NEW YORK – In a recent study led by researchers at the University of California, Los Angeles, proteomics firm IsoPlexis demonstrated the capabilities of its expanded single-cell analysis platform.
In the work, which was detailed in a paper published last month in Nature Communications, scientists used the company's platform to profile the cytokines produced by both T cells and natural killer (NK) cells in the blood of melanoma patients receiving adoptive cell transfer (ACT) therapy.
This was the first published work applying the firm's IsoLight platform to cells beyond T cells, which has been its area of focus to date. IsoPlexis is currently expanding its offerings to target a variety of different immune cell types, said Sean Mackay, the company's co-founder and CEO.
The IsoLight system uses microchips featuring arrays of thousands of microchambers that isolate individual cells from samples of interest. These chambers are then sealed with a slide patterned with groups of antibodies in a number of different spatially isolated lines. This allows the researchers to identify proteins based on the color of fluorescence produced upon binding and the location on the slide where the binding event occurs. In this way, they can multiplex well beyond the levels allowed by fluorescence readout alone.
Thus far, IsoPlexis' published work has focused largely on measuring the cytokines produced by T cells in patients undergoing ACT therapy. In particular, researchers are using the IsoLight platform to explore how multifunctional T cells — defined as T cells expressing multiple cytokines and chemokines — impact the effectiveness of ACT treatment.
Studies in infectious disease have found that multifunctional T cells are more effective at controlling viral infections, which suggests that a similar dynamic could be at play in cancer immunotherapies. Research using IsoPlexis' technology has found evidence to support this notion. A 2018 study published in Blood, for instance, found that the polyfunctionality of T cells introduced to patients undergoing chimeric antigen receptor (CAR) T cell therapy for non-Hodgkin lymphoma correlates with treatment response.
In the Nature Communications work, researchers led by Antoni Ribas, director of the tumor immunology program at UCLA's Jonsson Comprehensive Cancer Center, were investigating whether a modified version of the cytokine interleukin-2 (IL-2) could improve patient response to ACT therapy.
IL-2 is commonly used in ACT therapy to drive expansion and function of the transferred T cells, however, IL-2 treatment has high toxicity and it stimulative effects are not long lasting. Additionally, the molecule is known to cause expansion of regulatory T cells, which have been shown to tamp down the immune response in the tumor microenvironment.
Looking to address these issues, Ribas and his colleagues tested whether a modified version of IL-2, called NKTR-214, might prove more effective at boosting the impact of ACT therapy. Testing the molecule in mouse models of melanoma, they found that mice treated with NKTR-214 had more polyfunctional T cells in their spleens and tumors than mice treated with unmodified IL-2.
They also looked at T cell and NK cell polyfunctionality in the blood of melanoma patients treated with NKTR-214 as part of a phase I trial and found that it boosted polyfunctionality in these cells, as well.
From IsoPlexis' perspective, the analysis of NK cells is significant in that it demonstrates the ability of its platform to look beyond T cells and to effectively analyze NK cells circulating in patient blood.
"This is the first time that has been published," Mackay said, adding that it marks an important step for the company's efforts to move into cancer immunology more broadly.
"A lot of the developers of cancer immunology drugs are focused on things beyond T cells, like natural killer cells and what the mechanistic impact of those cells are on the tumor response, as well as cells like macrophages and monocytes," he said. "While we are using the same core platform, the types of cells we are starting to analyze are becoming much more broad, and this is one of the first peer-reviewed publications to highlight some of that."
The NK cell analysis used the same cytokine chip that IsoPlexis has offered for T cell research. However, Mackay said the company is developing new cytokine chips specific for macrophages and monocytes that will better address those cells' specific cytokine repertoires.
The company is also releasing this quarter a panel aimed at bulk cell analysis of cytokines. While IsoPlexis has focused primarily on single-cell analysis, Mackay said it has received requests from customers interested in doing comparisons of single-cell and bulk cytokine profiles.
Later this year, the firm will expand beyond cytokine research with the release of chips for single-cell phosphoproteomic analysis. California Institute of Technology researcher James Heath, whose lab originally developed the technology underpinning the IsoLight platform, has published work demonstrating the system's potential for phosphoproteomic experiments. In a 2016 study, for instance, Heath and his colleagues found that the platform could detect changes in protein phosphorylation patterns in glioblastoma cells as early as 2.5 days after drug treatment.
Mackay said that many of the company's customers are interested in using phosphoproteomic analyses to better understand the mechanisms of resistance to cancer immunotherapies.
"They are trying to understand the pathways that lead to resistance and can they get in front of those resistance pathways, drug them, avoid them, so that the tumors have more difficulty escaping from immunotherapy," he said.
He said he also expects demand for the phosphoproteomic chips from researchers interested in profiling intracellular signaling in different immune cell types.
In January IsoPlexis closed an additional $20 million as part of its Series C financing round, bringing the total figure for the round to $45 million, funds that Mackay said it plans to use in these efforts to diversify its product portfolio.
Beyond IsoPlexis' technology, the Nature Communications study provides a broader look at the use of single-cell proteomics tools in cancer immunotherapy, where demand for such tools have been strongest thus far.
In addition to the IsoLight platform, the study also made use of Fluidigm's Helios mass cytometry platform to look at the immune cell populations in the spleens and tumors of mice treated with ACT therapy in combination with either NKTR-214 or IL-2.
"What we saw [in the NKTR-214 treated mice] was a huge expansion of T cells in the spleen and then migration to the tumor," said Giulia Parisi, a postdoctoral fellow in Ribas' lab and first author on the study.
Using the mass cytometry data they were able to further analyze the behavior of specific T cell subpopulations, finding three main groups, CD8 T cells, CD4 T cells, and regulatory T cells, and observing that the NKTR-214-treated mice saw upregulation of CD8 T cells versus regulatory T cells while the opposite happened in the group treated with IL-2.
Parisi said this indicated the improved effectiveness of NKTR-214 for boosting the ACT treatment.
"It's pretty well established not only in adoptive cell transfer but also in PD-1 or CAR T-cell therapy, you really want in your tumor microenvironment to have a good population of cytotoxic T cells and a low population of regulatory T cells," she said. "I think now we have a good understanding of which subset of cells are detrimental and which can help with a good immune response."
Parisi noted that the Helios was key to gathering this data in that it allowed the researchers to more finely characterize the cells present.
"You can have a panel with 20, 30, 40 antibodies, so you can have really good characterization of your sample," she said.