Abstract RNA Sequencing (RNA-Seq) can facilitate prompt and precise management of many illnesses, including infection, especially when coupled with PCR. To further optimize RNA-Seq for the purposes of creating a RNA-Seq-informed PCR test, identifying reliable RNA primer targets is paramount. Essential criteria for constructing RNA primer targets include high gene expression or stability of transcripts. We hypothesize that free energy evaluation and motif analysis can demonstrate gene expression level or stability of transcripts to construct ideal RNA primers for RNA-Seq-informed PCR that can expedite the diagnosis and treatment of infection. Whole blood samples were collected from patients diagnosed with COVID-19 in the ICU at Rhode Island Hospital, stored in Paxgene tubes to preserve the integrity of the specimens, and submitted for RNA-Seq by a commercial sequencing service (Azenta/Genewiz). After quality assurance and quality control (QA/QC) measures are performed, we used RNAfold from the ViennaRNA Package to calculate the minimum free energy (MFE) and ensemble free energy (EFE) values to compare the stability among different secondary structures constructed from RNA-Seq read sequences. Energy parameters for calculations were set at 37 degree Celsius and standard physiological conditions at pH 7.4. In our analysis, we utilized multiple statistical tools, such as analysis of variance, Games-Howell post hoc tests, and a negative binomial regression. Of the 676 reads that aligned with SARS-CoV-2, there were 137 unique sequences among all patients. Among the unique read sequences, the average MFE was -30.46 kcal/mol and the average EFE was -32.94 in kcal/mol. There were 6 genes with these unique read sequences, two of which -- nucleocapsid ( N ) gene and spike ( S ) gene -- coded for integral structural proteins. Notably, the mean MFE and EFE of N gene were significantly different from ORF1ab gene (p = 2.81e-7; p = 0.005) and ORF6 gene (p = 0.023; p = 0.03). Our motif analysis demonstrated 7 motifs that corresponded to destabilization of the RNA, and a single motif (MEME-28) that corresponded to stabilization of the RNA. Within the nucleocapsid ( N ) gene, we found that reads from different regions also differed in stability. Our results demonstrate that the stability of reads from different genes varies in SARS-CoV-2 infections. Free energy evaluation and motif analysis are viable approaches to determine RNA structure stability or levels of differential gene expression. Stable transcripts that are highly expressed can be ideal RNA primers to be used in conjunction with PCR to expedite the management of infection. 

pdf/stability-and-motif-analysis-of-rna-seq-reads-from-covid-19-y3xejrkqujuf.pdf

Stability and Motif Analysis of RNA-Seq Reads from COVID-19 Patients


 Background: MUC16 is a large glycoprotein overexpressed on cell membrane of solid tumors, especially in almost 80% of ovarian cancer. The extracellular part of MUC16 could be cleaved as CA125, which is well known as a diagnosis biomarker for ovarian cancer. Despite recent advances in therapies such as PARP inhibitors, advanced ovarian cancer remains a disease of high unmet need. Herein a novel bispecific antibody targeting MUC16 and CD3, LBL-033, is developed to specifically kill MUC16 positive tumor cells by engaging T cells.
 Methods: LBL-033 was constructed using our unique T cell engager platform, which is a 2:1 format bispecific antibody with two arms targeting MUC16 membrane-proximal domain with high affinity and one arm targeting CD3 with fine-tuned affinity. The binding affinity of LBL-033 to MUC16 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-033 was investigated in C57BL/6-huCD3e mice implanted with MC38-huMUC16 cells. GLP-compliant repeat-dose toxicologic study was evaluated in cynomolgus monkeys.
 Results: The binding affinity of LBL-033 to MUC16 and CD3 protein was 2.67 nM and 29.6 nM, respectively. The epitope that anti-MUC16 binds was identified at the C-terminal of MUC16 extracellular domain; and the activity was not affected in the presence of soluble CA-125. In CD3 reporter gene assay, LBL-033 could activate the NFAT reporter element through MUC16 antigen-dependent CD3 cross-linking with an EC50 value of 1.068 nM. LBL-033 was shown a potent TDCC activity in OVCAR3 cells with T cell activation. In C57BL/6-huCD3e transgenic mice implanted with MC38-huMUC16 tumor cells, LBL-033 was shown a dose dependent anti-tumor activity. In addition, LBL-033 had a significant synergistic effect, when combined with anti-PD-1. LBL-033 was shown favorable pharmacokinetic properties with a good safety profile in cynomolgus monkeys.
 Conclusion: LBL-033, a novel bispecific antibody targeting CD3 and MUC16 with a unique epitope, which is located at the MUC16 membrane-proximal domain, with affinity differentiation between anti-MUC16 and anti-CD3. It is shown a great anti-tumor efficacy in animal models, with a good safety profile in monkeys. These data support LBL-033 as a novel therapeutic bispecific antibody for ovarian cancer and other MUC16 positive tumors. A dose escalation study in humans will begin in Q1 2023.
 Citation Format: Jianming Sun, Xiao Huang, Yurong Qin, XingXing Fang, Min Chen, Huan Lin, Jing Guan, Jing Huang, Tongjun Liu, Baohui Wang, Guojin Wu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2965.

Abstract 2965: LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16

Introduction Cystitis glandularis (CG) is a rare chronic bladder hyperplastic disease that mainly manifests by recurrent frequent urination, dysuria and gross hematuria. The current lack of unified diagnosis and treatment criteria makes it essential to comprehensively describe the inflammatory immune environment in CG research. Methods Here, we performed scRNA-sequencing in CG patients for the first time, in which four inflamed tissues as well as three surrounding normal bladder mucosa tissues were included. Specifically, we isolated 18,869 cells to conduct bioinformatic analysis and performed immunofluorescence experiments. Results Our genetic results demonstrate that CG does not have the classic chromosomal variation observed in bladder tumors, reveal the specific effects of TNF in KRT15 epithelial cells, and identify a new population of PIGR epithelial cells with high immunogenicity. In addition, we confirmed the activation difference of various kinds of T cells during chronic bladder inflammation and discovered a new group of CD27-Switch memory B cells expressing a variety of immunoglobulins. Discussion CG was regarded as a rare disease and its basic study is still weak.Our study reveals, for the first time, the different kinds of cell subgroups in CG and provides the necessary basis for the clinical treatment of cystitis glandularis. Besides, our study significantly advances the research on cystitis glandularis at the cellular level and provides a theoretical basis for the future treatment of cystitis glandularis.

/pdf/single-cell-rna-sequencing-reveals-the-immune-luz9f90b.pdf

Single-cell RNA sequencing reveals the immune microenvironment and signaling networks in cystitis glandularis

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Abstract 2965: LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16

Single-cell RNA sequencing reveals the immune microenvironment and signaling networks in cystitis glandularis