Enhanced Efficacy of Simultaneous PD-1 and PD-L1 Immune Checkpoint Blockade in High-Grade Serous Ovarian Cancer.

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC. SIGNIFICANCE: This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Wan C ，Keany MP ，Dong H ，Al-Alem LF ，Pandya UM ，Lazo S ，Boehnke K ，Lynch KN ，Xu R ，Zarrella DT ，Gu S ，Cejas P ，Lim K ，Long HW ，Elias KM ，Horowitz NS ，Feltmate CM ，Muto MG ，Worley MJ Jr ，Berkowitz RS ，Matulonis UA ，Nucci MR ，Crum CP ，Rueda BR ，Brown M ，Liu XS ，Hill SJ ... -  《-》

Sensitizing tumors to anti-PD-1 therapy by promoting NK and CD8+ T cells via pharmacological activation of FOXO3.

Stimulating antitumor immunity by blocking programmed death-1 (PD-1) or its ligand (programmed death-ligand 1 (PD-L1) is a promising antitumor therapy. However, numerous patients respond poorly to PD-1/PD-L1 blockade. Unresponsiveness to immune-checkpoint blockade (ICB) can cast significant challenges to the therapeutic options for patients with hard-to-treat tumors. There is an unmet clinical need to establish new therapeutic approaches for mitigating ICB unresponsiveness in patients. In this study, we investigated the efficacy and role of low-dose antineoplastic agent SN-38 or metformin in sensitizing unresponsive tumors to respond to ICB therapy. We assessed the significant pathological relationships between PD-L1 and FOXO3 expression and between PD-L1 and c-Myc or STAT3 expression in patients with various tumors. We determined the efficacy of low-dose SN-38 or metformin in sensitizing unresponsive tumors to respond to anti-PD-1 therapy in a syngeneic tumor system. We deciphered novel therapeutic mechanisms underlying the SN-38 and anti-PD-1 therapy-mediated engagement of natural killer (NK) or CD8+ T cells to infiltrate tumors and boost antitumor immunity. We showed that PD-L1 protein level was inversely associated with FOXO3 protein level in patients with ovarian, breast, and hepatocellular tumors. Low-dose SN-38 or metformin abrogated PD-L1 protein expression, promoted FOXO3 protein level, and significantly increased the animal survival rate in syngeneic mouse tumor models. SN-38 or metformin sensitized unresponsive tumors responding to anti-PD-1 therapy by engaging NK or CD8+ T cells to infiltrate the tumor microenvironment (TME) and secret interferon-γ and granzyme B to kill tumors. SN-38 suppressed the levels of c-Myc and STAT3 proteins, which controlled PD-L1 expression. FOXO3 was essential for SN38-mediated PD-L1 suppression. The expression of PD-L1 was compellingly linked to that of c-Myc or STAT3 in patients with the indicated tumors. We show that SN-38 or metformin can boost antitumor immunity in the TME by inhibiting c-Myc and STAT3 through FOXO3 activation. These results may provide novel insight into ameliorating patient response to overarching immunotherapy for tumors.

Chung YM ，Khan PP ，Wang H ，Tsai WB ，Qiao Y ，Yu B ，Larrick JW ，Hu MC ... -  《Journal for ImmunoTherapy of Cancer》

CXCL13 shapes immunoactive tumor microenvironment and enhances the efficacy of PD-1 checkpoint blockade in high-grade serous ovarian cancer.

Most patients with high-grade serous ovarian cancer (HGSC) lack an effective response to immune checkpoint blockade, highlighting the need for more knowledge about what is required for successful treatment. As follicular cytotoxic CXCR5+CD8+ T cells are maintained by reinvigoration by immune checkpoint blockade in tumors, we attempted to reveal the relationship between CXCR5+CD8+ T cells and the tumor microenvironment to predict immunotherapy responses in HGSC. 264 patients with HGSC from two cohorts and 340 HGSC cases from The Cancer Genome Atlas cohort were enrolled. Ex vivo and in vivo studies were conducted with human HGSC tumors and murine tumor models. The spatial correlation between CXC-chemokine ligand 13 (CXCL13), CXCR5, CD8, and CD20 was evaluated by immunohistochemistry and immunofluorescence. Survival was compared between different subsets of patients using Kaplan-Meier analysis. The therapeutic effect of CXCL13 and programmed cell death-1 (PD-1) blockade was validated using human HGSC tumors and murine models. High CXCL13 expression was associated with prolonged survival. Tumors with high CXCL13 expression exhibited increased infiltration of activated and CXCR5-expressing CD8+ T cells. Incubation with CXCL13 facilitated expansion and activation of CXCR5+CD8+ T cells ex vivo. CXCR5+CD8+ T cells appeared in closer proximity to CXCL13 in tumors and chemotaxis towards CXCL13 in vitro. The combination of CXCL13, CXCR5, and CD8+ T cells was an independent predictor for survival. In addition, CXCL13 was associated with clusters of CD20+ B cells. CD20+ B cells predicted better patient survival in the presence of CXCL13. Histological evaluation highlighted colocalization of CXCL13 with tertiary lymphoid structures (TLSs). TLSs carried prognostic benefit only in the presence of CXCL13. CXCL13 in combination with anti-PD-1 therapy retarded tumor growth in a CD8+ T-cell-dependent manner, resulting in increased infiltration of cytotoxic CD8+ T cells and CXCR5+CD8+ T cells. These data define a critical role of CXCL13 in shaping antitumor microenvironment by facilitating the maintenance of CXCR5+CD8+ T cells in TLSs and support a clinical investigation for a combination of CXCL13 and PD-1 blockade therapy in HGSC.

Yang M ，Lu J ，Zhang G ，Wang Y ，He M ，Xu Q ，Xu C ，Liu H ... -  《Journal for ImmunoTherapy of Cancer》

Immunotyping in tubo-ovarian high-grade serous carcinoma by PD-L1 and CD8+ T-lymphocytes predicts disease-free survival.

PD-L1 immune checkpoint inhibitor expression was evaluated in high-grade serous carcinoma (HGSC) ovary in the context of the overall immune landscape to determine its prognostic value. Consecutive cases of HGSC, 50 who underwent upfront surgery followed by adjuvant chemotherapy (HGSC-U) and 50 who underwent neoadjuvant chemotherapy (NACT) followed by interval debulking surgery (HGSC-PC) were selected. In HGSC-PC cases, the pre-NACT ascitic fluid cell blocks were included. Tumor-infiltrating lymphocytes (TILs) were scored, hotspots chosen for tissue microarray construction and immunohistochemistry performed and scored for CD4 and CD8 lymphocyte subsets, CD68+ tumor-associated macrophages (TAMs), PD-1 and PD-L1 expression. HGSC-post-chemotherapy showed increased TILs, predominantly CD8+T-lymphocytes, compared to HGSC-U. HGSC showed PD-L1 expression on tumor cells and/or TAMs in 60% cases with a linear correlation to CD4+, CD8+ TIL levels. Concordant PD-L1 expression was seen in matched pre- and post-NACT tumor cells. HGSC-PC showed higher expression of PD-L1. There was no association of PD-L1 cumulative proportion score or tumor cell score with outcome. Taking a cutoff for PD-L1 CPS at 10%, immunotype I (PD-L1+/CD-8+), corresponding to tumors with adaptive immune evasion, showed worst disease-free survival compared to all other immunotypes (p = 0.03) and was more significant (p = 0.01) when compared to immunotype III (PD-L1+/CD8-). Immunotyping based on PD-L1/CD8+ expression correlates to prognosis and outcome.

Bansal A ，Srinivasan R ，Rohilla M ，Rai B ，Rajwanshi A ，Suri V ，Chandra Saha S ... -  《-》

The immune suppressive factors CD155 and PD-L1 show contrasting expression patterns and immune correlates in ovarian and other cancers.

We recently showed that tumors with an immunologically 'cold' phenotype are enriched for expression of stemness-associated genes and PVR/CD155, the ligand of the immunosuppressive molecule TIGIT. To explore the therapeutic implications of this finding, we investigated the relationship between PVR/CD155 expression, tumor-infiltrating lymphocytes (TIL), and prognosis in high-grade serous ovarian cancer (HGSC) and other cancers. Expression of CD155, TIGIT, PD-1, PD-L1, and other immune markers in HGSC was assessed by high-dimensional flow cytometry, multi-color histological imaging, and/or gene expression profiling. The prognostic significance of PVR/CD155 and CD274/PD-L1 expression was assessed bioinformatically in HGSC and 32 other cancers in The Cancer Genome Atlas. T cells from HGSC frequently co-expressed TIGIT and PD-1, and the ratio of TIGIT to PD-1 expression increased markedly after in vitro expansion with a clinically relevant protocol. CD155 was commonly expressed on malignant epithelium in HGSC and showed a negative or non-significant association with TIL. In contrast, PD-L1 was predominantly expressed by tumor-associated macrophages and positively associated with TIL. These contrasts between CD155 and PD-L1 were seen across HGSC patients, across metastatic sites within individual patients, and even within individual tumor deposits. PVR/CD155 and CD274/PD-L1 exhibited divergent prognostic associations across diverse cancer types in TCGA, including HGSC. CD155 and PD-L1 exhibit contrasting expression patterns, TIL associations and prognostic significance, suggesting they represent non-redundant immunosuppressive mechanisms. The CD155/TIGIT pathway represents a compelling immunotherapeutic target for HGSC and for immunologically cold tumors in general.

Smazynski J ，Hamilton PT ，Thornton S ，Milne K ，Wouters MCA ，Webb JR ，Nelson BH ... -  《-》