Evaluating CAR T-Cell Therapy Toxicity

Understand, Predict, Monitor: The Case for Evaluating CAR T-Cell Therapy Toxicity with More Comprehensive Protein Panels

Introduction to CAR T-Cell Therapy and Associated Toxicities

The advent of CAR T-cell therapy has revolutionized cancer treatment. It is particularly successful in managing relapsed/refractory B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. Despite its success, CAR T-cell therapy is associated with certain toxicities, including Cytokine Release Syndrome (CRS) and Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS), which need to be diagnosed early, graded accurately, and managed effectively.

Understanding CRS and ICANS: The Role of Cytokine Profiling in CAR T-Cell Therapy

CRS is the most common adverse event following CAR T-cell therapy, characterized by a systemic inflammatory response triggered by elevated cytokines from activated T cells, monocytes, macrophages, and other antigen-presenting cells. Key immune mediators involved in CRS include pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ, IL-2), anti-inflammatory cytokines (IL-10, IL-1Ra), CXC chemokines (CXCL9, CXCL10), CC chemokines (MIP-1α, MIP-1β), and growth factors (GM-CSF). The surge in cytokines may lead to endothelial cell activation and compromised vascular integrity. Additionally, CRS can result from tumor cell pyroptosis, a form of inflammatory cell death, where CAR T-cells release perforin, granzyme, TNF-α, and IFN-γ upon antigen recognition. This process leads to the release of cellular contents that further activate macrophages and amplify cytokine production, particularly IL-1β and IL-6 (1,2,3).

The vast array of cytokines involved in CRS highlights the importance of broadly measuring all of them, as well as additional markers, to better understand, monitor, and predict CRS. ICANS is the second most common adverse event and can occur concurrently with or after CRS.

Despite the longstanding recognition of CRS and ICANS as common toxicities following CAR T-cell therapy, there are still significant gaps in understanding their pathophysiology and the different mechanisms influenced by CAR T-cell design, dosing, infusion schedule, tumor burden and distribution, and other patient-specific factors. Furthermore, a growing catalogue of adoptive cell therapies utilizing non-T cell effectors, such as NK cells, may ultimately be found to create their own patterns of toxicities based on their different cellular biology.

Using Olink Panels to Predict CAR T-Cell Therapy Toxicities and Biomarkers

Recent studies have highlighted the potential of using comprehensive cytokine panels, such as those provided by Olink, to better understand and predict these toxicities. For example, Sarén et al. conducted a phase II study where patients with B-cell lymphoma or leukemia received third-generation CD19-directed CAR T-cells (4). They observed that patients who developed grade 2-3 CRS had elevated levels of several proteins, such as IFN-γ, TNF, CXCL10, and CCL3, before CAR T-cell infusion and up to three weeks after infusion, underscoring the importance of a comprehensive approach to cytokine measurement for both predicting adverse events and guiding therapy.

Similarly, Diorio et al. used Olink panels to measure soluble proteins in the serum of pediatric and young adult patients undergoing CAR T-cell therapy. They identified novel biomarkers, including IL-18, which was associated with the development of ICANS, suggesting that IL-18 could be a target for future therapeutic development or provide drug repurposing opportunities. The study also identified other pre-infusion CRS biomarkers, such as MILR1 and FLT3, which outperformed clinical standards. This suggests that a comprehensive cytokine panel could be useful not only in measuring known cytokines associated with CAR T-cell therapy but also in identifying other proteins that could provide valuable insights into patient risk and treatment response (5).

Investigating Hematological Toxicities in CAR T-Cell Therapy Patients

Apart from CRS and ICANS, increasing recognition of hematological toxicities, such as cytopenia, has opened up new avenues of research. The pathological mechanism of late cytopenia is not fully understood. Building on this, Rejeski et al. examined the mechanisms underlying prolonged neutropenia following CAR T-cell therapy. Their multicenter observational study, involving 344 patients, utilized Olink panels to explore the connection between neutrophil recovery phenotypes and clinical outcomes (6). They discovered that certain proteins, including inhibitory soluble T cell checkpoint ligands, IL-15, IL-18, MCP-1/CCL2, IFN-γ, were upregulated in patients with unfavorable phenotypes. Stimulatory T cell ligand TNFSF14 (LIGHT) and angiogenic markers such as EGF were down-regulated, reflecting that immune dysregulation and macrophage activation are accompanied by T-cell exhaustion and endothelial dysfunction. These findings suggest that a comprehensive cytokine panel could improve the prediction and management of hematological toxicities.

Expanding Cytokine Measurement for Personalized Medicine in CAR T-Cell Therapy

These studies demonstrate that while it is essential to measure key cytokines already known to be associated with CAR T-cell therapy, expanding the scope to include additional markers can provide deeper insights into the mechanisms of toxicity, the potential for prediction with risk models and ultimately the possibility for personalized interventions. You can learn more about this here.

Why Proximity Extension Assays (PEA™) are Critical for CAR T-Cell Therapy Research

The advent of next-generation multiplex immunoassays, such as Proximity Extension Assay (PEA™), addresses major multiplexing challenges, ensuring high data quality across various panel sizes. PEA is a simple yet sophisticated innovation based on a dual-recognition immunoassay: Two antibodies, each tagged with oligonucleotides, bind the target analyte. When the antibodies are in proximity, a unique double-stranded DNA barcode is generated and can be quantified to reflect the target protein’s initial concentration.

Another key differentiator is the ability to extensively control and monitor all steps of the reaction in each individual sample, bringing an unprecedented degree of confidence to multiplexing. In addition, the microfluidics approach permits analysis of extremely small sample volumes, down to 1 μL, which is ideal for precious samples that are limited or difficult to extract.

These advantages make PEA technology particularly valuable for broad quantification that is still accurate and precise. For instance, the Olink Target 48 panels enable the absolute quantification of 89 proteins from just 2 µL of sample, including all the proteins discussed in this blog post and many more. This makes it an ideal tool for understanding, predicting, and monitoring cell therapy-associated toxicities. Learn more about the Target 48 panels here.

References:

1. Zhang Y, Qin D, Shou AC, Liu Y, Wang Y, Zhou L. Exploring CAR-T Cell Therapy Side Effects: Mechanisms and Management Strategies. J Clin Med. 2023;12(19):6124. doi:10.3390/jcm12196124. PMCID: PMC10573998. PMID: 37834768. https://www.mdpi.com/2077-0383/12/19/6124
2. Shaikh S, Shaikh H. CART Cell Therapy Toxicity. [Book]. Available from: https://pubmed.ncbi.nlm.nih.gov/37276287/
3. Hughes AD, Teachey DT, Diorio C. Riding the storm: managing cytokine-related toxicities in CAR-T cell therapy. Semin Immunopathol. 2024;46(3-4):5. doi:10.1007/s00281-024-01013-w. PMCID: PMC11252192. PMID: 39012374. https://link.springer.com/article/10.1007/s00281-024-01013-w
4. Sarén T, Ramachandran M, Gammelgård G, et al. Single-cell RNA analysis reveals cell-intrinsic functions of CAR-T cells correlating with response in a phase II study of lymphoma patients. Clin Cancer Res. 2023. doi:10.1158/1078-0432.CCR-23-0178. https://pubmed.ncbi.nlm.nih.gov/37540566/ 
5. Diorio C, Hernandez-Miyares L, Espinoza DA, et al. Quadriparesis and paraparesis following chimeric antigen receptor T-cell (CART) therapy in children and adolescents. Blood. 2024. doi:10.1182/blood.2024023933. https://www.sciencedirect.com/science/article/abs/pii/S0006497124016860
6. Rejeski K, Perez A, Iacoboni G, et al. Severe hematotoxicity after CD19 CAR-T therapy is associated with suppressive immune dysregulation and limited CAR-T expansion. Sci Adv. 2023. doi:10.1126/sciadv.adg3919. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10516499/