Science Spotlight

Remodeling the tumor microenvironment for improved CAR T-cell therapy

From the Riddell and Srivastava Labs, Clinical Research Division

Adoptive transfer of engineered T cells is a strategy for producing potent anti-tumor immune responses in cancer patients. These therapies have produced dramatic clinical outcomes, particularly against blood-borne cancers. Applications of T-cell therapy against solid tumors, however, have faced several major hurdles, including poor trafficking of T cells to solid tumor sites, and suppression of therapeutic T-cell responses within the solid tumor microenvironment (TME). Dr. Shivani Srivastava, formerly a postdoctoral fellow in the Riddell Lab in the Clinical Research Division and now leading her own laboratory in the Human Biology Division at Fred Hutch, and colleagues developed an animal model for testing approaches to enhance the efficacy of T-cell therapies against lung cancer. Their work, recently published in Cancer Cell, reveals that immunogenic chemotherapy and checkpoint inhibitors combine favorably with engineered T-cell therapy by remodeling the TME, leading to improved tumor control.

Immunogenic chemotherapy activates tumor macrophages to recruit therapeutic T cells for enhanced efficacy of anti-tumor immunotherapies.
Immunogenic chemotherapy activates tumor macrophages to recruit therapeutic T cells for enhanced efficacy of anti-tumor immunotherapies. Image provided by Dr. Srivastava

Chimeric antigen receptors (CARs) are protein fusions consisting of antibody fragments linked to T cell receptor and co-receptor signaling domains, such that recognition of extracellular antigens delivers powerful stimulatory signals to the CAR T cells that express them. ROR1 is a surface protein found to be expressed at high levels in non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC), but not in vital adult tissues, making it an ideal therapeutic target. CAR T-cell therapies targeting ROR1 are currently being tested in clinical trials here at Fred Hutch, but early evidence indicates that CAR T cells exhibit poor infiltration and function within patient tumors. Dr. Srivastava adapted a genetically-engineered mouse model (GEMM) of NSCLC to study mechanisms of resistance to ROR1-targeted CAR T-cell therapy and test strategies for overcoming them.

To mimic human disease, Dr. Srivastava engineered murine NSCLC to express the ROR1 antigen. Using this system, the group observed that, despite conferring modest tumor control, adoptively-transferred ROR1-specific CAR T cells did not efficiently infiltrate tumors and acquired signs of dysfunction, similar to what they had observed in patients. Having established a faithful preclinical model, the investigators hypothesized that the poor efficacy of CAR T-cell therapy in this setting might be caused by suppressive factors within the TME. Oxaliplatin is a chemotherapeutic agent previously shown to induce ‘immunogenic cell death’, leading to innate immune cell activation and enhanced anti-tumor T-cell responses in similar NSCLC GEMMs. Whole tumor transcriptional analysis of ROR1+ lung tumors treated with oxaliplatin revealed signatures consistent with pro-inflammatory remodeling of the TME, including upregulation of genes involved in T-cell recruitment. Indeed, when they transferred CAR T cells into animals pre-treated with oxaliplatin, the therapeutic cells homed more efficiently to tumors.

To hone in on cell type-specific changes within the TME, the group performed single cell RNA sequencing followed by unsupervised clustering analysis on tumors in this system. CAR T cells were enriched in oxaliplatin-treated tumors and showed elevated activation-associated transcripts compared to CAR T cells prior to infusion, indicating that they were becoming activated in tumors. They also observed striking changes in macrophage phenotypes between the treatment groups. Namely, two clusters of macrophages appeared sequentially after oxaliplatin administration: cluster 2, expressing macrophage activation gene signatures, was predominant in tumors early after oxaliplatin administration and prior to CAR T cell infusion; cluster 1, expressing Nos2, which encodes the potent macrophage effector molecule nitric oxide synthase (iNOS) and is a marker of highly pro-inflammatory macrophages, was predominant in tumors 10 days after receiving both oxaliplatin and CAR T cells. Interestingly, the appearance of iNOS + macrophages was dependent on CAR T cell-derived IFNg, indicating cross-talk between these populations. The early- and late-appearing macrophage clusters expressed different sets of T cell-recruiting chemokines. Using a complement of chemokine receptor-deficient CAR T cells, they showed that cluster 2-derived chemokines were important for initial CAR T-cell recruitment to tumors, leading CAR T cells to produce IFNg and activate cluster 1 macrophages to produce distinct chemokines, which were responsible for later recruitment of CAR T cells to tumors.

Having characterized a complex interplay between the TME and therapeutic T cells following immunogenic chemotherapy, the group dissected the functional outcomes of this combination. Importantly, they observed high levels of a checkpoint receptor, programmed death 1 (PD-1), on CAR T cells and elevated expression of its ligand, PD-L1, on tumor macrophages after oxaliplatin, indicating that this could be a mechanism of CAR T-cell suppression in this setting. Indeed, administration of a PD-L1-blocking antibody increased the frequency of CAR T cells in oxaliplatin-pretreated tumors. Thus, they included PD-L1 blockade in their therapeutic regimen. In this context, oxaliplatin pre-treatment lead to increased tumor infiltration, lowered expression of inhibitory checkpoint molecules, and boosted cytokine production capacity by CAR T cells, ultimately leading to extended survival of tumor-bearing animals.

“In this work, we identified a chemotherapy regimen that activates production of T cell-recruiting chemokines in tumors, resulting in increased CAR-T cell infiltration into tumors and improved response to immune checkpoint blockade,” said Dr. Srivastava. “This is a strategy that can be readily translated to the clinic and, if successful, could be broadly applied to a number of different solid tumors as a general strategy to convert ‘cold’ tumors to ‘hot’ ones that are more permissive to T cell infiltration.”  Based on these observations, the authors were able to modify the protocol of the ROR1-targeted CAR T-cell clinical trial and obtain pre-and post-treatment biopsy samples from a TNBC patient who received oxaliplatin conditioning therapy. Satisfyingly, the biopsies revealed T-cell recruitment and changes within the macrophage compartment that were consistent with their observations in the preclinical system.

Moving forward, the newly minted Srivastava group will examine strategies for further improving outcomes of CAR T-cell therapy. Though they achieved prolonged survival in this study, “tumor-infiltrating CAR-T cells eventually lose function and all mice still ultimately succumb to disease,” explained Dr. Srivastava. “We are working on understanding what drives CAR-T cell dysfunction in the tumor microenvironment and developing ways to engineer CAR-T cells to potentially resist these inhibitory mechanisms. The goal is to combine strategies aimed at improving both CAR-T cell trafficking to and function within tumors in order to induce more durable tumor regression.”

This work was funded by the National Institutes of Health, Juno Therapeutics, and the Cancer Research Institute. 

UW/Fred Hutch Cancer Consortium members Shivani Srivastava, Scott Furlan, Jennifer Specht, Sylvia Lee, McGarry Houghton, Robert Pierce, Raphael Gottardo, David Maloney, and Stanley Riddell contributed to this work.

Srivastava S, Furlan SN, Jaeger-Ruckstuhl CA, Sarvothama M, Berger C, Smythe KS, Garrison SM, Specht JM, Lee SM, Amezquita RA, Voillet V, Muhunthan V, Yechan-Gunja S, Pillai SPS, Rader C, Houghton AM, Pierce RH, Gottardo R, Maloney DG, Riddell SR. Immunogenic Chemotherapy Enhances Recruitment of CAR-T Cells to Lung Tumors and Improves Antitumor Efficacy when Combined with Checkpoint Blockade. Cancer Cell. 2020 Dec 1:S1535-6108(20)30597-3.doi: 10.1016/j.ccell.2020.11.005. 

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