SUMMARY The high expression across multiple tumor types and restricted expression in normal tissues make B7-H3 an attractive target for immunotherapy. We generated chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR-Ts) and found that B7-H3.CAR-Ts controlled the growth of pancreatic ductal adenocarcinoma, ovarian cancer and neuroblastoma in vitro and in orthotopic and metastatic xenograft mouse models, which included patient-derived xenograft. We also found that 4–1BB co-stimulation promotes lower PD-1 expression in B7-H3.CAR-Ts, and superior antitumor activity when targeting tumor cells that constitutively expressed PD-L1. We took advantage of the cross-reactivity of the B7-H3.CAR with murine B7-H3, and found that B7-H3.CAR-Ts significantly controlled tumor growth in a syngeneic tumor model without evident toxicity. These findings support the clinical development of B7-H3.CAR-Ts.

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Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by a paucity of tumor-proximal CD8 þ T cells and resistance to immunotherapeutic interventions. Cancer-associated mechanisms that elicit CD8 þ T-cell exclusion and resistance to immunotherapy are not well-known. Here, using a Kras-and p53-driven model of PDA, we describe a mechanism of action for the protumorigenic cytokine IL35 through STAT3 activation in CD8 þ T cells. Distinct from its action on CD4 þ T cells, IL35 signaling in gp130 þ CD8 þ T cells activated the transcription factor STAT3, which antagonized intratumoral infiltration and effector function of CD8 þ T cells via suppression of CXCR3, CCR5, and IFNg expression. Inhibition of STAT3 signaling in tumor-educated CD8 þ T cells improved PDA growth control upon adoptive transfer to tumor-bearing mice. We showed that activation of STAT3 in CD8 þ T cells was driven by B cell-but not regulatory T cell-specific production of IL35. We also demonstrated that B cell-specific deletion of IL35 facilitated CD8 þ T-cell activation independently of effector or regulatory CD4 þ T cells and was sufficient to phenocopy therapeutic anti-IL35 blockade in overcoming resistance to anti-PD-1 immunotherapy. Finally, we identified a circulating IL35 þ B-cell subset in patients with PDA and demonstrated that the presence of IL35 þ cells predicted increased occurrence of phosphorylated (p)Stat3 þ CXCR3 À CD8 þ T cells in tumors and inversely correlated with a cytotoxic T-cell signature in patients. Together, these data identified B cell-mediated IL35/gp130/ STAT3 signaling as an important direct link to CD8 þ T-cell exclusion and immunotherapy resistance in PDA.

Pancreatic cancer is an aggressive malignancy, often diagnosed at metastatic stages. Several studies have implicated systemic factors, such as extracellular vesicle release and myeloid cell expansion, in the establishment of pre-metastatic niches in cancer. The Rab27a GTPase is overexpressed in advanced cancers, can regulate vesicle trafficking, and has been previously linked to non-cell autonomous control of tumor growth and metastasis, however, the role of Rab27a itself in the metastatic propensity of pancreatic cancer is not well understood. Here, we have established a model to study how Rab27a directs formation of the pre-metastatic niche. Loss of Rab27a in pancreatic cancer cells did not decrease tumor growth in vivo, but resulted in altered systemic myeloid cell expansion, both in the primary tumors and at the distant organ sites. In metastasis assays, loss of Rab27a expression in tumor cells injected into circulation compromised efficient outgrowth of metastatic lesions. However, Rab27a knockdown cells had an unexpected advantage at initial steps of metastatic seeding, suggesting that Rab27a may alter cell-autonomous invasive properties of the tumor cells. Gene expression analysis of gene expression revealed that downregulation of Rab27a increased expression of genes involved in epithelial-to-mesenchymal transition pathways, consistent with our findings that primary tumors arising from Rab27a knockdown cells were more invasive. Overall, these data reveal that Rab27a can play divergent roles in regulating pro-metastatic propensity of pancreatic cancer cells: by generating pro-metastatic environment at the distant organ sites, and by suppressing invasive properties of the cancer cells.

The high expression across multiple tumor types and restricted expression in normal tissues make B7-H3 an attractive target for immunotherapy. We generated chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR-Ts) and found that B7-H3.CAR-Ts controlled the growth of pancreatic ductal adenocarcinoma, ovarian cancer and neuroblastoma in vitro and in orthotopic and metastatic xenograft mouse models, which included patient-derived xenograft. We also found that 4-1BB co-stimulation promotes lower PD-1 expression in B7-H3.CAR-Ts, and superior antitumor activity when targeting tumor cells that constitutively expressed PD-L1. We took advantage of the cross-reactivity of the B7-H3.CAR with murine B7-H3, and found that B7-H3.CAR-Ts significantly controlled tumor growth in a syngeneic tumor model without evident toxicity. These findings support the clinical development of B7-H3.CAR-Ts. (B) Expression of B7-H3 in five human PDAC cell lines stained with the 376.96 mAb as assessed by flow cytometry. (C and D) PDAC cell lines labeled with GFP were co-cultured with NT, B7-H3.CAR-28z-Ts, or B7-H3.CAR-BBz-Ts at the T cell to tumor cell ratio of 1 to 5. On day 5, PDAC (GFP + ) and B7-H3.CAR-Ts (CD3 + ) were enumerated by flow cytometry. Representative flow-cytometry plots (C) and quantification of residual tumor cells (D) are illustrated (n = 4). Error bars denote SD. (E and F) Summary of IFN-g (E) and IL-2 (F) released by NT, B7-H3.CAR-28z-Ts, and B7-H3.CAR-BBz-Ts in the culture supernatant after 24 h of co-culture with the indicated cell lines as measured by ELISA (n = 4). Error bars denote SD. (G) The Raji cell line was engineered to express h4Ig-B7-H3 via retroviral gene transfer. Cells were sorted based on different expression level of 4Ig-B7-H3, and single clones were expanded. B7-H3 expression of the clones was assessed by staining with the 376.96 mAb and analyzed by flow cytometry. (H and I) Representative flow-cytometry plots of Raji cells (CD4 À CD8 À ) and T cells (CD4 + or CD8 + ) (H) and summary of residual tumor cells (I) 5 days after Raji wildtype (WT) and Raji 4Ig-B7-H3 clones were co-cultured with NT, CD19.CAR-Ts, B7-H3.CAR-28z-Ts, or B7-H3.CAR-BBz-Ts at 1 to 1 ratio (n = 5). Error bars denote SD; **p < 0.01, one-way ANOVA with Holm-Sidak test adjusted p value. See also Figure S1. (A) Schema of the PDAC orthotopic xenograft model infused with CAR-Ts on day 12 after tumor inoculation. (B) Representative bioluminescence images of FFluc-Panc-1 tumor growth in the PDAC orthotopic model shown in (A). (legend continued on next page) (C) Representative ultrasound imaging measurement of the FFluc-Panc-1 tumor on day 50 after tumor implantation in mice treated with CD19.CAR-Ts in the PDAC orthotopic model shown in (A). The arrow indicates the tumor. Dimensions of the tumor were 6.85 3 5.85 mm. (D) Bioluminescence kinetics of FFluc-Panc-1 tumor growth (5 mice/group) in the PDAC orthotopic model in (A). (E) Schema of the PDAC orthotopic xenograft model infused with CAR-Ts on day 28 after tumor inoculation. (F and...