Keeping cancer immunotherapy patients safe with protein biomarkers

“This is personal to me. The early detection of cancer is the way to go.” In the July 2024 edition of the Mendelspod podcast, Alamar Biosciences founder Dr. Yuling Luo told Theral Timpson about how cancer took his mother and many other members of his family. Alamar was born from that loss, with the goal of improving protein detection to enable early diagnosis of disease, particularly cancers. The two studies below build on that mission by identifying early signatures of toxicity associated with cancer therapies.

The Promise of Precision Oncology

Liquid biopsy is best known for telling you whether you likely have cancer. Proteomics can potentially provide additional biomarker signatures of cancer detection, as well as clinically meaningful and personalized information, including progression monitoring and treatment response assessment (Pando-Caciano et al., 2024).

But what if a routine fluid biosample (e.g., blood or urine) could also inform your:

• Risk of developing organ toxicity upon receiving cancer immunotherapy
• Likelihood of developing severe immune reactions upon CAR-T treatment
• Risk of survival after cancer diagnosis

In this blog, we dive into a publication and a preprint that address the first two; in the next blog, we will explore the third bullet.

A Two-Protein Urine Signature for Diagnosing ICI-Related Kidney Injury

Immune checkpoint inhibitor (ICI) therapy is a cornerstone treatment for many cancers, but as many as 60% of patients receiving ICI develop immune-related adverse events (irAEs). On one hand, irAEs are typically associated with a response to therapy, but they can also be acutely severe. One out of five patients with ICI-associated irAEs report acute kidney injury (AKI), while only 2 to 5% are specifically diagnosed with acute interstitial nephritis (AIN). AKIs result in the temporary suspension of ICI therapy to determine the cause, and invasive biopsies are required for AIN diagnosis (see figure below). Not only can this disruption delay the reintroduction of therapy by up to four weeks, but patients are also subjected to further invasive biopsies that can cause excessive bleeding. Furthermore, delays in diagnosis put over 50% of patients at risk of permanent renal damage. The need for a non-invasive and rapid diagnostic for AIN cannot be overstated.

To address this need, Jamie Lin and colleagues at the MD Anderson Cancer Center turned to the NULISAseq Inflammation 250 Panel to identify low-abundance markers associated with AIN in patient urine and plasma. “We’ve been waiting for an assay with this sensitivity,” Dr. Lin said in a recent webinar on immuno-oncology. “This is really a big breakthrough for urine analysis,” she added about the NULISA platform.

The team found that urine proteomics outperformed plasma results in distinguishing ICI-AIN from non-AIN cases (Long et al., 2025). In the urine biomarker analysis, they identified 10 key proteins, two of which confirmed previous findings (CXCL9 and TNF-α), while eight were novel (IL-5, Fas, TNFSF4, CD274, IL-20, TNFSF15, TSLP, TREM1, and CCL1). The careful curation and multiplexing of important immune-related markers in the panel thus provided a previously undescribed signature and instilled confidence in the results, effectively recapitulating the literature.

Using machine learning and computational approaches, the team refined their signature to two proteins, IL-5 and Fas, that achieved an area under the curve of 0.94, indicating a remarkably sensitive and selective diagnosis of ICI-AIN. Notably, these two markers complemented each other in sensitivity and specificity, with IL-5 showing 100% sensitivity and Fas displaying 100% specificity.

With further validation, the two-protein signature could eventually become a bedside diagnostic tool for rapidly identifying patients receiving ICI who likely require glucocorticoid co-therapy to alleviate AIN.

“I’m personally excited to see where the NULISA platform is going to take us. We expect to see big changes in the nephrology world with this assay,” Dr. Lin concluded in her webinar.

• Figure: Current (left, red box) and proposed (right, yellow box) standard of care in identifying and treating acute kidney injury (AKI) in patients receiving immune checkpoint inhibitor (ICI) therapy. Adapted from Dr. Jamie Lin’s webinar, April 30, 2025. 

Tracking Cytokine Release Syndrome and Neurotoxicity Biomarkers in CAR-T Cell Therapy

CAR-T therapy is another application of cancer immunotherapy in dire need of irAE biomarkers. Like ICI, irAEs are a hallmark of CAR-T infusion and are indicative of patient response. Severe reactions often manifest as neurotoxicity (ICANS) and cytokine release syndrome (CRS), which can occur independently or in tandem. CRS symptoms typically appear within a week after conditioning and CAR-T cell infusion, while ICANS onset occurs later, after effector immune cells infiltrate the blood-brain barrier (BBB) (Morris et al., 2022). The lack of sensitive techniques for exploring early-stage inflammatory changes means that the underlying contributions to immune activation, BBB collapse, and response severity are poorly understood.

A collaborative study between the Cleveland Clinic and Alamar Biosciences uncovered important signals of irAEs during the administration of anti-CD19 CAR-T cell therapy. In the most comprehensive characterization of immune response to CAR-T cell therapy to date, the team utilized the NULISAseq Inflammation Panel 250 in a longitudinal cohort of 80 patients with B-cell lymphoma (LBCL), involving 480 plasma samples over six time points (baseline and up to 16 days post-anti-CD19 CAR-T cell infusion) and including ASTCT consensus criteria scores for ICANS and CRS.

The data read like a contour map, displaying the peaks and valleys of effector immune cell activation, exhaustion, and resolution pathways (Kirkpatrick et al., 2024). The superior sensitivity of the NULISA platform is on constant display, revealing novel markers previously unreported in CAR-T immune response, including several chemokines (CX3CL1, CXCL1, CCL4, CXCL10), Th2 T-cell effector cytokines (IL-4, IL-5, IL-13), and T-cell exhaustion proteins (CTLA4, PDCD1, CD274, LAG3). These results also confirm the known role of IL-17 in mediating immune injury, which is implicated in permeabilizing the BBB (Rahman et al., 2018). Over days 1 to 9, this crescendo was notably more profound in the severe irAE groups. In contrast, these groups showed downregulated growth and repair mediators, including PDGFA, EGF, and brain-derived neurotrophic factor (BDNF); limited access to the latter may worsen ICANS.

Overall, this study opens the door to a better understanding of the causative mechanisms of acute immune injury in CAR-T cell therapy and informs several mitigation strategies, pending further validation. We look forward to the peer-reviewed publication of this preprint.

Why Multiplexed, Ultra-Sensitive Proteomics Matters for Catching Immunotherapy Toxicity Early

In both RNA detection with Advanced Cell Diagnostics and protein detection with Alamar Biosciences, Dr. Luo recognized that the key to unlocking the potential of measuring these biomolecules lies in enhancing the signal-to-noise ratio by suppressing background interference. These innovations are essential in oncology, both for detecting single RNA molecules at the single-cell level (ACD) and for achieving unprecedented sensitivity and dynamic range for proteins in biofluids (Alamar). In just two years, equipping researchers with the NULISA platform’s biofluid protein sensitivity has paid dividends for the early detection of immune-related adverse events associated with cancer immunotherapy.

Multiplexing enables researchers and clinicians to move beyond single-analyte assays and lower-plex panels that systematically rule in or out certain conditions. Rather, the approach supports the discovery of unexpected signatures like the IL-5/Fas uremic pair, or mapping the dozens of interacting cytokines, chemokines, and exhaustion markers that cycle across the CAR-T time course. Measuring 250 proteins at once, with attomolar-to-femtomolar sensitivity, allowed both findings to emerge as data-driven discoveries rather than confirmations of a single suspected candidate.

Be sure to look out for the next post in this cancer biomarker series, which will focus on how NULISA is uncovering novel signatures of patient outcomes, including risk stratification and therapy response.

In short: cancer immunotherapies save lives, but they can also trigger serious immune-related adverse events (irAEs). Two recent studies identified promising signatures of therapy-related irAEs using Alamar’s NULISAseq Inflammation Panel 250. First, a simple urine test can identify immune checkpoint inhibitor (ICI)-related kidney injury with 0.94 AUC accuracy, potentially replacing invasive biopsies. Second, a longitudinal blood-biomarker profile can track the severity of cytokine release syndrome (CRS) and neurotoxicity (ICANS) in CAR-T cell therapy. Together, these findings position protein biomarkers as a tool for catching immunotherapy’s side effects early, reducing their negative impacts and shortening therapy disruption windows.

Key Takeaways

• Immune-related adverse events (irAEs) are a real, common risk of cancer immunotherapies, and biomarkers to catch them early have been lacking.
• The NULISAseq™ Inflammation Panel 250 revealed a two-protein urine signature (IL-5 and Fas) for diagnosing ICI-related kidney injury with a 0.94 AUC, potentially replacing invasive biopsies.
• Cytokine release syndrome and neurotoxicity are common irAEs in CAR-T cell therapies. A Cleveland Clinic collaboration profiled 480 plasma samples from 80 lymphoma patients and identified multiple previously unreported biomarkers that track the severity of these irAEs over time.
• Together, the two studies establish NULISA as a tool for catching immunotherapy’s side effects early, providing a new layer of context for tracking patient safety and monitoring, beyond cancer detection alone.
• Both findings are research results pending further validation; neither signature is yet a clinically approved diagnostic test.

Frequently Asked Questions

What is an immune-related adverse event (irAE)?

An irAE is a side effect caused by the immune system being activated or dysregulated by a cancer immunotherapy, such as an immune checkpoint inhibitor (ICI) or CAR-T cell therapy. IrAEs range from mild to severe and can affect organs including the kidneys and brain.

What is acute interstitial nephritis (AIN), and why is it hard to diagnose?

AIN is a specific type of kidney inflammation caused by ICI therapy that requires an invasive kidney biopsy for confirmation. Only 2 to 5% of patients with ICI-related kidney injury are specifically diagnosed with AIN, and the biopsy process can delay treatment decisions by weeks while carrying a risk of bleeding.

How was a urine test for AIN identified?

Researchers at MD Anderson Cancer Center used the NULISAseq Inflammation Panel 250 to measure low-abundance proteins in urine and identified a two-protein signature, IL-5 and Fas, that together distinguish ICI-related AIN from other causes of kidney injury with a 0.94 AUC. IL-5 provides 100% sensitivity, and Fas provides 100% specificity.

What are CRS and ICANS in CAR-T cell therapy?

Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are the two most common severe immune-related adverse events after CAR-T cell infusion. CRS typically appears within the first week after infusion, while ICANS tends to occur later, after immune cells cross the blood-brain barrier.

What did the Cleveland Clinic and Alamar Biosciences study find?

Using the NULISAseq Inflammation Panel 250 across 480 plasma samples from 80 lymphoma patients, the study identified multiple previously unreported biomarkers, including specific chemokines, T-cell effector cytokines, and T-cell exhaustion proteins, that track the severity of CRS and ICANS over time after CAR-T infusion.

Are these biomarker signatures available as clinical diagnostic tests today?

No. Both the urine IL-5/Fas signature and the CAR-T plasma biomarkers are research findings that require further validation before they could become approved clinical diagnostics. The NULISAseq Inflammation Panel 250 is currently for research use only.

References

1. Kirkpatrick, R. et al. (2024) ‘Novel Biomarkers of Immune Toxicity from CAR-T Cell Therapy Using Ultrasensitive NULISA™ Proteome Technology’, medRxiv, p. 2024.12.18.24319239. doi:10.1101/2024.12.18.24319239. Preprint; not yet confirmed peer-reviewed as of this writing.
2. Long, J.P. et al. (2025) ‘Urine proteomics defines an immune checkpoint-associated nephritis signature’, Journal for ImmunoTherapy of Cancer, 13(1), p. e010680. doi:10.1136/JITC-2024-010680.
3. Morris, E.C. et al. (2022) ‘Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy’, Nature Reviews Immunology, 22(2), pp. 85–96. doi:10.1038/S41577-021-00547-6.
4. Pando-Caciano, A. et al. (2024) ‘Unlocking the promise of liquid biopsies in precision oncology’, The Journal of Liquid Biopsy, 3, p. 100151. doi:10.1016/J.JLB.2024.100151.
5. Rahman, M.T. et al. (2018) ‘IFN-γ, IL-17A, or zonulin rapidly increase the permeability of the blood-brain and small intestinal epithelial barriers: Relevance for neuro-inflammatory diseases’, Biochemical and Biophysical Research Communications, 507, pp. 274–279. doi:10.1016/j.bbrc.2018.11.021.

The NULISAseq Inflammation Panel 250 and the biomarker signatures described here are for research use only and are not intended for use in diagnostic procedures.

This Blog was originally published May12, 2025. It was updated and re-published on September 11, 2026.