CAR-T Characterization
Human
Ideally suited for new CAR-T development and QC, this panel measures 8 essential components of CAR-T biology in 780 genes and facilitates the development of robust product release assays with a streamlined workflow goaled at reducing vein to vein time. Developed in partnership with several large pharma and industry experts, this panel is the gold standard assay for use in development, manufacturing and delivery of CAR-T cell therapies.
Benefits
01
Confidently profile CAR-T products throughout your manufacturing process with this highly reproducible and automated assay.
02
Optimize CAR-T method development and create manufacturing acceptance criteria.
03
Curated panel content includes: Signaling pathways influencing T-cell differentiation and activity, phenotypic and metabolic switches, T-cell subsets associated with differentiation and activation, T-Cell Receptor diversity and immune cell profiling.
04
Useful for measuring metabolic fitness and persistence and monitoring post-infusion exhaustion and toxicity.
Details & data
CAR-T Therapy Workflow
Understanding each step of the CAR-T workflow is critical to ensuring quality and efficacy of the final CAR-T product. The nCounter CAR-T Characterization Panel can be used throughout development and manufacturing as a standardized panel of genes for optimizing methods, developing manufacturing acceptance criteria, as well as understanding the host influences beyond manufacturing.
Leukapheresis
What is the viability & purity of the leukapheresis product?- Profile cell phenotype
- Measure TCR diversity
- Quantify contaminating cell types
Selection & Activation
What is the cell activation state?- Profile signaling pathways
- Measure antigen presentation & processing
Gene Transfer
Are the cells expressing the transgene & CAR?- Quantify transgene & CAR expression with customized probes
Cell Expansion
Are the CAR-T cells fit & expanding?- Measure metabolic fitness
- Track differentiation status
- Profile cell phenotype
Infusion
Is there a sustained response?- Confirm persistence
- Measure overall TCR diversity
- Profile cell phenotypes
- Monitor exhaustion
- Identify potential toxicities
CAR-T Panel Functional Annotations
This panel includes 780 genes covering T-cell biology and the pathways and processes important to T-Cell research and development, especially signaling pathways influencing T-cell differentiation and activity, phenotypic and metabolic switches, T-cell subsets associated with differentiation and activation state, T-Cell Receptor diversity and immune cell profiling.
| Essential CAR-T Biology | Description | Pathways and Processes |
|---|---|---|
| Phenotype | Multiple subtypes of T-cells and the phenotypic changes that accompany them can be distinguished via the cytokines and pathways that maintain, promote, and modulate their activity. | Notch, Wnt signaling, Tfh, TGF-beta, Th1, Th17, Th2, Th9, Treg, Innate-like T-cells, Vitamin A (RA) Signaling |
| Cell Types | Other cell types can contaminate the population of active CAR-T cells. Similarly, measurement of B-cell populations may aid in assessment of whole blood post-infusion measurement of tumor burden in B-cell lymphomas. | Immune cell profiling |
| TCR Diversity | The TCR diversity of CAR-T cells can provide information on the number of clones present after leukapheresis, manufacturing, and infusion. | TCR Content |
| Activation | T-cell activation is primarily mediated by antigens presented to the TCR complex and modulated by costimulatory molecules. Downstream of the TCR, multiple pathways induce transcriptional changes that lead to the production of chemokines and cytokines. Cell surface receptors signal a change in phenotype. | Chemokine Signaling, Costimulatory Molecules, Interleukin Signaling, TCR signaling, JAK-STAT, MAPK and PI3K Signaling, Myc targets, NFAT, Antigen processing & presentation, T-cell activation markers |
| Metabolism | Fundamental changes in T-cell metabolism are induced upon activation to enable rapid cell division and thus expansion of relevant clones. These changes can be observed across basic metabolic pathways, including carbohydrate and fatty acid metabolism. | Glycolysis, Mitochondrial biogenesis, Fatty Acid Metabolism Glutamine metabolism, Circadian Clock, One-carbon metabolism, Oxidative phosphorylation, mTOR, Cell Cycle, Autophagy |
| Persistence | Ongoing presence of a T-cell and cytotoxic T-cell population can be measured by via cell type profiling. By measuring molecules involved in T-cell migration we can assess the ability of T-cells to home in on their target antigens. | T-cell migration, T-cell cell type profiling |
| Exhaustion | T-cell exhaustion can be induced by costimulatory molecules, other cell-cell interactions, and cell death via apoptosis. | T-cell exhaustion markers, Apoptosis, Interactions with Non-Lymphoid Cells, Costimulatory Molecules |
| Toxicity | Toxicity is correlated with certain cytokine and chemokine profiles. These signals can induce a pro-inflammatory environment in various tissues and lead to off-target toxicities of CAR-T treatment. | NK cell cytotoxicity, NKT Receptors, NF-kB, Type I interferon signaling Type II interferon signaling, Interleukin signaling, Chemokine signaling |
Immune Cell Profiling and TCR Diversity Features
Genes included in the CAR-T Characterization Panel provide unique cell profiling and TCR diversity data to measure the relative abundance of immune cell types1 and shifts in TCR populations2 . The tables below summarize the cell type and TCR diversity content in the panel, as qualified through biostatistical approaches and selected literature in the field of immunology.
| Cell Type | Description | Panel Genes | # Genes |
|---|---|---|---|
| B Cells | B cells are the primary mediators of the humoral immune response, bearing antigen-specific B cell receptors and producing antibodies that can enable the immune system to respond to a broad variety of antigens. B cells can also function as MHC class II antigen presenting cells to stimulate T cell immunity. | BLK, CD19, CD20, TNFRSF17 | 9 |
| T Cells | T-cells mediate cell-based immunity by recognizing primarily peptide antigens displayed on MHC class I or class II and either producing cytokines or directly killing the presenting cell. | CD3D, CD3E, CD3G, CD6, SH2D1A | 6 |
| TH1 | CD4+ T cell subset that produces IL2 and Interferon-gamma to promote cellular immunity by acting on CD8+ T Cells, NK Cells and Macrophages. | TBX21 | 1 |
| Regulatory T Cells (Tregs) | CD4+ T Cells that suppress effector B and T Cells and play a central role in suppression of the immune response and tolerance to self-antigens. | FOXP3 | 1 |
| CD8+ T Cells | A subset of T cells that are capable of binding cognate-antigen expressing cells via class I MHC and directly lysing them via perforin and granzymes. | CD8A, CD8B | 2 |
| Exhausted CD8+ T Cells | T-cells overstimulated by antigen can develop an "exhausted" phenotype, in which they are no longer effective in targeting antigen-bearing cells. | CD244, EOMES, CD223 | 4 |
| Cytotoxic Cells | All cells capable of cytotoxic activity, which can include T, NKT, and NK-cells. | CTSW, GNLY, GZMA, GZMB, GZMH, CD161, CD94, KLRK1, PRF1 | 10 |
| Dendritic Cells | Professional antigen presenting cells that internalize, process, and present antigens to lymphocytes via MHC class I and class II along with costimulatory signals to initiate cellular immune responses. | CCL13, CD209, HSD11B1 | 3 |
| Macrophages | Pluripotent cells with critical roles in initiating innate and adaptive immune responses, phagocytosing abnormal cells, and regulating wound healing and tissue repair. | CD163, CD68, CD84 | 4 |
| Mast Cells | Mast cells release histamine containing granules and other signals in order to promote inflammation and regulate allergic responses. | MS4A2, TPSAB1 | 4 |
| Neutrophils | Neutrophils are highly abundant cells that respond early to sites of infection or inflammation, phagocytose cellular debris, and promote downstream immunity. | CSF3R, CD16, S100A12 | 6 |
| Natural Killer (NK) Cells | Cytotoxic cells of the innate immune system that are a significant source of interferon-gamma and are capable of directly killing targeted cells via detection of a loss in MHC surface expression. | NKP46, XCL2 | 2 |
| NK CD56dim cells | The amount of CD56 present on an NK cell is indicative of its age and differentiation state; CD56 dim cells are mature NK cells, more commonly found in peripheral blood than secondary lymphoid tissues, and have the greatest cytolytic activity. | IL21R, KIR2DL3, KIR3DL1, KIR3DL2 | 3 |
TCR Diversity Content
| Chain Type | Constant Chains | Variable Chains |
|---|---|---|
| Alpha | TRAC | TRAV_45 probes, 46 genes TRAV8-2 and 8-4 covered by 1 probe |
| Beta | TRBC1/2 | TRBV_46 probes, 48 genes TRBV6-3 and 6-2 covered by 1 probe TRBV12-4 and 12-3 covered by 1 probe |
| Gamma | TRGC1, TRCG2 | TRGV_5 probes, 6 genes TRGV3 and 5 covered by 1 probe |
| Delta | TRDC | TRDV_3 probes, 3 genes |
| Immune Cell Markers |
|---|
| CD3D/E/G, CD4, CD8A/B, PTPRC (CD45), CD45R0, CD45RA, SELL (CD62L), CCR7, CD28, CD40LG, IL2RA (CD25), NCR1 (NKp46) |
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