Bamlanivimab reduces nasopharyngeal SARS-CoV-2 RNA levels but not symptom duration in non-hospitalized adults with COVID-19

Importance: The antiviral activity and efficacy of anti-SARS-CoV-2 monoclonal antibody (mAb) therapies to accelerate recovery from COVID-19 is important to define. Objective: To determine safety and efficacy of the mAb bamlanivimab to reduce nasopharyngeal (NP) SARS-CoV-2 RNA levels and symptom duration. Design: ACTIV-2/A5401 is a randomized, blinded, placebo-controlled platform trial. Two dose cohorts were enrolled between August 19 and November 17, 2020 for phase 2 evaluation: in the first, participants were randomized 1:1 to bamlanivimab 7000 mg versus placebo, and in the second to bamlanivimab 700 mg versus placebo. Randomization was stratified by time from symptom onset (≤ or >5 days) and risk of progression to severe COVID-19 ('higher' vs 'lower'). Setting: Multicenter trial conducted at U.S. sites. Participants: Non-hospitalized adults ≥18 years of age with positive SARS-CoV-2 antigen or nucleic acid test within 7 days, ≤10 days of COVID-19 symptoms, and with oxygen saturation ≥92% within 48 hours prior to study entry. Intervention: Single infusion of bamlanivimab (7000 or 700 mg) or placebo. Main Outcomes and Measures: Detection of NP SARS-CoV-2 RNA at days 3, 7, 14, 21, and 28, time to improvement of all of 13 targeted COVID-19 symptoms by daily self-assessment through day 28, and grade 3 or higher treatment emergent adverse events (TEAEs) through day 28. Secondary measures included quantitative NP SARS-CoV-2 RNA, all-cause hospitalizations and deaths (composite), area under the curve of symptom scores from day 0 through day 28, plasma bamlanivimab concentrations, plasma and serum inflammatory biomarkers, and safety through week 24. Results: Ninety-four participants were enrolled to the 7000 mg cohort and 223 to the 700 mg cohort and initiated study intervention. The proportion meeting protocol criteria for 'higher' risk for COVID-19 progression was 42% and 51% for the 7000 and 700 mg cohort, respectively. Median time from symptom onset at study entry for both cohorts was 6 days. There was no difference in the proportion with undetectable NP SARS-CoV-2 RNA at any post-treatment timepoints (risk ratio compared to placebo, 0.82-1.05 for 7000 mg dose [overall p=0.88] and 0.81-1.21 for 700 mg dose [overall p=0.49]), time to symptom improvement (median of 21 vs 18.5 days, p=0.97, for 7000 mg bamlanivimab vs placebo and 24 vs 20.5 days, p=0.08, for 700 mg bamlanivimab vs placebo), or grade 3+ TEAEs with either dose compared to placebo. Median NP SARS-CoV-2 RNA levels were lower at day 3 and C-reactive protein, ferritin, and fibrinogen levels significantly reduced at days 7 and 14 for bamlanivimab 700 mg compared to placebo, with similar trends observed for bamlanivimab 7000 mg. Viral decay modeling supported more rapid decay with bamlanivimab compared to placebo. Conclusions and Relevance: Treatment with bamlanivimab 7000 mg and 700 mg was safe and compared to placebo led to more rapid reductions in NP SARS-CoV-2 RNA and inflammatory biomarkers, but did not decrease time to symptom improvement. The clinical utility of mAbs for outcomes other than hospitalizations and deaths is uncertain. Trial Registration: ClinicalTrials.gov Identifier: NCT04518410

Hepatitis C Virus Core Antigen as an Alternative to RNA in the Assessment of Response to Treatment with Direct-acting Oral Antivirals

Background: Affordable and effective diagnostic and treatment monitoring algorithms are urgently needed to achieve the global elimination of hepatitis C virus (HCV) infection. Methods: A total of 274 patients were treated with direct-acting antivirals (DAAs) in the Spanish Hospital of Albacete between 2004 and 2020. This study compared the enzyme-immunoassay technique for HCV core antigen (HCVcAg) with the determination of RNA of HCV (HCV RNA) by polymerase chain reaction (PCR) in monitoring treatment with DAA, setting the lower limit of detection of HCVcAg < 3 fmol/L and RNA < 10 IU/mL. In all cases, the P value of differences associated with the contrast test was less than or equal to 0.05. Results: We evaluated the viral loads of our patients before treatment, during their treatment, and after its completion. The HCV RNA quantification at diagnosis was 2309327 IU/mL. The mean HCVcAg load was 5972 fmol/L. There was a strong correlation between HCVcAg levels and RNA levels with a Spearman rho of 0.832 (P < 0.01). The HCVcAg sensitivity at diagnosis was 99%, but the specificity could not be calculated because there were no true negatives or false positives at this point. Twelve weeks after treatment, in patients with treatment failure, we obtained a mean of 19084 IU/mL for RNA, while for HCVcAg, the mean was 103 fmol/L. At this time point, we also found a strong correlation between HCVcAg levels and HCV RNA levels with a Spearman rho of 0.775 (P < 0.01). Finally, the virological cure was achieved in 99% of our patients. The results for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 100%, 99.87%, 86.33%, and 100%, respectively. Conclusions: HCVcAg determination is an excellent alternative to HCV RNA in the assessment of treatment response. This is particularly relevant in lower- and middle-income countries and resource-limited settings where the high cost of labor, equipment, and reagents can prohibit molecular testing.

Treating Iron Overload Disorders with a Novel Therapeutic Antibody Targeting TMPRSS6

Iron is an essential element for almost all living organisms as it participates in a wide variety of metabolic processes. Disorders of iron metabolism are among the most prevalent human diseases, ranging from anemia to hemochromatosis. Excessive iron accumulations in major organs of iron overload patients can lead to high mortality. Hepcidin, a HAMP-encoded liver hormone, is the master regulator of iron homeostasis. By binding to the sole iron exporter ferroportin and causing internalization and degradation of the complex, hepcidin inhibits cellular iron efflux, thereby lowers plasma iron levels. Inappropriately suppressed/low hepcidin production is central to iron overload. Transmembrane protease serine-6 (TMPRSS6), a type II transmembrane serine protease primarily expressed in liver, downregulates hepcidin expression through BMP-SMAD pathway. TMPRSS6 deficiencies have been shown to cause hepcidin overexpression in both TMPRSS6-mutant mice and in patients with iron-refractory iron deficiency anemia (IRIDA). Therefore, TMPRSS6 is a viable therapeutic target for iron overload disorders. Here we report the generation of an anti-TMPRSS6 antibody through a hybridoma campaign using a DNA-based immunization approach, followed by humanization and sequence optimization. Lead antibody, hzMWTx-003 selectively binds human TMPRSS6 with low nanomolar affinity (KD: 7.6nM), and is cross-reactive to rodent (mouse and rat) and monkey (cynomolgus and rhesus) TMPRSS6. Single-dose injection of hzMWTx-003 was able to significantly elevate serum hepcidin and liver HAMP RNA levels in wildtype mice, resulting in significantly reduced serum iron level. The Hbb th3/+ mouse model of β-thalassemia, like its human counterpart, is characterized by iron overload, ineffective erythropoiesis and splenomegaly. Treatment of Hbb th3/+mice with MWTx-003 effectively increased hepcidin expression at both protein and RNA levels, leading to significantly reduced serum iron and liver non-heme iron content. MWTx-003 also dramatically improved anemia and ineffective erythropoiesis, and alleviated splenomegaly in these mice. CMC development of hzMWTx-003 confirms outstanding biophysical properties. Preliminary studies in cynomolgus monkey using GLP-grade material demonstrated good pharmacokinetics of hzMWTx-003 and expected pharmacodynamic response where reduction of serum iron could be sustained for 21 days after single dose administration. A single dose toxicology study in cynomolgus monkey revealed no safety concerns, and no production of anti-idiotype antibodies was detected. In summary, anti-TMPRSS6 antibody MWTx-003 represents a promising therapy for iron overload disorders such as β-thalassemia, and potentially other diseases where iron restriction is beneficial. Disclosures Chen: Mabwell Therapeutics Inc: Current Employment. Wang: Mabwell Therapeutics Inc: Current Employment. Zheng: Mabwell (Shanghai) Bioscience Co. Ltd: Current Employment. Huang: Mabwell Therapeutics Inc: Current Employment. Mao: Mabwell (Shanghai) Bioscience Co. Ltd.: Current Employment. Ouyang: Mabwell (Shanghai) Bioscience Co. Ltd.: Current Employment. Du: Mabwell Therapeutics Inc: Current Employment.

Detection of CCND1 Overexpression By RNA-Seq from Tna Samples As a Surrogate for t(11:14) Translocation Traditionally Measured By FISH in Multiple Myeloma Patients for Improved Patient Care

Background: Multiple myeloma (MM) is a blood cancer type affecting plasma cell in bone marrow. MM is heterogenous in nature but t(11;14)(q13;q32) translocation is a common prognostic marker among MM patients. One of the most frequent oncogenic drivers involved in this chromosomal rearrangement is CCND1 (Cyclin D1) gene translocation downstream to the immunoglobulin heavy chain (IGH), which results on overexpression of CCND1, thus promoting abnormal cell proliferation. Oncogenic CCND1 RNA levels can result from translocations such as t(11;14), gene amplifications, increased transcription rates and/or RNA stability. Indeed, CCND1 RNA overexpression has a favorable prognostic value for patients treated with high doses of chemotherapies but important challenges remain in accurate detection of CCND1 RNA levels. Currently, FISH is the gold standard method for detecting t(11:14) translocations at the DNA level. However, it cannot detect CCND1 overexpression. Therefore, a method that can detect CCND1 overexpression levels, as well as in frame transcripts has clinical implications. In the current study we leveraged in-use NeoGenomics Heme TNA single tube NGS assay to enable the detection of CCND1 RNA overexpression as a complementary test to FISH testing. Methods: We performed RNA sequencing from 32 healthy donors and on fixed cell pellets from 94 CD138-enriched BM samples from MM patients and from using the amplicon based (Qiagen, inc) NGS assay. We developed pipeline for gene expression by TPM count (transcript per million) for CCND1, and further normalized to the "housekeeping" gene GUSB. We validated the normalization to GUSB by comparing to normalization using the geometric mean of four housekeeping genes (GUSB, PGD, RPL5 and RPL19) showing a high correlation (R 2&gt;0.95). A commercially available qRT-PCR assay was used as orthogonal method to further confirm the linearity of the quantitative gene expression signal in NGS. The analytical cutoff was determined from normalized TPM calculation from 32 healthy volunteers following CLSI guideline (CLSI_EP17-A2) and further updated from MM-PCE samples with t(11:14) translocations from a CLIS-validated FISH assay . Results: From 94 CD138-enriched BM samples, 26 had t(11:14) translocations, or CCND1 gains as detected by FISH, 15 samples were confirmed negative by FISH and 32 normal volunteers with no suspected disease. Also, we determined the analytical cutoff for CCND1 overexpression based on the CLSI guidelines to be 2.37 times the expression level of GUSB ("housekeeping" gene) using normal volunteers (n=32) (sensitivity 86% and specificity 77%). We found specificity to be low, so further evaluated the threshold using a ROC curve analysis with multiple tests. Using Fischer's exact test, we found CCND1 expression 3.27 times the GUSB expression to yield higher specificity of 86.5 % and sensitivity for 78.9%. Further, we used 26 FISH positive and 32 normal samples to build a new model and determined the cutoff for CCND1 overexpression to be 4.15 times GUSB expression, which resulted lower sensitivity but higher specificity (75% sensitivity and 100% specificity). When we evaluated 15 FISH negative samples with this cutoff we observed CCND1 was not overexpressed in six samples, but 9 samples did have some degree of overexpression. Overexpression was confirmed by qRT-PCR. Two CCND1 high- and low- expressing normal samples (MM-PCE 27 and 48) were further evaluated using alternative extraction methods to test the dependencies on extractions and the data showed concordant to each other for overexpression. Interestingly, 1 sample (MM-PCE-27) showed very high overexpression without t(11:14) translocation event (~100 fold over expressed). Cytogenetic studies were discordant with FISH as well for this sample, showing abnormalities related to chr7q,13q,12p but no indication of any chr11 related event. Conclusions: In this study we evaluated our existing NGS assay for CCND1 overexpression using TNA as a surrogate for traditional FISH, while demonstrating the accuracy of the RNA quantitation by NGS using qRT-PCR. We developed an RNA-seq based CCND1 expression assay that could be used to complement traditional FISH testing especially if there is limited specimen. The confirmation of overexpression in FISH negative samples may suggest new ways to improve MM patients risk stratification and treatment. Disclosures Ghosal: NeoGenomics Laboratories: Current Employment. Alarcon: NeoGenomics Laboratories: Current Employment. Koo: Neo Genomics Laboratories: Current Employment. Kang: Neo Genomics Laboratories: Current Employment. Ramesh: Neo Genomics Laboratories: Current Employment. Gyuris: Neo Genomics Laboratories: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Thomas: NeoGenomics Laboratories, Inc.: Current Employment. Fabunan: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Nam: NeoGenomics Laboratories, Inc.: Current Employment. Petersen: Neo Genomics Laboratories: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Yamahata: Neo Genomics Laboratories: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Ye: Neo Genomics Laboratories: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.

Elevated N6-Methyladenosine RNA Levels in Peripheral Blood Immune Cells: A Novel Predictive Biomarker and Therapeutic Target for Colorectal Cancer

Effective biomarkers for the diagnosis of colorectal cancer (CRC) are essential for improving prognosis. Imbalance in regulation of N6-methyladenosine (m6A) RNA has been associated with a variety of cancers. However, whether the m6A RNA levels of peripheral blood can serve as a diagnostic biomarker for CRC is still unclear. In this research, we found that the m6A RNA levels of peripheral blood immune cells were apparently elevated in the CRC group compared with those in the normal controls (NCs) group. Furthermore, the m6A levels arose as CRC progressed and metastasized, while these levels decreased after treatment. The area under the curve (AUC) of the m6A levels was 0.946, which was significantly higher than the AUCs for carcinoembryonic antigen (CEA; 0.817), carbohydrate antigen 125 (CA125; 0.732), and carbohydrate antigen 19-9 (CA19-9; 0.771). Moreover, the combination of CEA, CA125, and CA19-9 with m6A levels improved the AUC to 0.977. Bioinformatics and qRT-PCR analysis further confirmed that the expression of m6A modifying regulator IGF2BP2 was markedly elevated in peripheral blood of CRC patients. Gene set variation analysis (GSVA) implied that monocyte was the most abundant m6A-modified immune cell type in CRC patients’ peripheral blood. Additionally, m6A modifications were negatively related to the immune response of monocytes. In conclusion, our results revealed that m6A RNA of peripheral blood immune cells was a prospective non-invasive diagnostic biomarker for CRC patients and might provide a valuable therapeutic target.

Joint changes in RNA, RNA polymerase II, and promoter activity through the cell cycle identify non-coding RNAs involved in proliferation

Proper regulation of the cell cycle is necessary for normal growth and development of all organisms. Conversely, altered cell cycle regulation often underlies proliferative diseases such as cancer. Long non-coding RNAs (lncRNAs) are recognized as important regulators of gene expression and are often found dysregulated in diseases, including cancers. However, identifying lncRNAs with cell cycle functions is challenging due to their often low and cell-type specific expression. We present a highly effective method that analyses changes in promoter activity, transcription, and RNA levels for identifying genes enriched for cell cycle functions. Specifically, by combining RNA sequencing with ChIP sequencing through the cell cycle of synchronized human keratinocytes, we identified 1009 genes with cell cycle-dependent expression and correlated changes in RNA polymerase II occupancy or promoter activity as measured by histone 3 lysine 4 trimethylation (H3K4me3). These genes were highly enriched for genes with known cell cycle functions and included 57 lncRNAs. We selected four of these lncRNAs—SNHG26, EMSLR, ZFAS1, and EPB41L4A-AS1—for further experimental validation and found that knockdown of each of the four lncRNAs affected cell cycle phase distributions and reduced proliferation in multiple cell lines. These results show that many genes with cell cycle functions have concomitant cell-cycle dependent changes in promoter activity, transcription, and RNA levels and support that our multi-omics method is well suited for identifying lncRNAs involved in the cell cycle.