The Center for Immunotherapy & Precision Immuno-Oncology's (CITI) Discovery Lab is one of three collaborative CITI fee-for-service platforms dedicated to the advancement of cancer and immunologic research using high-throughput technologies. The lab provides services for a spectrum of research projects ranging from immuno-oncology to transplantation, as well as cardiac, metabolic, neurologic, and autoimmune diseases.
The Discovery Laboratory offers cutting-edge high-throughput technologies, immunologic testing, and next generation sequencing (NGS)-based platforms and possesses the expertise to guide investigators through the fast-paced landscape of cutting-edge genomic technologies to advance patient-centric research.
Dr. Raghvendra Srivastava, PhD, Scientific Director of the CITI Discovery Lab, leads this laboratory and works in collaboration with academic, clinical and industry leaders, engaging in translational research in Cleveland and across the United States. The Discovery Lab performs in-house HLA class I/II typing, single cell RNA library preparations and quality control, neoantigen screening assays, immune repertoire (TCR/BCR) sequencing services (in-house and send-out), NGS library preparation for immuno-sequencing projects, and multiplex cytokine/chemokine assays using the Luminex platform. The Discovery Lab also supports all bioanalytical requirements to sequence custom-made libraries.
A unique facet of the CITI Integrated Platforms is the seamless harmonization with computational genomic and transcriptomic data analysis, as the Discovery lab works together with the CITI Computational Immunology Platform (CIP) team.
To initiate a project request or consultation for CITI platform services, visit Cleveland Clinic's "CITI Integrated Platforms: Immunomonitoring/Discovery/Computational" in Agilent's iLab.
As Scientific Director of the CITI Discovery lab, Dr. Srivastava leads translational immuno-oncology-based fee-for-service assays, including, but not limited to, HLA and TCR sequencing and neo-antigen screening methods. In 2020, he moved to the Cleveland Clinic from the Memorial Sloan Kettering Cancer Center, where he worked on single cell sequencing projects as a Senior Research Scientist in Dr. Tim Chan’s lab. Raghu earned his PhD in cancer immunology at the Centre for Cellular and Molecular Biology in Hyderabad, India. He started his career as a Project Assistant at the Central Drug Research Institute, where he focused on structural immunology in the design and synthesis of biologically active peptides for antibacterial and anti-inflammatory application. He then served as a research associate at the University of Pittsburgh, where he contributed to the discovery of novel immunological mechanisms for EGFR targeted drugs.
The Discovery Lab provides services such as RNA or DNA extraction, quantification, and quality control to ensure that NGS sample content achieves the desired sequencing depth and base pair coverage. It houses instruments such as the Thermo Fisher Scientific Qubit for nucleic acid quantification and the Agilent Tapestation and Agilent 2100 Bioanalyzer for nucleic acid quality control. The DL also houses the BioMérieux eMAG® system, a molecular biology platform for high throughput nucleic acid extraction of clinical grade samples – an automated system that can process 48 samples simultaneously and is used in the clinical laboratory for the extraction of DNA and RNA from patient tissue.
The Discovery Lab utilizes the MiSeq Illumina sequencing system for next generation DNA sequencing. It integrates cluster generation, sequencing, and data analysis, allowing up to 30 million reads in a single run and combining proven sequencing by synthesis (SBS) technology with a workflow from DNA to data analysis in just a few hours. This sequencing platform allows for flexibility and quick access for targeted re-resequencing, 16S metagenomics, and small genome sequencing and has a broad range of applications in immunotherapy.
Human Leukocyte Antigen (HLA) Typing is being performed in-house using CareDx AlloSeq Assign software for data output. This HLA sequencing assay permits investigators to characterize and identify HLA genotypes for research use only.
T Cell Receptor (TCR) Sequencing is being performed in partnership with Adaptive Biotechnologies Corporation via their DNA-based ImmunoSEQ assay. The Discovery Lab also offers in-house TCR sequencing using Integrated DNA Technologies' ArcherDx Immunoverse with RNA as input material. Archer Immunoverse Technology utilizes an FFPE-optimized strategy for TCR beta/gamma, TCR alpha/delta, and Ig heavy chain sequencing in FFPE archived tissues. It can be used to profile tumor-infiltrating lymphocytes and to compare TCR diversity in the tumor and in circulation. The Discovery Lab also assists with dynamic visualization and a full data export in order to best interpret complex research questions.
The Discovery Lab also collaborates with the Broad Institute of MIT and Harvard, as well as the Yale Center for Genome Analysis, for the following services:
Whole Exome Sequencing and Whole Genome Sequencing, Whole Transcriptome Sequencing (mRNA) for fresh/frozen tissue, and Transcriptome Capture for formalin fixed paraffin embedded (FFPE) tissue samples.
Luminex 200 Multi-Analyte Bioassay Detection System
DL utilizes the Thermo Fisher Luminex™ 200™ System to analyze soluble factors, including protein and gene expression profiling, for laboratory and clinical trial specimens, which can reveal novel biomarkers that help to understand disease mechanisms, immune cell activation states, cellular crosstalk, and resistance mechanisms.
ELISPOT & ImmunoSpot
CTL's ImmunoSpot® is a software tool for analyzing ELISPOT assay data. Its flexible yet user-friendly environment provides a wide array of powerful features geared towards maximizing productivity and minimizing the amount of user effort required. ImmunoSpot® employs an intuitive, five-step analysis procedure designed to automate as much of the counting and documentation process as possible without sacrificing flexibility. This same procedure also allows multiple plate images to be loaded simultaneously for analysis. This combination of automation and versatility permits genuine "walk-away" plate processing, allowing ImmunoSpot® to process massive amounts of diverse plate data without any human intervention.
The Discovery Lab performs single cell RNA library preparation and quality control on a variety of cancer tissues, core biopsies, and blood to prepare genomic libraries for gene expression and utilizes the 10X Genomics' Chromium Controller. This high throughput state-of-the-art technology can interrogate cellular heterogeneity and gene expression, dissect intrinsic variation in TCR repertoire, define cellular states, and spatially resolve single cells to interrogate biological mechanisms.
Cancer neoantigen-specific T cells play a dominant role in determining the efficacy of PD-1, CTLA-4, and PD-L1 immune checkpoint blockade. The Discovery Lab performs the screening and validation of neoantigens by utilizing a tandem minigene approach to clone and prepare transcripts of detected mutations in patients. The lab transfects RNA containing mutations into patients' dendritic cells and detects neoantigen-specific autologous T cells, which can recognize mutations in tumors and destroy cancerous cells. The neoantigens identified through these protocols are used as peptide vaccines for patients with advanced cancer.