The success of cell-based immunotherapies is transforming precision medicine, even though the full clinical potential of these therapies has yet to be exploited. Cell therapies are highly complex, heterogeneous, edited living drugs making their behavior tricky to characterize, predict and optimize.
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, making it virtually impossible to reproducibly develop an identical “drug”. Having the ability to drive better outcomes by profiling the complete function of each cell would be a significant advantage for bioengineers—a challenge the IsoPlexis single-cell proteomic system has been designed to address.
In a recent Genetic Engineering & Biotechnology News webinar, Jonathan Chen, co-inventor and Director of New Collaborations at IsoPlexis, and James R. Heath, Ph.D., President of the Institute for Systems Biology and IsoPlexis’ co-founder, discussed the history of the technology and how it has begun to redefine what is possible by stratifying patient response to novel immunotherapy regimens for cancer, autoimmune and infectious diseases.
To view the on demand webinar, visit:
Single Cell Proteomics and the Immune System
Over the past decade, Dr. Heath’s research team has developed single-cell characterization methods that led to the formation of the IsoPlexis technology. The Polyfunctional Strength Index (PSI™) has progressed from an initial report of an interesting metric to a compelling demonstration of the detailed resolution of effective therapies and responses.
The 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 aggregated and simplified to provide a comprehensible visualization of the potent cell subsets and the cytokine types that drive them.
Most immunotherapy revolves around T cells. But immune system monitoring is deceptively tricky. Current technologies are inadequate to assess critical quality, function and variability attributes that drive efficacy, potency, and safety especially as the mechanism for both clinical effects and cytotoxicity can be heavily mediated by cytokines (functional proteins through which immune cells send and receive signals; tracking them to the cells that secrete them is not a simple endeavor).
Take cancer immunotherapy as an example: blood contains many proteins and immune cell phenotypes, of which anti-tumor immune cells make up a small subset. Even important cell subsets, such as effector T cells, have a large clonal diversity. The few clones that might have a therapeutic impact are mixed along with all other cells, illustrating the need for a single cell technology to locate the cells of interest.
The secreted cytokines reflect immune cell functions. In an immunotherapy, a T cell is tasked to target and kill cancer cells, recruit other immune cells of specific types, promote or mediate inflammation, replicate, or induce other immune cells to replicate—upwards of about 20 distinct functions. The challenge is to monitor different T-cell populations along with their functional and anti-tumor performance. Phenotyping, for example using flow cytometry, only provides for population statistics, not functional performance.
A single cell secretes few copies of a protein. To address this technical hurdle, the IsoPlexis microchip contains a parallelized series of microchambers that are encoded with a full copy of a multiplexed antibody array. This allows single cells to be captured and the small number of multiple protein copies secreted to become sufficiently concentrated for their automatic measurement with a standard immunofluorescence sandwich assay based on antibody capture and detection.
Using a straightforward workflow, the technology isolates thousands of immune cells and identifies polyfunctional cells (that secrete multiple cytokines). 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.
Data through Time
Early single-cell proteomics data from melanoma patients treated with engineered T cells that had MART-1 TCR specificity revealed high functional heterogeneity of MART-1 cytolytic T lymphocytes despite the cells being homogenous in phenotypes.1 Polyfunctionality was confined to a small subgroup of cells.
The cytokine panel was expanded and melanoma patients re-evaluated over the course of a clinical trial.2 After deconstructing the secreted proteins into biological function buckets, this study showed that the secreted protein response from tumor-antigen-specific T cells was highly coordinated, and that 5-10% of the T cells were polyfunctional and dominated the anti-tumor immune response.
T cells can perform 20-30 separate functions; the most effective ones perform all or most of the functions whereas the least effective may not perform any functions. A simple metric was developed to understand the data. This metric has evolved to become the PSI.
In 2017, Dr. Rong Fan from Yale University and an IsoPlexis co-founder, along with scientists at IsoPlexis, tested the index on samples from various CAR-T studies. They found a correlation of the single-cell PSI with patient response and non-response. To date, PSI has been repeated used to assess the function of the immune response in multiple clinical trials focusing on immunotherapy, autoimmune and inflammatory diseases, and vaccine development.
Through its innovative technology, IsoPlexis has democratized PSI measurements and increased the cytokine panel size to approximately 40, allowing for higher data multiplexing. Importantly, all data underlying the PSI calculation, the protein secretion signature from each individual cell, are retained and allow for deeper dives into how cells evolve over time and a history of unique cytokine contribution.
In a 2018 publication in Blood, pre-infusion CD4+ and CD8+ CAR-T cell samples from 20 of 22 non-Hodgkin lymphoma patients in a CAR-T clinical trial were profiled.3 This study revealed that PSI could be used as a quality-control metric for CAR-T cells to predict response. No other immune profiling metrics provided the same type of deep useful data.
This study also uncovered unexpected findings. CD4+ T-cells with definitive cytokine signatures were more associated with response relative to CD8+ T-cells. Additionally, it was found that levels of IL-15 and product PSI acted synergistically for an even more compelling metric to predict response.
At the 2018 American Association for Cancer Research annual meeting, Parisi et al. presented a preclinical study on a pmel-1 mouse model examining the effects of IL-2 and NKTR-214, a PEGylated IL-2, on the polyfunctionalities of T cells. PSI demonstrated that combination therapy with adoptive cell transfer and NKTR-214 provided a robust antitumor response in the aggressive B16F10 melanoma model.4
The metric was also used to detect subtle differences in CAR-T bioprocessing methods. These 2018 data by Srivastava et al. demonstrated that a modified manufacturing method significantly improved the overall quality of CAR-T cell products and allowed a deeper understanding of the drivers of this polyfunctional upregulation for better cell product characterization.5
In discovery and optimization, potent polyfunctional cell subsets have revealed key differences between therapeutic options in combination cell therapy, and cell therapy bioprocessing methods. In the future, PSI will certainly be applied to a host of other large clinical problems and be built upon to increase its utility even further.
1. C. Ma et al. A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells, Nat Med. 2011 Jun;17(6):738-43. doi: 10.1038/nm.2375
2. C. Ma et al., Multifunctional T-cell analyses to study response and progression in adoptive cell transfer immunotherapy, Cancer Discov. 2013 Apr;3(4):418-29. doi: 10.1158/2159-8290.CD-12-0383. Epub 2013 Mar 21
3. J. Rossi et al., Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL, Blood. 2018 Aug 23; 132(8): 804–814. doi: 10.1182/blood-2018-01-828343: 10.1182/blood-2018-01-828343
4. G. Parisi et al., Enhanced expansion and tumor targeting of adoptively transferred T cells with NKTR-214, 2018 American Association for Cancer Research annual meeting
5. S. Srivastava et al., Abbreviated T-cell activation on the automated Clinimacs Prodigy device enhances bispecific CD19/22 Chimeric Antigen Receptor T-cell viability and fold expansion, reducing total culture duration, 2018 American Society of Hematology annual meeting