Single-Cell Functional Biomarkers for Each Stage of the Cell Therapy Development Pipeline

In CAR-T cell therapy, the patient’s own cells or donor cells are modified and re-introduced to target cancer. Single-cell functional proteomics is key to unlocking the potential of CAR-T therapies. Recent studies have demonstrated that single-cell functional profiling and polyfunctionality—the ability of an individual cell to secrete a range of cytokines simultaneously— can reveal predictive insights across the entire cell therapy development pipeline, from confirming the function of gene edits and identifying prime candidates to optimizing cell therapy manufacturing and revealing predictive biomarkers for patient outcomes.

The following cases demonstrate how IsoPlexis’ Single-Cell CAR-T Cell Protocol has been utilized by leading researchers to improve CAR-T therapies at every stage of the development pipeline.

Predictive Functional Biomarkers for Patient Response

Throughout the CAR-T development process, predictive biomarkers that allow researchers to stratify patient response to therapy and identify those with an increased risk of adverse side eff­ects are needed. IsoPlexis’ polyfunctional biomarker is a powerful predictive metric which has correlated in various studies to therapy response.

A 2018 publication in Blood by Rossi et al. demonstrated a significant association between the single-cell polyfunctionality of an anti-CD19 CAR-T cell product before treatment and the objective response in patients with non-Hodgkin lymphoma, where traditional methods such as flow cytometry and bulk ELISA were unable to do so. The results of the study “highlight the potential to predict whether cancer patients will respond to CAR-T cell therapy before treatment, as well as to improve both pre-infusion product potency testing and guide cell product optimization.”1

Confirming the Function of Engineered Cells

Predictive functional insights can be used to accelerate preclinical development of engineered cell therapies. When engineering new cell therapies, it is critical that researchers ensure that the edits function as intended. Transcriptomics alone may miss post-translational modifications and protein interactions that may affect the function of these novel therapies. In a study published in Cell Stem Cell, researchers Zhu, et al. used IsoPlexis’ single-cell proteomics to evaluate the function of CRISPR-edited NK cells and found that the edited cells showed improved in vivo persistence, potency, polyfunctionality, and anti-tumor activity against acute myeloid leukemia.2 With this technology, researchers were able to confirm that the CRISPR-edited NK cells performed the desired function without causing unwanted effects. This type of single-cell functional profiling empowers researchers to make informed decisions at this critical stage of the cell therapy development pipeline.

Functional Biomarkers of Persistence in Pre-Clinical Therapy Development

Cell therapies need to be durable and persistent in order to promote positive clinical outcomes. Single-cell proteomics is critical during CAR-T development and testing to drive lead choice enhancement and provide predictive metrics for product quality.

Relapse is a challenge for patients with difficult to treat cancers such as multiple myeloma, and CAR-T-based approaches targeting CD19 have been met with disease resistance and immune evasion caused by loss of antigen expression. In a study published in Blood Advances, researchers Schmidt, et al. developed trimeric CAR-T cells capable of binding to multiple antigens. To examine the proliferative capacity of these constructs, the authors then looked at polyfunctionality using single-cell functional proteomics. The researchers found a positive association of PSI with the functional enhancement of trimeric CAR-T cells and improved anti-tumor activity in both in vitro and in vivo models. IsoPlexis’ single-cell metrics revealed functional drivers that correlated to CAR-T cell persistence in solid tumor, providing a critical metric for lead candidate identification.3

Functional Quality Metrics for Cell Therapy Process Optimization

The ability of IsoPlexis’ single-cell proteomics to reveal product quality is equally crucial for optimization of cell therapy manufacturing. The identification of critical quality indicators of potency in the manufacturing and bioprocessing process is a significant challenge that scientists face in the development of these novel therapies.

A recently published paper in Nature Medicine explored how IsoPlexis’ platform helps researchers overcome critical challenges in cell therapy manufacturing by providing deep insights into product quality and potency4. In a Phase 1 clinical trial in patients with relapsed B cell acute lymphoblastic leukemia, IsoPlexis’ single-cell functional proteomics provided meaningful product quality insights to predict CAR potency in vivo. Researchers identified a functional driver of relapse suggesting possible improvements in CAR manufacturing that could prevent antigen+ resistance.

This recent publication from Nature Medicine further highlights how IsoPlexis is providing critical predictive metrics for the product characterization and optimization of CAR-T therapies and manufacturing workflows. These high-impact studies demonstrate how IsoPlexis’ single-cell proteomics accelerates and optimizes the development and use of CAR-T therapies across each stage, from preclinical to clinical trials and use in patients.


  1. Rossi J, et al. Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL. Blood 2018; 132 (8): 804–814.
  2. Zhu H, et al. Metabolic Reprogramming via Depletion of CISHin Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity. Cell Stem Cell 2020; 27: 1-14.
  3. Schmidt A, et al. Rational design of a trimeric APRILbased CAR-binding domain enables efficient targeting of multiple myeloma. Blood Advances 2019; 3(21): 3248-60.
  4. Spiegel J, et al. CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. Nature Medicine
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