell line with high titer wild-type adenovirus infection. Both of these options make the process even more complex. To address this issue, we have developed a stable cell line-based production with the use of only one rHSV-RepCap virus. Using this new methodology in small-scale production, we achieved â¼1-6 E + 04 vg/cell of AAV9 in the top producer clones. Large-scale production in 10-CS (10-Cell Stack) of one of the top producing clones resulted in â¼1-2 E + 13 vg/10-CS with 50% of full capsid ratio after purification. This method could potentially be adapted to suspension cells. The major advantage of this novel methodology is that by using the rHSV-RepCap virus, high titer AAV can be produced with any GOI containing a stable adherent or suspension producer cell line. The use of this AAV production platform could be beneficial for the treatment of many diseases.
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Dependovirus , Vectores Genéticos , Línea Celular , Dependovirus/genética , Terapia Genética , Vectores Genéticos/genética , TransfecciónRESUMEN
Targeted inhibiting insulin-like growth factor 1 is an effective approach for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is considered as a potential therapeutic protein. However, producing high quality of such non-IgG proteins in mammalian cells is still a challenge in biopharmaceutical development. Here, we report a rapid production process by using transient gene transfection in HEK 293E cells. A set of constructs combining several expression promoters, leader sequences, and 5' un-translated regions were generated and optimized, from which the best vector with expression level at ~50mg/L was selected for production at 2L cell culture scale. Comparison study in downstream purification methods led to development of a scalable, non-affinity chromatography strategy through Super Q, Fast Flow Q, and Heparin columns. The product was characterized in purity (99%), isoelectric point, molecule weight, glycosylation, and stability by using SEC-HPLC, SDS-PAGE, isoelectric focusing and mass spectrometry. The highly purified product shows IGF-1 binding activity and inhibits IGF-1-induced cell proliferation. This process not only provides a remarkable high expression at ~50mg/L and pure glycosylated mammalian rhIGFBP7, also highlights that transient gene expression technology is practical to be used for production and early development of recombinant non-IgG therapeutic proteins.
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Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/efectos de los fármacos , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Proliferación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Vectores Genéticos , Glicosilación , Células HEK293 , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Datos de Secuencia Molecular , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
Ch14.18 is a mouse-human chimeric monoclonal antibody to the disialoganglioside (GD2) glycolipid. In the clinic, this antibody has been shown to be effective in the treatment of children with high-risk neuroblastoma, either alone or in combination therapy. Extensive product characterization is a prerequisite to addressing the potential issues of product variability associated with process changes and manufacturing scale-up. Charge heterogeneity, glycosylation profile, molecular state and aggregation, interaction (affinity) with Fcγ receptors and functional or biological activities are a few of the critical characterization assays for assessing product comparability for this antibody. In this article, we describe the in-house development and qualification of imaged capillary isoelectric focusing to assess charge heterogeneity, analytical size exclusion chromatography with online static and dynamic light scattering (DLS), batch mode DLS for aggregate detection, biosensor (surface plasmon resonance)-based Fcγ receptor antibody interaction kinetics, N-glycoprofiling with PNGase F digestion, 2-aminobenzoic acid labeling and high performance liquid chromatography and N-glycan analysis using capillary electrophoresis. In addition, we studied selected biological activity assays, such as complement-dependent cytotoxicity. The consistency and reproducibility of the assays are established by comparing the intra-day and inter-day assay results. Applications of the methodologies to address stability or changes in product characteristics are also reported. The study results reveal that the ch14.18 clinical product formulated in phosphate-buffered saline at a concentration of 5 mg/ml and stored at 2-8°C is stable for more than five years.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica/inmunología , Gangliósidos/inmunología , Neuroblastoma/terapia , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Relación Dosis-Respuesta Inmunológica , Estabilidad de Medicamentos , Humanos , Ratones , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Reproducibilidad de los ResultadosRESUMEN
Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.
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Codón de Terminación , Escherichia coli/genética , Interleucina-15/genética , Ingeniería de Proteínas , Proliferación Celular/efectos de los fármacos , Escherichia coli/metabolismo , Expresión Génica , Humanos , Interleucina-15/metabolismo , Interleucina-15/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance criteria based on comparability and consistency in the four parameters of the model. The assay is precise, accurate and robust and can be fully validated. Applications of the assay were established including process development support, release of the rHuIL-15 product for pre-clinical and clinical studies, and for monitoring storage stability.