Chimeric antigen receptor (CAR) T cell therapy has been successful in clinical trials against hematological cancers, but has experienced challenges in the treatment of solid tumors. One of the main difficulties lies in a paucity of tumor-specific targets that can serve as CAR recognition domains. We therefore focused on developing VHH-based, single-domain antibody (nanobody) CAR T cells that target aspects of the tumor microenvironment conserved across multiple cancer types. Many solid tumors evade immune recognition through expression of checkpoint molecules, such as PD-L1, that down-regulate the immune response. We therefore targeted CAR T cells to the tumor microenvironment via the checkpoint inhibitor PD-L1 and observed a reduction in tumor growth, resulting in improved survival. CAR T cells that target the tumor stroma and vasculature through the EIIIB+ fibronectin splice variant, which is expressed by multiple tumor types and on neovasculature, are likewise effective in delaying tumor growth. VHH-based CAR T cells can thus function as antitumor agents for multiple targets in syngeneic, immunocompetent animal models. Our results demonstrate the flexibility of VHH-based CAR T cells and the potential of CAR T cells to target the tumor microenvironment and treat solid tumors.

The clinical application of cytokine therapies for cancer treatment remains limited due to severe adverse reactions and insufficient therapeutic effects. Although cytokine localization by intratumoral administration could address both issues, the rapid escape of soluble cytokines from the tumor invariably subverts this effort. We find that intratumoral administration of a cytokine fused to the collagen-binding protein lumican prolongs local retention and markedly reduces systemic exposure. Combining local administration of lumican-cytokine fusions with systemic immunotherapies (tumor-targeting antibody, checkpoint blockade, cancer vaccine, or T cell therapy) improves efficacy without exacerbating toxicity in syngeneic tumor models and the BrafV600E/Ptenfl/fl genetically engineered melanoma model. Curative abscopal effects on noncytokine-injected tumors were also observed as a result of a protective and systemic CD8+ T cell response primed by local therapy. Cytokine collagen-anchoring constitutes a facile, tumor-agnostic strategy to safely potentiate otherwise marginally effective systemic immunotherapies.

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In non-small cell lung cancer (NSCLC), response to immune checkpoint blockade (ICB) is associated with programmed cell death ligand 1 expression that is induced by interferon--producing, tumor-infiltrating CD8 + T cells. However, not all tumors with a CD8 + T cell infiltrate respond to ICB, and little is known about the mechanisms governing ICB resistance in T cell-infiltrated NSCLC. We used an orthotopic NSCLC mouse model to study ICBrefractory CD8 + T cell responses. Single-cell RNA sequencing of the NSCLC mouse tumors revealed that lung cancerspecific tumor-infiltrating CD8 + T cells exhibited clonal expansion but lacked expression of genes associated with effector and exhausted T cell responses, indicating that they underwent a differentiation program distinct from conventional T cell exhaustion. This lung cancer-specific T cell dysfunction program was established early during priming in the mediastinal lymph node and was characterized by robust proliferation but a failed up-regulation of effector and exhausted T cell characteristics. Intriguingly, CD8 + T cells from patients with NSCLC expressed an analogous gene expression program, which appeared distinct from conventional T cell exhaustion. Administration of recombinant interleukin-2 (IL-2) and IL-12 was sufficient to restore effector T cell differentiation and induce control of KP lung tumors. These findings imply that a CD8 + T cell differentiation trajectory, activated during T cell priming in the mediastinal lymph node, limits the response of CD8 + T cells to ICB and thereby may contribute to failure of ICB in a subset T cell-infiltrated NSCLC.