Chemokines expressed by engineered bacteria recruit and orchestrate antitumor immunity

Abstract

Tumors use multiple mechanisms to actively exclude immune cells involved in antitumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16^K42A) offer therapeutic benefit in multiple mouse tumor models, an effect mediated via recruitment of CD8⁺ T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells, using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16^K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive antitumor immune responses, offering a new cancer immunotherapy strategy.

INTRODUCTION

Surmounting the obstacles tumors use to suppress immune cell infiltration has proven to be an elusive target. Immune infiltration, particularly by CD8⁺ cytotoxic T lymphocytes and, more specifically, memory CD8⁺ T cells, within tumors is widely considered a positive prognostic factor (1–4). Immune cells undergo chemotaxis in response to chemokines (5, 6); however, although chemokines may be an attractive therapeutic target in cancer (7), conventional drug delivery methods fail to generate tumor-localized chemokine gradients that sufficiently overcome immune cell exclusion (8, 9). Thus, despite the importance of tumor infiltration by immune cells in treatment outcomes, new approaches are needed to actively attract leukocytes into the tumor microenvironment.

The chemokine CXCL16 was first identified for its ability to recruit activated T cells with extralymphoid homing potential (10, 11). Although some reports have suggested that tumor cell–derived CXCL16 may contribute to tumor invasion and metastasis (12–14), CXCL16 and its receptor, CXCR6 (also known as Bonzo), are positively correlated with T cell infiltration and with increased survival in patients with colon and lung cancer (15, 16). Moreover, recent reports suggest that the CXCL16/CXCR6 axis plays a critical role in generating antitumor immunity and that the manipulation of this axis has therapeutic potential (17–19). However, a method to specifically deliver CXCL16 to the tumor microenvironment to recruit activated T cells has not been available, making the assessment of its potential for directly manipulating antitumor T cell responses in vivo difficult to discern.

Bacteria are increasingly recognized as a component of the tumor microenvironment, with genetic manipulations of bacteria enabling renewed consideration for bacterial cancer therapy (20–22). A previously generated synthetic gene circuit in bacteria, termed the synchronized lysis circuit (SLC), enables repeated delivery of tumor-localized therapeutics following synchronized lysis (23–25). Here, we first use the SLC and engineer bacteria that produce tumor-localized CXCL16 to recruit activated CD8⁺ T cells and promote antitumor immunity. We then combine the expression of CXCL16 with CCL20 to recruit innate and adaptive immune cells involved in both the priming and response phases of tumor immunity, augmenting the overall antitumor immune response and enhancing therapeutic efficacy, a heretofore unavailable therapeutic approach.

RESULTS

Mutated human CXCL16 variant in probiotic Escherichia coli has bioactivity in vitro

We first hypothesized that SLC-mediated release of CXCL16 in tumors would promote infiltration of activated T cells and support antitumor immunity. Toward this aim, we expressed the bioactive mature region (Asn³⁰-Pro¹¹⁸) of human CXCL16 (hCXCL16) on a previously described (25) high–copy number plasmid in the probiotic E. coli Nissle 1917 (EcN) and confirmed that the release of hCXCL16 was SLC dependent (Fig. 1A). The release of hCXCL16 in tumors in vivo was also SLC dependent, with minimal detection in tumor homogenates from untreated tumors or those treated with EcN expressing hCXCL16 without SLC (−SLC), but with significantly greater detection when SLC was present (+SLC) (Fig. 1B). To identify the optimal variant of CXCL16, we generated mouse CXCL16 (mCXCL16) and previously described (26) activating (hCXCL16^K42A) and inactivating (hCXCL16^R73A) mutants of human CXCL16 (Fig. 1C). For functional assessment of the probiotic-derived CXCL16 variants, we developed a chemotaxis assay in which activated T cells were assayed for their migration in response to the lysate of wild-type or variant-expressing EcN strains. Compared to wild-type lysate, activated mouse CD4⁺ and CD8⁺ T cells significantly migrated in response to lysates of EcN strains expressing mCXCL16 and activating hCXCL16^K42A but not wild-type hCXCL16 or inactivating hCXCL16^R73A (Fig. 1D). Consistent with previous characterization of the hCXCL16^K42A mutation (26), activated human T cells displayed a similar response to the lysate of the hCXCL16^K42A-expressing EcN strain (fig. S1). These data suggest that E. coli–derived hCXCL16^K42A potently attracts mouse and human activated T cells, potentially easing translational and preclinical studies and warranting an assessment of this strain in vivo using murine tumor models…