Mesenchymal Stromal Cell Delivery of Oncolytic Immunotherapy Increased Polyfunctionality and Efficacy of CAR-T Cells: Published in Molecular Therapy

  • IsoPlexis’ technology demonstrates an association of enhanced tumor antigen-specific polyfunctionality of HER.2 CAR-T cells promoted by IL-12 and PD-L1-engineered adenoviruses-infected Mesenchymal stromal cells (CAd12_PD-L1 MSCs ) with in vitro and in vivo improved antitumor activity in solid tumor.
  • IsoPlexis single-cell functional phenotyping reveals augmented polyfunctionality of HER.2 CAR-T cells in response to tumor antigen, from 34% of the CD4+ and 40% of CD8 CAR-T cells in the CAd12_PD-L1 MSCs treatment group compared to 5% CD4 and 16% CD8+ CAR-T cells in the HER.2 only group.
  • Interestingly, the polyfunctional CD4+ HER.2 CAR-T cells showed the majority cytokine signatures of co-secreting IFN-g, Granzyme B, and CCL11, whereas polyfunctional CD8 HER.2 CAR-T cells had greater cytolytic activity with IFN-g, Granzyme B, TNF-a, and perforin co-secretions when coculture with tumor cells and CAd12_PD-L1 MSCs, which correlates with improved in vivo HER.2 CAR-T cell antitumor activity by CAd12_PD-L1 MSCs.
  • IsoPlexis single-cell functional proteomics provides the unique mechanistical insights for CAd12_PD-L1 MSCs in enhancing HER.2 CAR-T cell polyfunctionality with effector cytokines and demonstrates the combination treatment of oncolytic immunotherapy with CAR-T cell therapy in promoting superior antitumor functions for solid tumor.

Resolving Challenges of Oncolytic Immunotherapy Delivery to Disrupt the Tumor Microenvironment with Functional Single-Cell Proteomics

The complexities of the tumor microenvironment (TME) can limit the infiltration of CAR-T cells, and creating a barrier to effectively treating solid tumors with adoptive cell therapies. The TME produces immune-inhibitory ligands, cytokines, and chemokines, which inhibit cytotoxic T cell responses. Thus, the identification of therapies which can disrupt the TME while promoting functional drivers of T cell persistence is a significant goal of research in solid tumor. Oncolytic immunotherapy with engineered adenoviruses (OAd) can disrupt the TME and stimulate CAR-T cells, enhancing the host anti-tumor immune response. Unfortunately, intralesional administration of oncolytic virotherapy is challenging for many tumor locations, and intravenous administration is impossible in some cases due to patients’ innate or adaptive responses to the therapy. To overcome these challenges, cell carrier systems such as mesenchymal stromal cells (MSCs) can be used to deliver the OAd to tumors. A recent study in Molecular Therapy highlighted IsoPlexis’ single-cell proteomics in identifying improved methods for treating solid tumors.

To test the effects of treatment with combinatorial Ad vector (CAd) MSCs and clinically validated tumor-directed HER.2 CAR-T cells, McKenna, et al. infected MSCs with CAd and co-cultured the MSCs with two non-small cell lung cancer cell lines (A549, H1650).1 After 48 hours of co-culture, the researchers added either non-transduced (NTR) cells or CAR-T cells. Residual tumor amount was then quantified at 48, 72, and 96 hours post-treatment with the NTR or CAR-T cells. Compared to a control of group of cancer cells that were co-cultured with uninfected MSCs and without CAR-T cells, treatment with CAd MSCs combined with CAR-T enhanced CAR-T function, leading to a reduction in tumor cells.1 Additionally, compared to treatment with HER.2 CAR-T alone, the combination of CAd MSCs with HER.2 CAR-T cells significantly reduced tumor cell viability.

Single-Cell Secreted Proteomics Identifies Upregulated T Cell Polyfunctionality Associated with Improved Anti-Tumor Function of CAR-T Cells Treated with CAd-Infected MSCs

McKenna, et al. used IsoPlexis’ functional proteomics to investigate the mechanisms through which CAd-infected MSCs improve HER.2 CAR-T cell efficacy. The Single-Cell Secretome solution was used to measure the polyfunctionality of individual T cells. In previous studies, polyfunctionality has been associated with anti-tumor activity and has predicted clinical outcomes. IsoPlexis’ single-cell secreted proteomics found that CAd MSC-treated HER.2 CAR-T cells that were co-cultured with tumor cell lines were more polyfunctional than those that were not treated with CAd MSCs (34% and 40% of CD4 and CD8 cells, respectively). In comparison, the untreated HER.2 CAR-T cells had CD4 and CD8 polyfunctionality rates of only 5% and 16%. Infection with CAd MSCs was also associated with an increase in frequency of NTR CD8 T cells secreting proinflammatory cytokines.1 These results suggest that the combination therapy effectively enhanced the activity of HER.2 CAR-T cells by increasing the number of polyfunctional cells.

The IsoSpeak data informatics software was used to identify rare subsets of CD4 and CD8 populations. One subset of CD4 cells co-cultured with uninfected MSCs primarily secreted IL-8, while a second subset of CD4 cells that was co-cultured with CAd MSCs secreted up to 9 cytokines per cell, with the majority secreting IFN-γ, granzyme B, and CCL11 simultaneously. Thus, CAd MSCs increased the production of proinflammatory cytokines by CD4 CAR-T cells in response to the tumor cells. There was also a shift in CD8 CAR-T cell phenotype associated with treatment using CAd-MSCs. CD8 HER.2 CAR-T cells generally expressed proinflammatory cytokines following tumor exposure, but treatment with MSCs was not associated with phenotype. However, both CD4 and CD8 CAR-T cells treated with CAd MSCs showed enhanced secretion of CCL11, a chemokine associated with increased adhesion molecules and T cell chemotaxis.1  IsoPlexis’ functional single-cell proteomics revealed the mechanisms of anti-tumor activity through which the CAd-infected MSCs enhance the efficacy of CAR-T cells.

This anti-tumor activity correlated with the results from a subsequent in vivo mouse lung cancer model, demonstrating that IsoPlexis’ technology can provide insights with strong correlations to in vivo biology in less time. CAd MSCs combined with HER.2 CAR-T cells significantly reduced tumor growth compared to either treatment alone.1 While CAd MSCs alone did not reduce tumor expansion, the combination of CAd MSCs with the NTR T cell group prevented tumor growth from day 10 to day 22. Furthermore, only the mice that received both CAd MSCs and HER.2 CAR-T cells were able to prevent tumor growth upon second challenge with the same A549 tumor line.1 Analysis of blood, spleen, and lung T cells showed that CD8+ T cells in the CAd MSC plus CAR-T cell group exhibited reduced expression of the exhaustion marker Tim3 compared to the CAR-T only group, indicating that the CAd MSCs promoted T cell persistence. in vivo studies using an H1650 xenograft lung tumor model produced results that were consistent with the A549 line: combination treatment led to complete clearance of tumors within 2 weeks, with no negative effects observed.1

IsoPlexis Accelerates Unique in vivo Insights with Highly Multiplexed Functional Single-Cell Proteomics

The results of the study by McKenna, et al. revealed mechanisms behind anti-tumor activity with the unique combination of CAd MSCs and CAR-T. These accelerated insights correlated with in vivo biology and anti-tumor response in mouse models. Combination treatment improved T cell infiltration and enhanced effector cell function. Furthermore, combination treatment enhanced T cell polyfunctionality, and polyfunctionality was associated with improved anti-tumor activity. IsoPlexis uniquely revealed this with the Single-Cell Secretome solution, which highly multiplexes 32+ cytokines across thousands of single cells in tandem with walk-away automation, capturing a full range of functional proteins in less time than it takes traditional technologies to capture only a few. With minimal hands-on sample prep time and end-to-end automation of time-consuming workflow steps such as incubation, wash cycle, and data analysis, IsoPlexis’ platform for highly multiplexed single-cell and bulk volume multi-omics saves scientists valuable time and money, accelerating the clinical impact of research. The results of this study highlight a potentially powerful combination therapy for solid tumors and provide further validation for the use of the polyfunctionality metric to predict clinical outcomes and evaluate CAR-T product quality and function. IsoPlexis reveals mechanisms through unique functional data layers that are undetectable with other technologies, and in doing so accelerates insights into novel combination therapies that are proving to have high efficacy against difficult to treat tumors.

Read the full publication here.

Source:

  1. McKenna et al., Mesenchymal stromal cell delivery of oncolytic immunotherapy improves CAR-T cell antitumor activity, Molecular Therapy (2021)
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