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Chimeric Antigen Receptor (CAR) T-cell immunotherapy holds enormous potential for the treatment of cancer. The goal of cell therapy is to not only achieve tumor reduction but also to activate an anti-tumor immune response. But many challenges remain, despite the remarkable success of CAR-T immunotherapy for some patients. Unfortunately, it remains unclear how to optimize the therapy.

Re-engineered T cells are living products; this makes their behavior difficult to characterize and predict. Unlike a small molecule or antibody that always has the same structure, each time an autologous cell therapy is developed the starting material comes from a different patient. This makes it virtually impossible to reproducibly develop an identical “drug.” Even if therapy began with similar cells, the result would most likely have different potencies and activities.

This is because in vivo cellular expansion cannot be predicted uniformly across a group of patients—it depends on multiple factors including the tumor microenvironment, target antigen expression intensity, distribution, and organ site. Additionally, life-threatening
immunotoxicity sometimes results once CAR-T cells are re-infused into the patient, such as cytokine release syndrome and neurologic toxicity.

Isoplexis Figure 1
Identifying functional heterogeneity of phenotypically similar cells, on the IsoCode Chip, helps reveal subsets of highly potent polyfunctional anti-tumor T-cell subsets that correlate to in vivo patient response in published immunotherapy studies.

Today’s standard methods to evaluate quality and potency are inadequate to select products that provide better patient outcomes. Quality assays are mainly descriptive with some objective parameters like cytokine production, cytotoxicity, and proliferation.

Researchers and clinicians urgently need new approaches to understand the functional profile of cell therapy products to accelerate discovery and development, to achieve manufacturing consistency, and to predict which patients will derive the most value from these drugs while minimizing side-effects. Population level studies cannot provide the necessary insight into immune response heterogeneity at the single-cell level.

A New Approach Correlates Cell Therapy Potency to In Vivo Outcomes

IsoPlexis’ Polyfunctional Strength Index (PSI™) can be utilized to address these challenges, in particular to provide metrics that uniquely relate the CAR-T cell product response to in vivo preclinical and clinical outcome measures. Polyfunctional cells are recognized as key effector cells contributing to the development of potent and durable cellular immunity against viral infection, cancer, and other diseases.

To provide information about the sensitivity of the cellular response, the Isoplexis single-cell proteomics platform uniquely captures the full range of relevant cytokines from each immune cell connecting each cell to the many cytokines they secrete that orchestrate the immune system.

PSI is defined as the percentage of polyfunctional single cells in a sample that secretes two or more proteins, multiplied by the average signal intensity of the secreted proteins from individual functional groups from each cell. Each cell’s strength, across one thousand or more cells, is then aggregated and simplified to provide a comprehensible visualization of the potent cell subsets and the cytokine types that drive them.

The PSI metric has helped capture the potency of important and highly functional T-cell and other immune cell subsets. Using PSI, researchers have identified cytokine-based biomarkers, which objectively evaluate the quality of anti-tumor activity of CAR-T response, pre-therapy, in a manner that correlates with in vivo outcome.

This new approach helps researchers better understand how T cells functionally respond to immunotherapies by evaluating the CAR-T cell product quality in a scientifically rational manner. These types of data will enable more precise medicine and improve patient results in the future. PSI has also provided mechanistic insights to improve the decision-making process for choosing CAR-T cell and other immunotherapy lead candidates.

Discover: Revealing T-cell and Immunotherapy Mechanisms in Solid Tumors

Aldesleukin—recombinant Interleukin-2 (IL-2)—was the first cancer immunotherapy approved by the FDA for the treatment of metastatic renal cell carcinoma (1992) and metastatic melanoma (1998). However, the administration of high-dose IL-2 results in pleiotropic effects on the immune system including severe hypotension and vascular leak syndrome, which have significantly hampered its use.

IL-2 induces not only the desired expansion of tumor-killing CD8+ effector T cells, but also expands immunosuppressive CD4+ CD25+ regulatory T cells (Tregs), an undesirable side effect. To maximize the efficacy of IL-2 therapy while mitigating side effects, PEGylated IL-2 was generated and found to be profoundly more tolerable than its non-PEGylated counterpart.

PEGylated IL-2, NKTR-214, retains the same amino acid sequence as aldesleukin. The IL-2 core is conjugated to six releasable polyethylene glycol (PEG) chains. In the body, the PEG chains slowly release to generate active IL-2 conjugates to provide sustained signaling to preferentially activate and expand effector CD8+ T and NK cells over Tregs in the tumor.

At the AACR annual meeting in 2018, researchers from University of California Los Angeles, IsoPlexis, and Nektar Therapeutics presented a preclinical study on a pmel-1 mouse model, “Enhanced expansion and tumor targeting of adoptively transferred T cells with NKTR-214.” The research team examined the effects of IL-2 and NKTR-214 on the polyfunctionalities of T cells in both tumor infiltrating lymphocytes and the spleen. The study demonstrated that combination therapy with adoptive cell transfer (ACT) and NKTR-214 provided a robust antitumor response in the aggressive B16F10 melanoma model.

The PSI of the murine CD8+ T cells after ACT-NKTR-214 combination therapy was found to be significantly higher than the PSI of CD8+ T cells after ACT-IL-2 control treatment in both the spleen and tumor infiltrate. This type of insightful knowledge of T-cell potency of the treatment, driven by the PSI, which correlates to in vivo performance in mice, can be of crucial assistance in revealing lead choice in a variety of combination immunotherapy contexts.

Results suggest that PSI can help to reveal new mechanisms that enhance lead choice not only in immunotherapies against solid tumors but also when combining multiple complex immunotherapies to achieve eradication of tumors. With better understanding of the underlying mechanisms of combined immunotherapies, the design of immunotherapy strategies will improve, and further advances will be made to help treat and eradicate cancer.

Predict: Correlating Pre-Infusion Product to Clinical

Predicting patient response to cell therapy is a hurdle. Biomarkers that allow clinicians to stratify patients into responders and non-responders and identify those with an increased risk of adverse side effects, such as immunotoxicity, are needed. PSI is positioned to be a powerful predictive tool to determine objective response to cell therapy.

Isoplexis Figure 2
IsoPlexis’ systems uncover the polyfunctional subsets of cells which are contributors to response amongst the heterogeneity of patients. IsoPlexis’ unique ability to uncover differences in patient’s T-cell fitness uncovers the correlates to patient response, as published in Blood (2018).

In a 2018 publication in Blood by Rossi et al., pre-infusion CD4+ and CD8+ CAR-T cell samples from 20 of 22 patients in a CAR-T clinical trial were profiled. The study comprised a cohort of patients with aggressive refractory non-Hodgkin lymphoma. The PSI of the pre-infusion CAR-T cell products, which combined CD4+ and CD8+ CAR-T response, showed significant correlation with the objective response of patients. Specifically, the average PSI of the responder subgroup was more than twice as high as that of the non-responders, a difference that was statistically significant. PSI combined with CAR-T cell expansion or pretreatment serum IL-15 levels conferred additional significance.

PSI outperformed other pre-infusion metrics, including IFN-γ co-culture cytokine intensity, ratio of CD4+ to CD8+ T cells, and various T-cell phenotype frequencies, and was the only metric that statistically differentiated responding from non-responding patients. These results indicate the metric’s potential as a biomarker for guiding personalized CAR-T cell treatments and potentially predicting therapeutic efficacy.

While overall PSI correlated with clinical outcome, a study was undertaken to better understand which cellular and cytokine subsets of the CAR-T product PSI associated mostly strongly with outcome. Since CD8+ T cells are known to have effector and tumor-killing properties, it was hypothesized that the CD8+ subset would drive the increase in PSI.

However, the CD4+ CAR-T cell PSI was in fact more highly correlated with objective response of the patients than the CD8+ CAR-T cell PSI. Moreover, the higher PSI in responding patients’ CD4+ CAR-T samples was driven by multiple, non-redundant cytokines, including the effector/anti-tumor cytokines IFN-γ, and MIP-1α, stimulatory cytokine IL-8, and inflammatory cytokine IL-17A.

In addition, grade ≥3 cytokine release syndrome was associated with polyfunctional T cells, and both grade ≥3 neurologic toxicity and antitumor efficacy were associated with polyfunctional IL-17A–producing T cells suggesting that the combination of frequency and cytokine production levels of polyfunctional T cells in the product also are associated with toxicity resulting from treatment with CAR T cells.

This study showed the potential power of PSI as a pre-infusion biomarker for in vivo clinical outcome of CAR-T cell treatment in patients, where other tested metrics have not associated with outcome. Given the range of contributing cytokines noted, PSI is effective as an overall, aggregated cellular potency metric that does not require knowing the specific subsets that are most correlated with outcome. (For more information on the trial, visit www.clinicaltrials.gov and reference registration number #NCT00924326.)

PSI teases out subtle differences in response, further narrows down what drove differences in the index, and identifies additional biomarkers that strengthen the correlation with clinical outcome.

Optimize: Uncovering Subtle Differences in Bioprocessing for Manufacturing Optimization

As clinical applications for CAR-T cell therapy expand, cell manufacturing incorporating closed-system, automated instruments are supplanting traditional open-system, labor-intensive culture methods. One of these closed systems, the CliniMACS Prodigy (Miltenyi Biotec) was used to test the duration of T-cell activation/viral transduction, and total T-cell culture duration using an original and modified manufacturing method.

Data demonstrated that the modified manufacturing method which terminated T-cell activation/transduction by culture Day 3 resulted in reproducible and robust CAR-T cell production, even in relatively more sensitive patient cells. In addition, final product dose requirements were consistently met by culture Day 7 when using this method, augmenting process efficiency.

Specifically, the polyfunctional response to CD19 and CD22 antigen stimulation of CAR-T cell products generated was compared between the two methods. PSI revealed clear superiority of the new modified bispecific CAR-T manufacturing method in responding to CD19 and CD22 antigen stimulation as reported by Srivastava et al. at the 2018 American Society of Hematology Annual Meeting.

Both CD4+ and CD8+ samples produced with the modified method showed significant increases in PSI, clearly demonstrating that this method significantly improved the overall quality of the CAR-T cell products. Moreover, these data allowed a better understanding of the drivers of this polyfunctional upregulation for better characterization of the products, and demonstrated that PSI provides an effective method for characterizing cell therapy products and their potency.

A Powerful Unique Metric to Discover, Optimize, Predict

Isoplexis figure 3
IsoPlexis’ systems provide physiologically relevant readouts that empower decisions across the workflow from Lead Discovery, Optimizing Bioprocessing, and Biomarker correlates. These correlates then feed back into the funnel, driving more informed decisions through the drug development pipeline.

PSI is a powerful and unique tool with a wide range of applications in engineered immune cell therapy research and development. The metric can be used in preclinical discovery to understand correlations with in vivo response, in clinical outcome correlates, including the effectiveness of PSI as a potential pre-infusion biomarker for CAR-T cell therapy patient outcome, and product and manufacturing optimization, where PSI can reveal distinct differences in bioprocessing methods for process optimization.

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