Multiplex Human Cytokine ELISA Kit (Cytokines in CAR-T Cell Therapy)

INTENDED USE

EM10004 is designed for semi-quantitative and simultaneous determination of cytokines relevant to the cytokine releasing syndrom (CRS) and the immune response in chimeric antigen receptor (CAR) T cell therapy. The kit simultaneously determines the levels of Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interleukin-15 (IL-15), Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), Interferon-γ (IFN-γ), Monocyte Chemotactic and Activating Factor/Monocyte Chemoattractant Protein-1 (MCAF/MCP-1) in blood samples and cell culture supernatant. In combination with Anogen’s other quantitative cytokine ELISA kits, this multiplex ELISA kit is expected to be useful for the investigation of the roles of cytokines in immunotherapy and inflammation.

The kit is intended FOR LABORATORY RESEARCH USE ONLY and should not be used in any diagnostic or therapeutic procedures.

INTRODUCTION

CAR-T therapy is a significant breakthrough in cancer immunotherapy, particularly for certain blood cancers, and is considered one of the cornerstones of modern cancer care. It involves modifying a patient’s own T cells to recognize and attack cancer cells, offering high response rates and, in some cases, cures for patients with advanced or refractory cancers. The research on CAR-T therapy has now expanded to treat solid cancers and autoimmune diseases.

In CAR-T cell therapy, cytokines play important roles in the development of immune response, and are the main contributors in complications such as CRS and Immune Cell Activation Neurotoxicity (ICAN). CRS and ICAN are often manageable by treatment with anti-inflammatory cytokine antibody and corticosteroid, but severe CRS and ICAN can be life-threatening. Studying cytokine levels in the course of T cell therapy and other immunotherapy helps to understand their association with the complications.

The releasing of pro-inflammatory cytokines (such as IFN-γ and TNF-α) and damage-associated molecular patterns (DAMPs) is initiated upon CAR T-cells contacting with target (cancer) cells. These inflammatory signals activate nearby macrophages and other immune cells to release additional inflammatory cytokines such as IL-1β, IL-6, GM-CSF, MCP-1, etc., and accelerate immune cell expansion and activation, CRS cascade formation, and endothelial cell activation and leaking. Systemic inflammation and organ damage are the outcomes of this inflammatory process.

Cytokines take various roles in immune response and inflammation. IL-1β is involved in the initiation and amplification of inflammation by activating various immune cells, including T cells, B cells, and other myeloid cells. IL-1β and IL-6 induce the expression of adhesion molecules and both are potent cytokines in promoting endothelial cell activation. IL-6 plays an important role in acute phase response, innate and adaptive immunity, and is the key cytokine in CRS. High levels of IL-6 are strongly correlated with the severity of CRS. Blockage with anti IL-6R antibody has been proven to alleviate CRS and ICAN. IFN-γ activates macrophages, enhances antigen presentation, and promotes T-cell responses. Elevated IFN-γ levels are associated with the activation of inflammatory cascades and the systemic inflammatory symptoms of CRS. GM-CSF stimulates the production and activation of granulocytes and macrophages, and also implicated in the recruitment of immune cells to sites of inflammation.

The infiltration of immune cells in large amount through blood vessels can lead to tissue damage and organ dysfunction. MCP-1 is a chemokine that primarily attracts monocytes, macrophages, and other inflammatory cells to sites of inflammation. Chemokine IL-8 is involved with neutrophil recruitment and activation, and also induces the activation of endothelial cells. Endothelial cell activation increases the permeability of the Blood-brain barrier (BBB). Working in synergy with other pro-inflammatory cytokines, chemokines facilitate the passage of immune cells into the central nervous system and lead to neuro-inflammation, cerebral edema and neuronal dysfunction. It has been reported that elevated MCP-1 levels above a certain threshold may predict severe CRS development.

The balance between pro-inflammatory and anti-inflammatory cytokines is critical in the development of CRS and ICAN. IL-10 inhibits pro-inflammatory cytokine production thus helps reducing excessive immune responses. To counterbalance the inflammatory response during CRS, IL-10 levels often increase in the process to help resolve inflammation.

IL-15 plays a significant role in the expansion and survival of CAR-T cells and the activation of natural killer cells. It promotes the proliferation and survival of CD8+ cytotoxic T cells, which are critical for targeting and eliminating cancer cells. Another key feature of IL-15 is its ability to promote the development of memory T cells. Memory T cells can persist in the body for long periods, thus providing long-term immunity. In CAR-T therapy, promoting memory T cells is desirable because it can enhance the durability of the therapeutic effect and prevent cancer relapse. IL-15 is being investigated in clinical trials as an adjuvant for CAR-T therapies, especially for challenging cancers like solid tumors.

This ELISA assay is a 3.5-hour solid phase immunoassay readily applicable to measure the levels of eight cytokines relevant to the CRS of T cell therapy. It showed no cross reactivity with other proteins.

CITATIONS

1. Cytokine signaling in chimeric antigen receptor T-cell therapy Yuki Kagoya, International Immunology. 2024, 36(2):49–56
2. Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy Emma C. Morris, Nature Reviews Immunology. 2022, 22(2):85–96
3. Interleukin 15 in Cell-Based Cancer Immunotherapy. Yang Zhou et al., International Journal of Molecular Science. 2022, 23(13):7311
4. Mechanisms of cytokine release syndrome and neurotoxicity of CAR T-cell therapy and associated prevention and management strategies. Xinyi Xiao et al., Journal of Experimental & Clinical Cancer Research. 2021, 40(1):367
5. CAR-T cell therapy: current limitations and potential strategies. Robert C. Sterner, Blood Cancer Journal. 2021, 11(4):69
6. Neurotoxicity and Cytokine Release Syndrome After Chimeric Antigen Receptor T Cell Therapy: Insights Into Mechanisms and Novel Therapies. Elizabeth L Siegler et al., Frontiers in Immunology. 2020, 11:1973
7. Cytokine Release Syndrome: Current Perspectives. Hemant Murthy et al., Immuno Targets and Therapy. 2019, 8:43–52