SUMMARY Pancreatic ductal adenocarcinomas (PDA) erect physical barriers to chemotherapy and induce multiple mechanisms of immune suppression, creating a sanctuary for unimpeded growth. We tested the ability of T cells engineered to express an affinity-enhanced T cell receptor (TCR) against a native antigen to overcome these barriers in a genetically engineered model of autochthonous PDA. Engineered T cells preferentially accumulate in PDA and induce tumor cell death and stromal remodeling. However, tumor-infiltrating T cells become progressively dysfunctional, a limitation successfully overcome by serial T cell infusions that resulted in a near-doubling of survival without overt toxicities. Similarly engineered human T cells lyse PDA cells in vitro, further supporting clinical advancement of this TCR-based strategy for the treatment of PDA.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to most therapies, including immune checkpoint blockade. To elucidate mechanisms of immunotherapy resistance, we assessed immune parameters in resected human PDA. We demonstrate significant interpatient variability in T-cell number, localization, and phenotype. CD8+ T cells, Foxp3+ regulatory T cells and PD-1+ and PD-L1+ cells were preferentially enriched in tertiary lymphoid structures that were found in most tumors compared to stroma and tumor cell nests. Tumors containing more CD8+ T cells also had increased granulocytes, CD163+ (M2 immunosuppressive phenotype) macrophages, and FoxP3+ regulatory T cells. PD-L1 was rare on tumor cells, but was expressed by CD163+ macrophages and an additional stromal cell subset commonly found clustered together adjacent to tumor epithelium. The majority of tumoral CD8+ T cells did not express molecules suggestive of recent T-cell receptor (TCR) signaling. However, 41BB+PD-1+ T cells were still significantly enriched in tumors compared to circulation. Tumoral PD-1+ CD8+ T cells commonly expressed additional inhibitory receptors, yet were mostly T-bethi and Eomeslo, consistent with a less terminally exhausted state. Analysis of gene expression and rearranged TCR genes by deep sequencing suggested most patients have a limited tumor-reactive T-cell response. Multiplex immunohistochemistry revealed variable T-cell infiltration based on abundance and location, which may result in different mechanisms of immunotherapy resistance. Overall, the data support the need for therapies that either induce endogenous, or provide engineered, tumor-specific T-cell responses and concurrently relieve suppressive mechanisms operative at the tumor site.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapies, including immune-checkpoint blockade. We investigated two distinct strategies to modulate tumor-associated macrophages (TAM) to enhance cellular therapy targeting mesothelin in an autochthonous PDA mouse model. Administration of an antibody to colonystimulating factor (anti-Csf1R) depleted Ly6C low protumorigenic TAMs and significantly enhanced endogenous T-cell intratumoral accumulation. Despite increasing the number of endogenous T cells at the tumor site, as previously reported, TAM depletion had only minimal impact on intratumoral accumulation and persistence of T cells engineered to express a murine mesothelin-specific T-cell receptor (TCR). TAM depletion interfered with the antitumor activity of the infused T cells in PDA, evidenced by reduced tumor cell apoptosis. In contrast, TAM programming with agonistic anti-CD40 increased both Ly6C high TAMs and the intratumoral accumulation and longevity of TCR-engineered T cells. Anti-CD40 significantly increased the frequency and number of proliferating and granzyme B þ engineered T cells, and increased tumor cell apoptosis. However, anti-CD40 failed to rescue intratumoral engineered T-cell IFNg production. Thus, although functional modulation, rather than TAM depletion, enhanced the longevity of engineered T cells and increased tumor cell apoptosis, ultimately, anti-CD40 modulation was insufficient to rescue key effector defects in tumor-reactive T cells. This study highlights critical distinctions between how endogenous T cells that evolve in vivo, and engineered T cells with previously acquired effector activity, respond to modifications of the tumor microenvironment.

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