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Developing & Optimizing Acute Myeloid Leukemia Specific Therapeutic Antibodies [CITE-seq]

ABSTRACT: Developing & Optimizing Acute Myeloid Leukemia Specific Therapeutic Antibodies [CITE-seq]

PROVIDER: PRJNA909824 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR22571175.fastq.gz	Fastqsanger.gz
	SRR22571176.fastq.gz	Fastqsanger.gz
	SRR22571177.fastq.gz	Fastqsanger.gz
	SRR22571178.fastq.gz	Fastqsanger.gz
	SRR22571179.fastq.gz	Fastqsanger.gz

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Developing & Optimizing Acute Myeloid Leukemia Specific Therapeutic Antibodies

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Developing & Optimizing Acute Myeloid Leukemia Specific Therapeutic Antibodies [CITE-seq]

Project description:Following nearly five decades with few approved therapies for acute myeloid leukemia (AML), the last three years have brought stellar progress with the U.S. Food and Drug Administration approving a number of new therapies for AML patients. Despite these advances, there are several high-risk patient AML populations with dismal survival on the order of months including those with primary refractory disease, those whose AML developed from an antecedent hematologic disorder, and those with high risk cytogenetic or mutational profiles (such as TP53 mutations or a complex karyotype). Furthermore, therapeutic options are limited for those unfit to receive cytotoxic chemotherapy due to age or co-morbidities. These treatment challenges clearly illustrate the need for improved therapeutic approaches in AML. Here we identify that antibody therapies for AML can be enhanced via optimization of (1) Fab target selection, and (2) engineering the Fc portion of the antibody to maximally engage receptors on immune effector cells. We demonstrate that antibodies targeting a novel AML-specific cell surface marker known as U5 snRNP200 have therapeutic efficacy in AML. Finally, we performed a whole-genome CRISPR screen to identify mechanisms by which U5 snRNP200 traffic to the cell surface. This revealed a requirement for cell-surface CD32a to enable U5 snRNP200 cell membrane localization.

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Titration of 124 antibodies using CITE-Seq on human PBMCs

Project description:Single-cell RNA-sequencing (scRNA-Seq) is widely used to characterize immune cell populations. However, mRNA levels correlate poorly with expression of surface proteins, which are well established to define immune cell types. CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) utilizes oligonucleotide-tagged antibodies to simultaneously analyze surface phenotypes and transcriptomes. Considering the high costs of adding surface phenotyping to scRNA-Seq, we aimed to determine which of 188 tested CITE-Seq antibodies can detect their antigens on human peripheral blood mononuclear cells (PBMCs), a commonly interrogated cell population in immunology, and find the optimal concentration for staining. The recommended concentration was optimal for 76 antibodies, whereas staining quality of 7 antibodies improved when the concentration was doubled. 33 and 8 antibodies still worked well when the concentration was reduced to 1/5 or 1/25, respectively. 64 antigens were not detected at any antibody concentration. Optimizing the antibody panel by removing antibodies not able to detect their target antigens and adjusting concentrations of the remaining antibodies could enable a cost reduction of almost 50%. In conclusion, our data are a resource for building an informative and cost-effective panel of CITE-Seq antibodies and use them at their optimal concentrations in future CITE-seq experiments on human PBMCs.

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Strategies for optimizing CITE-seq for human islets and other tissues

Project description:Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor- matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITEseq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITEseq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues.

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Developing & Optimizing Acute Myeloid Leukemia Specific Therapeutic Antibodies

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