Your Comprehensive Guide to Tools for Immuno-Oncology Research

Immuno-oncology product resource guide Your comprehensive guide to tools for immuno-oncology research Cancer research Our goal is to support your immuno-oncology research with a comprehensive range of tools and technologies that maximize your time, budget, and data—helping to accelerate your path to discovery and translation to a clinical application. In this guidebook, you will discover educational resources and solutions for a number of immuno-oncology research approaches, including checkpoint inhibition, CAR T cell therapy, and cancer vaccine research. Learn about our capabilities, from leveraging innovative products and techniques to time-saving workflow applications. We’re committed to partnering with you for your next breakthrough. 2 I-O product resource guide thermofisher.com/immuno-oncology Immuno-oncology review 4 Key questions about I-O 4 General I-O workflow 7 Solutions by approach 8 Solutions for checkpoint inhibition 8 Solutions for CAR T cell therapy 12 Solutions for cancer vaccine research 18 Characterize and verify 20 Targeted genetic analysis 20 Targeted gene expression analysis 21 Cell analysis 23 Protein analysis 29 Ordering information 30 Contents 3 Immuno-oncology review What is I-O? Immuno-oncology (I-O), also known as cancer immunotherapy, is a rapidly growing field that studies the ways in which the body’s own immune system can fight cancer. Why does I-O research matter? I-O research aims to develop cancer immunotherapies that go beyond traditional methods such as surgery, chemotherapy, and radiation, by enabling the adaptive immune system to recognize and specifically attack cancer cells while leaving healthy ones undamaged. I-O research can potentially uncover ways to enable immunogenicity of all types of cancer and facilitate long-lasting, protective immunity against future recurrence [1-3]. Recent breakthroughs in checkpoint inhibition, chimeric antigen receptor (CAR) T cell therapy, and cancer vaccines illuminate the full capabilities of the immune system and how it may be harnessed to combat cancer. While every immune cell has a key part to play in the landscape of I-O, T cells and T cell–mediated responses are the focal points of I-O research today. What are some of the promising areas in I-O research? This handbook will provide an overview of three currently trending I-O research areas: checkpoint inhibition, CAR T cell therapy, and cancer vaccines. Figure 1 (adapted from Chen and Mellman [4]) shows where they correlate within the cancer-immunity cycle. Figure 1. Stages of the adapted cancer-immunity cycle [4] can be impacted by I-O approaches such as checkpoint inhibition, CAR T cell therapy, and cancer vaccines, as indicated by the icons. Checkpoint inhibition (e.g., B7/CTLA-4) CAR T cell therapy Periphery Tumor microenvironment Activated T cells circulate and infiltrate tumor tissue APCs prime and activate T cells, initiating immune response Antigen-presenting cells (APCs) process antigens and neoantigens Upon cell death, tumor cells release antigens and neoantigens T cells recognize and bind to malignant cells T cells destroy cancer cells Cancer vaccines Checkpoint inhibition (e.g., PD-1/PD-L1) 4 I-O product resource guide thermofisher.com/immuno-oncology I-O review Checkpoint inhibition Immune checkpoints are cell pathways crucial in maintaining a normal immune response and protecting tissues from damage when the immune system is activated [5,6]. Cancer cells dysregulate immune checkpoints and use them as a mechanism of immune resistance. Understanding the interactions between tumor and immune cells is one of the main approaches in I-O research [7,8]. There are natural mechanisms in place that serve to regulate T cell activity via interactions with the T cell receptor (TCR). For example, PD-1/PD-L1 is a coinhibitory pathway that “masks” cancer cells from T cell recognition, thereby preventing the attack by T cells. Antibodies that target the PD-1/PD-L1 pathway and bind to PD-1 suppress its coinhibitory function. The T cells then recognize the cancer cells and cytotoxic activity commences. Another example of T cell regulation is the B7/CTLA-4 pathway that plays a role during the priming of a T cell by an antigenpresenting cell (APC). Blocking of the CTLA-4 receptor by an antibody allows T cell activation, resulting in an anticancer immune response. There are multiple costimulatory and coinhibitory receptor–ligand interactions between APCs and T cells. For T cell activation or suppression, T cells must recognize their cognate antigens through TCRs and then respond to costimulatory (for activation) or coinhibitory (for suppression) receptor–ligand interactions, examples of which are shown in Figure 2 [6,9]. Adoptive cell therapy (ACT) and CAR T cell therapy ACT targets the immune system, enabling the body’s natural ability to fight the cancer, instead of directly targeting the cancer itself. This is accomplished by genetically modifying a subject’s own T cells to target antigens selectively expressed on cancer cells [10,11]. Successful applications of ACT include: • Tumor-infiltrating lymphocytes (TILs) are taken from tumor tissue, modified ex vivo, and infused in activated form back into the body to re-infiltrate the tumor and attack tumor cells. • CAR T cells are generated from the body’s own T cells and are engineered to express antibody-like chimeric antigen receptors for targeting specific cancer cells via surface proteins or intracellular proteins, inducing anticancer attack (Figure 3). Costimulatory checkpoint Coinhibitory checkpoint Antigen-presenting cells T cells Antigen-presenting cells T cells PD-L1 (B7-H1) PD-L2 PD1 B7-H3 (CD276) B7-H4 (VTCN1) 4-1BB L (CD137L) HVEM OX40L (CD252) GAL9 ? BTLA (CD272) TIM3 ? MHC II LAG3 ? PD-L1 or PD-L2 CD28 ICOSL (B7RP1) OX40 (CD134) CD80 or CD86 ICOS (CD278) 4-1BB (CD137) CD27 CD70 PVR (CD155) CD40L (CD154) CD40 IDO CTLA4 (CD152) CD80 or CD86 GITR (CD357) GITRL CD30 CD30L (TNFSF8) LIGHT (CD258) DR3 CD96 Nectin-2 (CD112) TL1A VISTA (B7-H5) ? ? DNAM1 (CD226) B7-CD28 family TNF/TNFR superfamily PVR (CD155) TIGIT Others Nectin-2 (CD112) Unknown VISTA (B7-H5) Figure 2. Multiple costimulatory and coinhibitory receptor–ligand interactions between APCs and T cells. One important family of membrane-bound molecules that bind both costimulatory and coinhibitory receptors is the B7-CD28 family (purple boxes); all of the B7 family members and their known ligands belong to the immunoglobulin superfamily. Another major category of signals arises from tumor necrosis factor (TNF) family members (green boxes), which regulate the activation of T cells in response to cytokines. I-O product resource guide thermofisher.com/immuno-oncology 5 A closer look: CAR T cells The TCR participates in the activation of T cells [2]. Its stimulation is triggered in response to cells expressing major histocompatibility complex (MHC) molecules with an antigen. Tumor-specific TCRs can be genetically engineered to recognize specific cancer cell populations. TCR technology is unique as it recognizes both intracellular and cell surface proteins, conferring a broad array of antigen targets. Limitations include patientspecific 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 [2,10,11]. They can be constructed by linking the variable regions of the heavy and light chains of the antibody to intracellular signaling chains (such as CD3-zeta, CD28, and 4-1BB) or other signaling factors. T cells that are engineered to express CARs are not limited by HLAs, since a CAR molecule recognizes an intact cell antigen on the surface of a cancer cell. However, they are limited by their inability to recognize mutated intracellular proteins. Cancer vaccines Vaccines represent another important I-O research area aimed at enabling the immune system to recognize cancer as a threat. This method is antigenbased, relying on the ability of the immune system to recognize the protein to induce the immune response. Scientists endeavor to identify new tumor-associated antigens, called neoantigens, released within the tumor microenvironment [12]. This aids in understanding how tumors form and spread, which informs the development of vaccines. Another method called dendritic cell (DC) therapy utilizes tumor fragments to activate extracted DCs. This activation turns DCs into APCs, which are then infused back into the subject to induce a secondary immune response, including antibody production [12]. Finally, new combination therapies and personalization methods are also being studied to further enrich the capabilities of anticancer immunity. Figure 3. CAR T cell engineering involves genetically modifying an individual’s own T cells to target antigens selectively expressed on cancer cells. PD-L1 PD-1 CAR T cell Tumor cell 6 I-O product resource guide thermofisher.com/immuno-oncology I-O review CAR T cell therapy Page 12—Find tools that span the entire workflow, from T cell isolation and activation to cell engineering and expansion, including scalable solutions to help get you from basic research to the clinic. Cancer vaccines Page 18—Find transfection solutions, gene-toprotein services, and genomic biomarker tools to develop vaccine therapies. General I-O workflow Thermo Fisher Scientific offers many research platforms and products to help you better understand the interplay between the immune system and cancer. Expand experimental capabilities I-O Clinical immunotherapy Cancer research Key approaches Figure 4. A growing focal area within cancer research, I-O encompasses a robust workflow. This starts with the biomarker discovery phase, continues into further research on targets of interest, including contextual studies within model systems, and finally may conclude with characterization and verification. This workflow is applicable across different approaches within I-O, including checkpoint inhibition, CAR T cell therapy, and cancer vaccine research. Biomarker discovery Targets Modification of model systems Characterization and verification Checkpoint inhibition Page 8—Find workflow tools for genomic biomarker discovery, verification, and protein expression, as well as immune checkpoint analysis by antibodies and assays. I-O review with our instruments, assays, and reagents to accelerate the development of the cancer immunotherapies of tomorrow. In the following chapters, explore our solutions by approach (Figure 4). I-O product resource guide thermofisher.com/immuno-oncology 7 Checkpoint inhibition (e.g., B7/CTLA-4) CAR T cell therapy Tumor microenvironment Periphery Activated T cells circulate and infiltrate tumor tissue APCs prime and activate T cells, initiating immune response Antigen-presenting cells (APCs) process antigens and neoantigens Upon cell death, tumor cells release antigens and neoantigens T cells recognize and bind to malignant cells T cells destroy cancer cells Cancer vaccines Checkpoint inhibition (e.g., PD-1/PD-L1) Genomic biomarker discovery and verification tools • Applied Biosystems™ Clariom™ D Pico assays—Get a deep view of the transcriptome to rapidly discover novel biomarkers. Analyze coding and long noncoding RNA as well as alternative splicing events from as little as 100 pg of total RNA with this microarray-based solution. • Applied Biosystems™ Clariom™ S Pico assays—Discover gene-level analysis of well-annotated genes across the transcriptome from as little as 100 pg of total RNA. Quickly identify important gene-level signatures and pathways and screen large numbers of samples with microarray-based highthroughput, automated formats. • Applied Biosystems™ TaqMan™ Array Human Immune Response Plate—Utilize a 96-well plate for quantitative gene expression analysis. Accurately analyze genes from 9 classes of immune system functions, including cell surface receptors, transcription factors, cytokines and cytokine receptors, and cell cycle and protein kinases. Solutions for checkpoint inhibition Find out more at thermofisher.com/checkpointinhibitor Identifying and validating predictive biomarkers for checkpoint immunotherapy is important to optimize therapeutic benefit, minimize toxicity risk, and guide combination therapy approaches. Discover a wide variety of solutions, from • Applied Biosystems™ TaqMan™ Array Human Immune Panel—Get quantitative gene expression analysis of a comprehensive set