Immuno-Oncology Product Resource Guide

Introduction

Thermo Fisher Scientific's goal is to support immuno-oncology research with a comprehensive range of tools and technologies designed to maximize researcher's time, budget, and data, thereby accelerating the path to discovery and translation to clinical applications. This guidebook provides educational resources and solutions for various immuno-oncology research approaches, including checkpoint inhibition, CAR T cell therapy, and cancer vaccine research. It details capabilities from innovative products and techniques to time-saving workflow applications, emphasizing a partnership approach for scientific breakthroughs.

Immuno-oncology Review

What is I-O?

Immuno-oncology (I-O), also known as cancer immunotherapy, is a rapidly growing field that studies how the body's immune system can fight cancer.

Why does I-O research matter?

I-O research aims to develop cancer immunotherapies that go beyond traditional methods like surgery, chemotherapy, and radiation. It focuses on enabling the adaptive immune system to specifically recognize and attack cancer cells while sparing healthy ones. I-O research can potentially uncover ways to make all types of cancer immunogenic and facilitate long-lasting, protective immunity against future recurrence. Recent breakthroughs in checkpoint inhibition, chimeric antigen receptor (CAR) T cell therapy, and cancer vaccines highlight the immune system's capabilities in combating cancer. While all immune cells play a role, T cells and T cell-mediated responses are central to current I-O research.

What are some of the promising areas in I-O research?

This guide overviews three trending I-O research areas: checkpoint inhibition, CAR T cell therapy, and cancer vaccines. These approaches correlate within the cancer-immunity cycle.

Checkpoint inhibition

Immune checkpoints are cell pathways crucial for maintaining normal immune responses and protecting tissues from damage during immune activation. Cancer cells exploit immune checkpoints as a mechanism of immune resistance. Understanding tumor-immune cell interactions is a primary approach in I-O research.

Adoptive Cell Therapy (ACT) and CAR T cell therapy

ACT targets the immune system to enable the body's natural ability to fight cancer. This is achieved by genetically modifying a subject's own T cells to target antigens selectively expressed on cancer cells. Successful ACT applications include Tumor-Infiltrating Lymphocytes (TILs) and CAR T cells, which are engineered to express chimeric antigen receptors to target specific cancer cells via surface proteins, inducing anticancer attack.

A closer look: CAR T cells

The T cell receptor (TCR) participates in T cell activation, triggered by cells expressing major histocompatibility complex (MHC) molecules with an antigen. Tumor-specific TCRs can be engineered to recognize specific cancer cell populations, recognizing both intracellular and cell surface proteins. Limitations include patient-specific Human Leukocyte Antigen (HLA) restrictions and the lack of unique tumor-specific antigens. CARs are fusion proteins combining intracellular T cell components and extracellular antigen-recognition domains from a monoclonal antibody. They are constructed by linking antibody variable regions to intracellular signaling chains (e.g., CD3-zeta, CD28, 4-1BB). CAR T cells are not limited by HLAs but cannot recognize mutated intracellular proteins.

Cancer vaccines

Vaccines are another I-O research area aimed at enabling the immune system to recognize cancer as a threat. This antigen-based method relies on the immune system's ability to recognize proteins to induce an immune response. Researchers identify new tumor-associated antigens, called neoantigens, released within the tumor microenvironment. Dendritic cell (DC) therapy utilizes tumor fragments to activate extracted DCs, turning them into APCs that induce a secondary immune response, including antibody production. Combination therapies and personalization methods are also being studied.

General I-O Workflow

Thermo Fisher Scientific offers research platforms and products to understand the interplay between the immune system and cancer. Instruments, assays, and reagents are available to accelerate the development of cancer immunotherapies.

Key approaches and solutions:

• Checkpoint inhibition: Tools for genomic biomarker discovery, verification, protein expression, and immune checkpoint analysis. (Page 8)
• CAR T cell therapy: Workflow tools for sequencing, cell engineering, T cell activation, and expansion, including scalable solutions. (Page 12)
• Cancer vaccines: Transfection solutions, gene-to-protein services, and genomic biomarker tools for vaccine therapy development. (Page 16)

Solutions for Checkpoint Inhibition

Identifying and validating predictive biomarkers for checkpoint immunotherapy is crucial for optimizing therapeutic benefit, minimizing toxicity, and guiding combination therapy. Thermo Fisher Scientific offers solutions for genomic biomarker discovery and verification, protein biology, and cell analysis to stratify responders and nonresponders.

Genomic biomarker discovery and verification tools

• Applied Biosystems™ Clariom™ D Pico assays: Microarray-based solution for deep transcriptome analysis, discovering novel biomarkers, analyzing coding/long noncoding RNA, and alternative splicing events from as little as 100 pg of total RNA.
• Applied Biosystems™ Clariom™ S Pico assays: Microarray-based solution for gene-level analysis of well-annotated genes across the transcriptome from as little as 100 pg of total RNA. Enables identification of gene signatures and pathways, and screening of large sample numbers.
• Applied Biosystems™ TaqMan® Array Human Immune Response Plate: 96-well plate for quantitative gene expression analysis of genes from 9 immune system function classes (e.g., cell surface receptors, transcription factors, cytokines).
• Applied Biosystems™ TaqMan® Array Human Immune Panel: 384-well microfluidic card for quantitative gene expression analysis of a comprehensive set of human immune response targets, including immune regulators, apoptosis markers, and ischemia markers.
• Applied Biosystems™ TaqMan® Gene Expression Assays: Gold standard for real-time PCR gene expression analysis, providing a fast, simple method for verifying gene expression biomarkers.
• Ion Torrent™ Oncomine™ Immune Response Research Assay: Targeted next-generation sequencing (NGS) gene expression assay to measure genes involved in tumor-immune system interactions and identify immunotherapy biomarkers.

Protein expression

• Gibco™ Expi293™ Expression System: Rapid, high-yield system for producing proteins from recombinant 293 cells in 5-7 days, delivering up to 6x more protein production compared to other transient 293 systems.
• Gibco™ ExpiCHO™ Expression System: Offers superior protein yields compared to alternative transient systems, saving time, incubator space, and plasticware costs. It provides scalable protocols for various workflow needs.

Antibodies

• Invitrogen™ antibodies: Detect key checkpoint targets with primary, secondary, and custom validated antibodies, including those for Foxp3, Human TIGIT, Human LAG3, Arginase-1, and Granzyme B.
• Invitrogen™ Flow Cytometry Panel Builder: Tool for designing multicolor flow cytometry panels to incorporate multiple I-O antibodies with minimal spectral overlap.

Quantitative protein analysis

• ELISAs, Invitrogen™ ProQuantum™ high-sensitivity immunoassays, Luminex® multiplex platforms, and next-generation immunoassays: Used for quantitative assessment of soluble proteins like cytokines, chemokines, and growth factors.
• Invitrogen™ ProcartaPlex™ Immuno-Oncology Checkpoint Markers Panel: Multiplexed analysis using Luminex® xMAP® technology for simultaneous detection and quantitation of multiple secreted proteins.
• Invitrogen™ QuantiGene™ Plex Assay: Utilizes Luminex xMAP bead technology for multiplexing to measure RNA from archival tumor sections and achieve quantitative measurement of biomarkers. Offers over 17,000 validated genes for pathway- and disease-themed panels.

Other assay tools

• Invitrogen™ CellTrace™ assays: For proliferation measurements based on DNA synthesis or cellular metabolism, reporting cell health, genotoxicity, and inhibition of tumor cell growth.
• Invitrogen™ Click-iT™ EdU assays: A simplified, robust assay for analyzing DNA replication in proliferating cells.
• Invitrogen™ PrimeFlow™ RNA assays: For RNA and protein expression analysis as cells change over time or in response to stimuli; assay for any protein when no antibody is available.

Solutions for CAR T cell therapy

Analyzing the immune repertoire to capture TCR rearrangement diversity can advance I-O research. Thermo Fisher Scientific offers workflow solutions from engineering to sequencing, including cell media and reagents.

Sequencing

• Next-generation sequencing (NGS) using the Ion GeneStudio™ S5 System series: Ion Torrent™ semiconductor sequencing for targeted NGS applications with speed and scalability. Systems offer sequencing of 2-130 million reads per run using different chips.
• Sanger sequencing with the Applied Biosystems™ SeqStudio™ Genetic Analyzer: For TCR sequencing (beta, gamma, alpha) and analysis to assess T cell repertoire diversity, supporting custom TCR sequencing applications for sequences up to 700 bp.
• Ion Torrent™ Oncomine™ TCR Beta-LR Assay: Rapid, long-read NGS assay for optimizing T cell function and manufacturing, and investigating markers for immune-mediated adverse events (IMAEs).

Engineering

• Gibco™ LV-MAX™ Lentiviral Production System: Cost-effective and scalable platform for lentiviral vector production, addressing challenges in adherent and suspension methods. It offers higher viral titers and over 50% cost reduction compared to PEI-mediated methods.
• Invitrogen™ TrueGuide™ Synthetic gRNA: Ready-to-transfect single guide RNA (sgRNA) designed and validated for consistent, high-efficiency genome editing with Invitrogen™ tools.
• Invitrogen™ Neon™ Transfection System: Next-generation electroporation device achieving up to 90% transfection efficiency in primary cells, stem cells, and difficult-to-transfect cells.
• Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit: PCR-based method to measure genome editing accuracy.
• Invitrogen™ GeneArt™ Gene-to-Protein Services: Fast, reliable protein expression and production from mammalian or insect cells, with short processing times.
• Invitrogen™ TrueCut™ Cas9 Protein v2: Offers 2x higher editing efficiency in difficult targets, including immune cell lines.

Translate your cell therapy to the clinic with cGMP-grade reagents

Thermo Fisher Scientific's Gibco™ Cell Therapy Systems (CTS™) products facilitate the transition from research to clinical applications and commercialization. CTS media and reagents are manufactured under cGMP for medical devices, with extensive safety testing and traceability documentation.

Cell expansion

• Gibco™ CTS™ OpTmizer™ T Cell Expansion Serum-Free Medium (SFM): Xeno-free formulation proven for clinical success, developed for human T lymphocyte growth and expansion.
• Gibco™ CTS™ Immune Cell Serum Replacement: Defined xeno-free formulation supporting human T cell expansion when supplemented with basal media.

Cell isolation and activation

• Gibco™ CTS™ Dynabeads™ CD3/CD28: Magnetic beads technology for ex vivo T cell isolation, activation, and expansion for immunotherapy.

Solutions for Cancer Vaccine Research

Cancer vaccine research is evolving with combination therapies and personalized approaches, showing promise beyond conventional treatments. Thermo Fisher Scientific offers solutions to facilitate this research journey.

Genomic biomarker discovery and verification tools

• Applied Biosystems™ OncoScan™ CNV Assay: Microarray-based whole-genome copy number assay for solid tumor samples, detecting somatic copy number aberrations (gains, losses, LOH, chromothripsis, aneuploidy).
• Applied Biosystems™ CytoScan™ HD Suite: Microarray-based whole-genome copy number assay for hematological malignancy samples, detecting somatic copy number aberrations.

Gene synthesis services

• Invitrogen™ GeneArt™ Gene Synthesis: Industry-leading gene synthesis services, optimized protein expression, and outsourced protein/cell line production. GeneOptimizer™ software enhances mRNA stability and translational efficiency.

Engineering

• Invitrogen™ PureLink™ Expi Endotoxin-Free Maxi Plasmid Purification Kit: Isolates endotoxin-free plasmid DNA using anion exchange membrane in 90 minutes.
• Invitrogen™ Lipofectamine™ 3000 Transfection Reagent: Achieves 10-fold higher efficiency into a broad spectrum of difficult-to-transfect cells.
• Invitrogen™ Invivofectamine™ 3.0 Reagent: Breakthrough reagent for in vivo RNAi delivery with improved performance and up to 85% knockdown.
• Invitrogen™ mMESSAGE mMACHINE™ SP6 Transcription Kit: Designed for in vitro synthesis of large amounts of capped RNA, mimicking eukaryotic mRNAs.

Characterize and Verify

Enhance the analysis of genes, proteins, and cells, from confirming targets and expression to understanding mechanisms of action.

Targeted genetic analysis

Confirm and quantify genetic changes for a wide range of selected DNA targets key to research. Tools include DNA isolation reagents and kits, purification systems, fluorometers, spectrophotometers, thermal cyclers, real-time PCR assays, DNA polymerases, electrophoresis systems, and Sanger sequencing instrumentation.

Targeted gene expression analysis

Confirm and quantify RNA targets at the gene, exon, or noncoding RNA level. Solutions include RNA isolation reagents and kits, decontamination solutions, purification systems, thermal cyclers, RT-PCR systems, electrophoresis systems, and NGS platforms.

Solution spotlight: genetic analysis tools

• Ion Torrent™ Oncomine™ Immune Response Research Assay: Monitors the tumor microenvironment (TME) for identification of biomarkers and study of mechanisms of action in combination therapy experiments. It detects lower expressors associated with T cell receptor (TCR) signaling and checkpoint inhibitors.
• Ion Torrent™ Oncomine™ Tumor Mutation Load (TML) Assay: An independent predictor for patient stratification for immunotherapy response. Covers 1.7 Mb across 409 cancer-driven genes, requires as little as 20 ng of tumor DNA, and offers a 3-day workflow with streamlined analysis.
• Ion Torrent™ Oncomine™ TCR Beta-SR Assay: Interrogates the CDR3 region of the TCR beta chain, compatible with FFPE DNA and RNA. Enables characterization of immune status and detection of T cell minimal residual disease (MRD). Offers a 2-day turnaround time with superior informatics.
• Ion Torrent™ Oncomine™ TCR Beta-LR Assay: Utilizes long-read sequencing technology to capture all three complementarity-determining regions (CDR1, CDR2, CDR3) of the TCR beta chain with high accuracy. Enables biomarker discovery, T cell characterization, and identification of variable gene polymorphisms.
• Ion GeneStudio™ S5 systems: Benchtop NGS systems with less than 45 minutes of hands-on time, featuring cartridge-loaded reagents and a straightforward user interface. Ideal for cancer or inherited disease research labs. Ion Torrent™ technology has been referenced in over 4,000 publications.
• Applied Biosystems™ TaqMan® Gene Expression Assays: Over 1.8 million predesigned assays covering more than 30 species, available in various formats. Considered the gold standard for gene expression quantification and referenced in tens of thousands of publications.

Cell analysis

Understand the impact of external and internal perturbations on cellular phenotypes and behavior. Tools include imaging systems, cell counters, flow cytometers, and quantitative imaging platforms.

• Imaging: Compact, all-in-one transmitted-light systems for cell, colony, and tissue identification and visualization (e.g., Invitrogen™ EVOSTM XL and XL Core Imaging Systems).
• Counting: Accurate and automated counting, monitoring of fluorescent protein expression, and viability studies (e.g., Invitrogen™ Countess™ II Automated Cell Counter).
• Visualization: Multicolor fluorescence detection in cells and tissues, plate scanning, z-stacks, and time-lapse studies (e.g., Invitrogen™ EVOSTM FL Imaging System).
• Flow cytometry: Run multicolor samples faster with acoustics-assisted focusing technology, clog-resistant engineering, and simplified sample prep (e.g., Invitrogen™ Attune™ NxT Flow Cytometer).
• Quantitative imaging: Integrate high-content screening and analysis for up to 500,000 phenotypic measurements (e.g., Thermo Scientific™ CellInsight™ CX5, CX7, and CX7 LZR HCS/HCA platforms).

Protein analysis

Analyze the identity, function, and level of expression of key proteins. Solutions cover isolation, separation, transfer, detection, quantification, and modification of proteins.

• Isolation and cleanup: Chromatography media, dialysis products, desalting products, concentrator devices, magnetic beads, and cell lysis solutions.
• Separation: Precast protein gels, pour-your-own gel systems, protein ladders, and gel tanks.
• Transfer: Buffers, membranes, and transfer systems for Western blotting.
• Detection: Western blotting reagents, chemiluminescent substrates, and imaging systems.
• Quantification: Total protein assays, immunoassays, mass spectrometry reagents, and microplate readers.
• Modification: Protein crosslinkers and bioconjugation reagents.

Thermo Fisher Scientific offers solutions to preserve protein samples during extraction and purification, increase Western blotting throughput and reproducibility, and obtain more information from samples using cutting-edge mass spectrometry reagents.

Ordering Information

Invitrogen™ PrimeFlow™ Probe Sets are needed separately for each target. Go to thermofisher.com/primeflow to view a complete listing of over 8,200 synthesized probe sets.

References

1. Corrigan-Curay J, Kiem HP, Baltimore D et al. (2014) T-cell immunotherapy: looking forward. Mol Ther 22:1564-1574.
2. Grupp SA, Kalos M, Barrett D et al. (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Eng J Med 368:1506-1518.
3. Batlevi CL, Matsuki E, Brentjens RJ et al. (2016) Novel immunotherapies in lymphoid malignancies. Nat Rev Clin Oncol 13:25-40.
4. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity 2013;39(1):1-10.
5. Junttila MR, de Sauvage FJ (2013). Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 501:46-354.
6. Topalian SL, Drake CG, Pardoll DM (2015) Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer cell 27(4):450-461.
7. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57-70
8. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646-674
9. Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 12:252–264.
10. Wesolowski R, Markowitz J, Carson WE (2013). Myeloid derived suppressor cells–a new therapeutic target in the treatment of cancer. J Immunother Cancer 1:10.
11. Rosenberg SA, Restifo NP (2015) Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348:62-68.
12. Carreno BM, Magrini V, Becker-Hapak M et al. (2015) Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells. Science 348(6236):803-808.
13. Maus MV et al. (2014) Adoptive immunotherapy for cancer or viruses. Annu Rev Immunol 32:189-225.
14. Rapoport AP et al. (2015) NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med 21:914-921.
15. Qasim W et al. (2007) Lentiviral vectors for T-cell-suicide gene therapy: preservation of T-cell effector function after cytokine-mediated transduction. Mol Ther 15:355-360.
16. Fath S, Bauer AP, Liss M et al. (2011) Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression. PLoS One 6(3):e17596.

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For Research Use or Non-Commercial Manufacturing of Cell-Based Products for Clinical Research. Caution: Not intended for direct administration into humans or animals. Not for use in diagnostic procedures. © 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. B-27 is a trademark of Southern Illinois University. Essential 8 is a trademark of Cellular Dynamics International, Inc. Cy and Ficoll are trademarks of GE Healthcare. Luminex and xMAP are registered trademarks of Luminex Corporation. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. RealTime StatMiner is a trademark of Integromics, S.L. NanoDrop is a trademark of NanoDrop Technologies LLC. TRIzol is a trademark of Molecular Research Center, Inc. COL06768 0618