CXCL9/10-engineered dendritic cells promote T cell activation and enhance immune checkpoint blockade for lung cancer.

Immune checkpoint blockade (ICB) with PD-1/PD-L1 inhibition has revolutionized the treatment of non-small cell lung cancer (NSCLC). Durable responses, however, are observed only in a subpopulation of patients. Defective antigen presentation and an immunosuppressive tumor microenvironment (TME) can lead to deficient T cell recruitment and ICB resistance. We evaluate intratumoral (IT) vaccination with CXCL9- and CXCL10-engineered dendritic cells (CXCL9/10-DC) as a strategy to overcome resistance. IT CXCL9/10-DC leads to enhanced T cell infiltration and activation in the TME and tumor inhibition in murine NSCLC models. The antitumor efficacy of IT CXCL9/10-DC is dependent on CD4+ and CD8+ T cells, as well as CXCR3-dependent T cell trafficking from the lymph node. IT CXCL9/10-DC, in combination with ICB, overcomes resistance and establishes systemic tumor-specific immunity in murine models. These studies provide a mechanistic understanding of CXCL9/10-DC-mediated host immune activation and support clinical translation of IT CXCL9/10-DC to augment ICB efficacy in NSCLC.

Dendritic Cell Vaccination in Non-Small Cell Lung Cancer: Remodeling the Tumor Immune Microenvironment.

Non-small-cell lung cancer (NSCLC) remains one of the leading causes of death worldwide. While NSCLCs possess antigens that can potentially elicit T cell responses, defective tumor antigen presentation and T cell activation hinder host anti-tumor immune responses. The NSCLC tumor microenvironment (TME) is composed of cellular and soluble mediators that can promote or combat tumor growth. The composition of the TME plays a critical role in promoting tumorigenesis and dictating anti-tumor immune responses to immunotherapy. Dendritic cells (DCs) are critical immune cells that activate anti-tumor T cell responses and sustain effector responses. DC vaccination is a promising cellular immunotherapy that has the potential to facilitate anti-tumor immune responses and transform the composition of the NSCLC TME via tumor antigen presentation and cell-cell communication. Here, we will review the features of the NSCLC TME with an emphasis on the immune cell phenotypes that directly interact with DCs. Additionally, we will summarize the major preclinical and clinical approaches for DC vaccine generation and examine how effective DC vaccination can transform the NSCLC TME toward a state of sustained anti-tumor immune signaling.

CCL21-DC in situ vaccination in murine NSCLC overcomes resistance to immunotherapy and generates systemic tumor-specific immunity.

BACKGROUND: Despite recent advances in immunotherapy, many patients with non-small cell lung cancer (NSCLC) do not respond to immune checkpoint inhibitors (ICI). Resistance to ICI may be driven by suboptimal priming of antitumor T lymphocytes due to poor antigen presentation as well as their exclusion and impairment by the immunosuppressive tumor microenvironment (TME). In a recent phase I trial in patients with NSCLC, in situ vaccination (ISV) with dendritic cells engineered to secrete CCL21 (CCL21-DC), a chemokine that facilitates the recruitment of T cells and DC, promoted T lymphocyte tumor infiltration and PD-L1 upregulation. METHODS: Murine models of NSCLC with distinct driver mutations (KrasG12D/P53+/-/Lkb1-/- (KPL); KrasG12D/P53+/- (KP); and KrasG12D (K)) and varying tumor mutational burden were used to evaluate the efficacy of combination therapy with CCL21-DC ISV plus ICI. Comprehensive analyses of longitudinal preclinical samples by flow cytometry, single cell RNA-sequencing (scRNA-seq) and whole-exome sequencing were performed to assess mechanisms of combination therapy. RESULTS: ISV with CCL21-DC sensitized immune-resistant murine NSCLCs to ICI and led to the establishment of tumor-specific immune memory. Immunophenotyping revealed that CCL21-DC obliterated tumor-promoting neutrophils, promoted sustained infiltration of CD8 cytolytic and CD4 Th1 lymphocytes and enriched progenitor T cells in the TME. Addition of ICI to CCL21-DC further enhanced the expansion and effector function of T cells both locally and systemically. Longitudinal evaluation of tumor mutation profiles revealed that CCL21-DC plus ICI induced immunoediting of tumor subclones, consistent with the broadening of tumor-specific T cell responses. CONCLUSIONS: CCL21-DC ISV synergizes with anti-PD-1 to eradicate murine NSCLC. Our data support the clinical application of CCL21-DC ISV in combination with checkpoint inhibition for patients with NSCLC.

Inhibition of Granulocytic Myeloid-Derived Suppressor Cells Overcomes Resistance to Immune Checkpoint Inhibition in LKB1-Deficient Non-Small Cell Lung Cancer.

LKB1 inactivating mutations are commonly observed in patients with KRAS-mutant non-small cell lung cancer (NSCLC). Although treatment of NSCLC with immune checkpoint inhibitors (ICI) has resulted in improved overall survival in a subset of patients, studies have revealed that co-occurring KRAS/LKB1 mutations drive primary resistance to ICIs in NSCLC. Effective therapeutic options that overcome ICI resistance in LKB1-mutant NSCLC are limited. Here, we report that loss of LKB1 results in increased secretion of the C-X-C motif (CXC) chemokines with an NH2-terminal Glu-Leu-Arg (ELR) motif in premalignant and cancerous cells, as well as in genetically engineered murine models (GEMM) of NSCLC. Heightened levels of ELR+ CXC chemokines in LKB1-deficient murine models of NSCLC positively correlated with increased abundance of granulocytic myeloid-derived suppressor cells (G-MDSC) locally within the tumor microenvironment and systemically in peripheral blood and spleen. Depletion of G-MDSCs with antibody or functional inhibition via all-trans-retinoic acid (ATRA) led to enhanced antitumor T-cell responses and sensitized LKB1-deficent murine tumors to PD-1 blockade. Combination therapy with anti-PD-1 and ATRA improved local and systemic T-cell proliferation and generated tumor-specific immunity. Our findings implicate ELR+ CXC chemokine-mediated enrichment of G-MDSCs as a potential mediator of immunosuppression in LKB1-deficient NSCLC and provide a rationale for using ATRA in combination with anti-PD-1 therapy in patients with LKB1-deficient NSCLC refractory to ICIs. SIGNIFICANCE: These findings show that accumulation of myeloid-derived suppressor cells in LKB1-deficient non-small cell lung cancer can be overcome via treatment with all-trans-retinoic acid, sensitizing tumors to immunotherapy.

Novel Kras-mutant murine models of non-small cell lung cancer possessing co-occurring oncogenic mutations and increased tumor mutational burden.

Conditional genetically engineered mouse models (GEMMs) of non-small cell lung cancer (NSCLC) harbor common oncogenic driver mutations of the disease, but in contrast to human NSCLC these models possess low tumor mutational burden (TMB). As a result, these models often lack tumor antigens that can elicit host adaptive immune responses, which limits their utility in immunotherapy studies. Here, we establish Kras-mutant murine models of NSCLC bearing the common driver mutations associated with the disease and increased TMB, by in vitro exposure of cell lines derived from GEMMs of NSCLC [KrasG12D (K), KrasG12DTp53-/-(KP), KrasG12DTp53+/-Lkb1-/- (KPL)] to the alkylating agent N-methyl-N-nitrosourea (MNU). Increasing the TMB enhanced host anti-tumor T cell responses and improved anti-PD-1 efficacy in syngeneic models across all genetic backgrounds. However, limited anti-PD-1 efficacy was observed in the KPL cell lines with increased TMB, which possessed a distinct immunosuppressed tumor microenvironment (TME) primarily composed of granulocytic myeloid-derived suppressor cells (G-MDSCs). This KPL phenotype is consistent with findings in human KRAS-mutant NSCLC where LKB1 loss is a driver of primary resistance to PD-1 blockade. In summary, these novel Kras-mutant NSCLC murine models with known driver mutations and increased TMB have distinct TMEs and recapitulate the therapeutic vulnerabilities of human NSCLC. We anticipate that these immunogenic models will facilitate the development of innovative immunotherapies in NSCLC.