Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells that expand in tumor bearing hosts in response to soluble factors produced by tumor and stromal cells. MDSC expansion has been linked to loss of immune effector cell function and reduced efficacy of immune-based cancer therapies, highlighting the MDSC population as an attractive therapeutic target. Ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and IL2-inducible T-cell kinase (ITK), is in clinical use for the treatment of B cell malignancies. Here, we report that BTK is expressed by murine and human MDSCs, and that ibrutinib is able to inhibit BTK phosphorylation in these cells. Treatment of MDSCs with ibrutinib significantly impaired nitric oxide production and cell migration. In addition, ibrutinib inhibited in vitro generation of human MDSCs and reduced mRNA expression of indolamine 2,3-dioxygenase, an immunosuppressive factor. Treatment of mice bearing EMT6 mammary tumors with ibrutinib resulted in reduced frequency of MDSCs in both the spleen and tumor. Ibrutinib treatment also resulted in a significant reduction of MDSCs in wildtype mice bearing B16F10 melanoma tumors, but not in X-linked immunodeficiency mice (XID) harboring a BTK mutation, suggesting that BTK inhibition plays an important role in the observed reduction of MDSCs in vivo. Finally, ibrutinib significantly enhanced the efficacy of anti-PD-L1 (CD274) therapy in a murine breast cancer model. Together, these results demonstrate that ibrutinib modulates MDSC function and generation, revealing a potential strategy for enhancing immune-based therapies in solid malignancies.

mAbs are used to treat solid and hematologic malignancies and work in part through Fc receptors (FcRs) on natural killer cells (NK). However, FcR-mediated functions of NK cells from patients with cancer are significantly impaired. Identifying the mechanisms of this dysfunction and impaired response to mAb therapy could lead to combination therapies and enhance mAb therapy. Cocultures of autologous NK cells and MDSC from patients with cancer were used to study the effect of myeloid-derived suppressor cells (MDSCs) on NK-cell FcR-mediated functions including antibody-dependent cellular cytotoxicity, cytokine production, and signal transduction Mouse breast cancer models were utilized to study the effect of MDSCs on antibody therapy and test the efficacy of combination therapies including a mAb and an MDSC-targeting agent. MDSCs from patients with cancer were found to significantly inhibit NK-cell FcR-mediated functions including antibody-dependent cellular cytotoxicity, cytokine production, and signal transduction in a contact-independent manner. In addition, adoptive transfer of MDSCs abolished the efficacy of mAb therapy in a mouse model of pancreatic cancer. Inhibition of iNOS restored NK-cell functions and signal transduction. Finally, nonspecific elimination of MDSCs or inhibition of iNOS significantly improved the efficacy of mAb therapy in a mouse model of breast cancer. MDSCs antagonize NK-cell FcR-mediated function and signal transduction leading to impaired response to mAb therapy in part through nitric oxide production. Thus, elimination of MDSCs or inhibition of nitric oxide production offers a strategy to improve mAb therapy. .

Background Tumor-associated macrophages (TAM) are expanded and exhibit tumor-promoting properties within the tumor microenvironment. Current methods to study TAM have not been replicated across cancer types and often do not include exogenous growth factors from the tumor, a key factor in TAM differentiation in vivo. Methods In this study, an in vitro method to generate monocyte- derived TAM using tumor- conditioned media (TCM) and a cytokine cocktail containing IL-4, IL-10, and M-CSF was utilized to study the phenotype, morphology, and function of TAM across multiple cancer types. TCM was generated from two breast cancer cell lines and an Epstein-Barr virus-positive lymphoma cell line. The properties of in vitro generated TAM were compared to in vitro generated M1 and M2- like macrophages and TAM isolated from patients with cancer. Results TAM generated in this fashion displayed an increase in CD163/CD206 co-expression compared to M2- like macrophages (87 and 36%, respectively). TAM generated in vitro exhibited increased transcript levels of the functional markers IL-6, IL-10, CCL2, c-Myc, iNOS, and arginase compared to in vitro generated M2-like macrophages. Functionally, in vitro generated TAM inhibited the proliferation of T cells (47% decrease from M1-like macrophages) and the production of IFN-γ by natural killer cells was inhibited (44%) when co-cultured with TAM. Furthermore, in vitro generated TAM secreted soluble factors that promote the growth and survival of tumor cells. Conclusions Limited access to patient TAM highlights the need for methods to generate TAM in vitro. Our data confirm that monocyte-derived TAM can be generated reliably using TCM plus the cytokine cocktail of IL-4, IL-10, and M-CSF. Given the ability of TAM to inhibit immune cell function, continued study of methods to deplete or deactivate TAM in the setting of cancer are warranted. Electronic supplementary material The online version of this article (10.1186/s40425-019-0622-0) contains supplementary material, which is available to authorized users.

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fc␥ receptors (Fc␥R) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on Fc␥R-mediated activities in monocytes. We found that ibrutinib did not affect monocyte Fc␥R-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed Fc␥R-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased Fc␥R-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte Fc␥R-mediated cytokine production could be rescued by IFN␥ priming because NK cells produce IFN␥ in response to antibody therapy. Pretreatment of monocytes with IFN␥ abrogated the effects of ibrutinib on Fc␥R-mediated cytokine production, suggesting that IFN␥ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit Fc␥R-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.

Triple-negative breast cancer (TNBC) is a leading cause of breast cancer death and is often associated with and mutation. Due to the lack of validated target molecules, no targeted therapy for TNBC is approved. Tissue factor (TF) is a common yet specific surface target receptor for cancer cells, tumor vascular endothelial cells, and cancer stem cells in several types of solid cancers, including breast cancer. Here, we report evidence supporting the idea that TF is a surface target in TNBC. We used cancer lines and tumor xenografts in mice, all with or mutations, derived from patients' tumors. We showed that TF is overexpressed on TNBC cells and tumor neovasculature in 50% to 85% of TNBC patients ( = 161) and in TNBC cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) from mice, but was not detected in adjacent normal breast tissue. We then describe the development of a second-generation TF-targeting immunoconjugate (called L-ICON1, for lighter or light chain ICON) with improved efficacy and safety profiles compared with the original ICON. We showed that L-ICON1 kills TNBC cells via antibody-dependent cell-mediated cytotoxicity and can be used to treat human and murine TNBC CDX as well as PDX in orthotopic mouse models. Thus, TF could be a useful target for the development of immunotherapeutics for TNBC patients, with or without and mutations. .