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How Single-Cell Functional Proteomics Accelerates Immunotherapy Development: Opportunities from Pre-Clinical to Clinical Stages

Many companies are racing to develop cancer immunotherapies–a market estimated to be worth $50 billion and expected to grow exponentially over the next several years. Some of the earliest such approved products have shown great promise, but one of the challenges to overcome is that these therapies have had mixed results in a limited number of patients.

Challenges Driving the Field

What makes a particular immunotherapy work on an individual? And how can we determine if a patient will respond to a certain treatment? In the age of targeted therapies, those are very basic questions, but they present big hurdles because most of the tools we have to measure biological differences between cells aren’t precise enough to accurately measure differences between single cells. Instead, they take an average from all the cells in a sample, masking the individual cell’s biology. A critical need that current technologies cannot provide is the ability to measure single cell function, revealing the most potent cell subsets which have correlated with outcome in multiple studies.

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Developing better methods to evaluate immunotherapies and correlate their levels to in vivo activity is crucial for this field. Single-cell functional proteomics will potentially make it easier to select the most promising candidate treatments, match them to the right patients, and combine them in the optimal fashion to get better results.

Single-cell functional proteomic analysis is among the most important advances in cancer treatment today. It is uniquely capable of discovering correlations to in vivo response in immunotherapeutic patients.

This technology addresses the core problem—that T cells, which play a major role in immunotherapies are heterogenous. There is strong evidence that the characteristics of unique subsets of cells determine the effectiveness and safety of such products. But until recently, little was known about what differentiates immune cell potency, cell product toxicity, and functional differences between patient responders and non-responders to immunotherapies.

“The ability of the IsoLight to provide detailed analyses from very few cells is key, given that we are limited to small sample quantities of blood.”

—Petter Brodin, Karolinska Institute†

†”IsoPlexis Launches First European Office & Initial IsoLight Single Cell Cytokine Detection System at Karolinksa Institute in Sweden.” Press Release 2019.

Single-cell functional proteomic analysis is a breakthrough technology which, for the first time, unlocks actionable potency, safety, and patient-difference metrics to help researchers fine-tune their immunotherapeutic candidates and figure out how to combine them. Scientists are finally able to correlate functional data from single cells with what is happening in vivo.

The effects are far-reaching, from lead selection to lead discovery, precision bioprocessing, and clinical biomarkers. IsoPlexis’ single-cell functional platform can determine the full range of cytokines secreted by individual cells. This technology is being used to uncover the functional protein profiles of individual cells and to guide therapeutic and biomarker development.

Next generation systems from IsoPlexis are the first to detect highly potent single-cell functional subsets. The company’s technology can measure the true cytokines secreted by single cells. Its system can run more than 1,000 cells on a single chip, which detects an unprecedented number of multiplexed cytokines per cell. The data are delivered via the company’s analytics software, IsoSpeak, which delivers advanced visualizations with ease, so researchers can focus on the most pressing questions they need to answer. IsoPlexis is working with many industry leaders in immunotherapy development, with numerous advances emerging from these studies. Their results address questions ranging from selection of lead immunotherapies, finding biomarkers for neuroinflammation and autoimmune reactions, and precision processes for manufacturing.

This report highlights how single-cell functional proteomic analysis can provide critical insights during pre-clinical, early clinical, and clinical studies. It will also specifically discuss the opportunities in two growing fields–cell therapies and checkpoint inhibitors.

The Introduction of Single-Cell Functional Proteomic Analysis

Cell therapy is a part of the new paradigm shift in personalized medicine. There is evidence that a range of factors influence therapy results, including patient disease characteristics, quality of T cells used, quality of assays used to predict efficacy and toxicity, and manufacturing processes. To address any of these factors we need better methods of studying these therapies and how they interact with immune biology.

“Quality assays are mainly descriptive with some objective parameters like cytokine production, cytotoxicity, and proliferation. Only recently, with PSI…can we start to differentiate products.”

—Marco L. Davila, Moffitt Cancer Center†

†”The Power of PSI in Evaluating CAR-T Therapy.” GENengnews. 2019.

The core challenge is that engineered T cell products are very heterogeneous, but the traditional methods for measuring proteins in cell samples (such as flow cytometry, bulk ELISA, and RNA seq) can only estimate overall levels of cytokines. They cannot distinguish the highly polyfunctional subsets of single cells, which are secreting many cytokines simultaneously within a sample.

Researchers first used the IsoPlexis platform in 2011 for quantification of T cell polyfunctional diversity via the simultaneous measurement of highly multiplexed effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs). The CTLs were actively responding to tumor and compared against a cohort of healthy donor controls.

They found “profound, yet focused, functional heterogeneity in active tumor antigen- specific CTL, with the major functional phenotypes quantitatively identified.”1

Since then, dozens of studies have documented the unparalleled power of the IsoPlexis single-cell proteomics platform in determining cellular fitness to accelerate development of immunotherapies from pre-clinical through clinical studies by correlating Polyfunctional Strength Index (PSITM) to response. Below, we highlight some of these studies.

Applications of Single-Cell Proteomic Analysis in Immunotherapy Development
Optimizing Lead Selection:

While chimeric antigen receptor (CAR) T cell and bispecific antibody immunotherapies have shown great promise as cancer treatments, the number of patients who benefit from these treatments is still relatively small. Traditional technologies have not been able to provide strong correlative data between these next generation therapies and patient response.

IsoPlexis’ single-cell proteomics system is now delivering the needed data by identifying polyfunctional cells and detecting the functional cytokines from each cell. The Polyfunctional Strength Index (PSITM) is derived by multiplying the number of cytokines secreted per cell with the amount of each cytokine, to identify the most potent immunotherapies.

“As a cancer research center with a key focus on CAR-T and other cell therapies, we feel that IsoPlexis will enable us to better characterize response and potentially predict whether cancer patients will respond to CAR-T therapy before treatment.”

—Christine Brown, City of Hope†

†”Early Access to IsoPlexis Single Cell Analytics Platform Enhances Cell Therapy Program at World Renowned Research and Treatment Center.” Press Release 2018.

PSI thus provides a new window on how T cells functionally respond to immunotherapies. Researchers have used it to find biomarkers and correlations between in vivo activity and protein levels for several years now, cementing its reputation as a breakthrough technology for guiding immunotherapy development.

Results from PSI help researchers better understand how T cells respond to immunotherapies and provides biomarkers and mechanistic insights to improve decision-making for choosing CAR-T cell and bispecific antibody lead candidates. As a result, researchers working on lead candidates to advance the next generation of cell therapies have identified cytokine-based biomarkers, which objectively evaluate the functional quality and potency of CAR-T cell response. These pre-therapy biomarkers have correlated with outcome.

For example, more than 90% of human tumors express ligands called natural killer group 2, member D (NKG2D) ligands (MICXA, MICB, ULBP1-6), but very little of these ligands is expressed in normal tissues. Researchers decided to explore whether targeting ligands can enhance antigen-specific T cell responses.3 They engineered bispecific T cell antibodies for these ligands, then used IsoPlexis’ platform to characterize the functional immune differences between therapies.

Results of this study (see Figure 1), showed that hNKG2D-OKT3 bispecifics induced a more potent polyfunctional T cell response than B2-OKT3 bispecific proteins from both CD4+ and CD8+ human T cells. Both profiles were dominated by effector cytokines. CD4+ and CD8+ cells show the same trend and similar functional profiles within their protein secretions. KG2D can recognize multiple ligands, including MICA, MICB, and ULBP1-6, whereas B2 bind only MICA. These results suggest that bispecifics capable of binding to multiple tumor antigens may yield better therapeutic effect.

Figure 1. Bispecific T cell antibodies were engineered for MICA (B2-OKT3, MICA x CD3) and NKG2D (hNKG2D-OKT3, NKG2D ligands x CD3). To test whether the engineered bispecific constructs could induce a potent antigen-specific T cell response, single cell PSI was computed for CD4+ and CD8+ T cells. hNKG2G-OKT3 was found to induce a more potent upregulation of Polyfunctional Strength Index (PSITM) than B2-OKT3 in both CD4+ and CD8+ cells by the activation of K562 cells. CD4+ and CD8+ cells show the same trend, with both profiles dominated by effector cytokine secretions.

Improving CAR-T therapy:

A distinguishing characteristic of cell therapy is utilizing immune cell function to make the treatment successful. There are limitations to traditional assays in evaluating the immune landscape within these therapies. Researchers and clinicians need better assays that can determine functional single-cell immune response, which have correlated to patient outcomes and toxicities.

Beyond determining cell fitness and potency, it is also important to detect signs of adverse effects, such as severe cytokine release syndrome (CRS) or neurotoxicity. Circulating blood levels of cytokines are monitored, but it would be advantageous to detect the signs of CRS and neurotoxicity earlier for the mitigation of these adverse effects.

IsoPlexis’ platform can extensively evaluate cells to provide a much more detailed and comprehensive profile of the cell therapy product and its effects in the patient. It provides a complete functional cytokine characterization of the CAR-T cell product and can potentially be used to better predict outcomes.

In another study, researchers correlated CAR-T cell product PSI with clinical outcome. These researchers evaluated CAR-T cell activity using IsoPlexis’ single-cell multiplexed cytokine analysis platform in lymphoma patients. This study also examined associations of a polyfunctional inflammation biomarker applied to CAR-T cell products, objective responses, and toxicities in non-Hodgkin lymphoma patients. The researchers found that highly polyfunctional T cells within CAR-T cell products were significantly associated with clinical response and that a subset of polyfunctional CD4+ cells producing IL-17A associated with neurotoxicity from CAR-T cell therapy.2

Combination Therapies:

Targeting the PD-1/PD-L1 pathway has led to tumor regression and long-term patient survival in clinical trials of a variety of cancers. Research also suggests that pairing anti-PD1/PD-L1 with anti-CTLA-4 are promising therapeutic combinations. These combinations have spurred the launch of more than 100 clinical trials in various stages. Immune checkpoint blockade combination therapies have shown remarkable success in treating multiple refractory cancers.

By revealing the mechanisms behind differences in response to combination immune therapies, IsoPlexis makes it possible to identify potential synergies while also identifying unwanted side effects. IsoPlexis’ single-cell protein analysis can thus provide data to reveal the mechanisms behind differences in response to combination immunotherapies. The discovery of highly functional CD8+ TILs (described below) reveals the functional diversity among these cells. These findings support further data-driven design and evaluation of new combination therapies.

“Current assays that rely on single-plex ELISA or even multiparametric flow cytometry don’t give you the level of resolution that the IsoPlexis platform can provide.”

—John Rossi, Kite Pharma†

†”2017 Top 10 Innovations.” The Scientist. 2017.

Solid tumors:

While immunotherapies have had unparalleled successes with blood cancers, challenges remain with solid tumors. Over time, it has become evident that to understand the mechanisms responsible for immunotherapy failure, it is necessary to uncover the functional immune interactions of immunotherapies at the single-cell level. In fact, a better understanding of this interaction could also inform patient to patient differences in response and adverse side effects.

Tumor-infiltrating T lymphocytes (TILs) are thought to correlate with patient survival. However, histology metrics, typically used to measure lymphocyte infiltration, have had mixed results in correlating with patient outcome. TILs are a highly heterogenous population, comprising unique subsets with different degrees of immune function. Cytokines and chemokines are particularly difficult to identify and detect in solid tumors with traditional methods, such as flow cytometry and bulk ELISA. However, researchers have uncovered key differences in response to immunotherapies using IsoPlexis’ multiplexed single-cell analysis, which measures over 30 key secreted proteins simultaneously from each live single cell, across more than 1,000 cells in parallel to produce a Polyfunctional Strength Index (PSITM). The PSI is computed as the percentage of polyfunctional cells (i.e., those with two or more proteins secreted per cell), multiplied by the mean fluorescence intensity of the proteins secreted by those cells. As noted earlier, PSI has been used to find key differences in response to immunotherapies across populations that are not detect- able through bulk analyses.

Per Figure 2, the PSI of CD8+ TILs can differentiate between patients who are responding to checkpoint immunotherapies and non-responding patients. Meanwhile, histology metrics, as mentioned above, did not reveal any difference between these two groups of patients in this study.

Figure 2. Single cell PSITM predicts clinical response to checkpoint inhibitor blockade therapy in patients with metastatic melanoma.

Bringing Immunotherapy to the Next Level

IsoPlexis’ single-cell system is helping to drive immunotherapy development through data that reveals the relationship between in vivo activity and levels of specific polyfunctional cytokines in unique subsets of cells. This alone is a breakthrough, but additionally, the capability is packaged with the bioinformatics software suite, IsoSpeak, which includes a fully automated workflow and bioinformatics pipeline. The single-cell mapping tools such as functional heatmaps, polyfunctional activation topology principal component analysis (PAT PCA), t-SNE, and PSI, allow exploration and reveal further insights about cellular activity across samples.

As IsoPlexis’ single cell system is incorporated from pre-clinical drug discovery and development through precision bioprocessing and manufacturing, and clinical biomarkers, we envision immunotherapeutic development becoming a predictable and productive field that leads to many industry firsts and an increasingly competitive market.

References
  1. Ma, C. et al. A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells. Nature Med. 2011;17:738-743.
  2. 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.
  3. Mackay, S. et al. Single-cell proteomic analysis of T cells stimulated by Bi-specific T cell Engagers shows a robust and unique polyfunctional secretion profile. ASH Annual Meeting, 2018

 

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