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. .

The rates of duplex formation for two octamers of DNA (5' d-CACGGCTC/5' d-GAGCCGTG and 5' d-CACAGCAC/5' d-GTGCTGTG), the homologous RNA, and both sets of hybrids in 1 M NaCl buffer have been measured using stopped-flow spectroscopy. In addition, the thermodynamic parameters, ΔH° and ΔS°, have been determined for the same sequences under the same buffer conditions using optical melting techniques. These data reveal a linear free energy relationship between the free energy of activation for denaturation and the change in free energy for formation of the duplexes. This relationship indicates that these duplex formation reactions occur through a common unstructured transition state that is more similar to the single strands in solution than to the ensuing duplex. In addition, these data confirm that the greater stability of RNA duplexes relative to that of homologous DNA and hybrid duplexes is controlled by the denaturation rate and not the duplex formation rate.

Regulatory T (Treg) cells are important suppressive cells among tumor-infiltrating lymphocytes (TIL). Treg cells express the well-known immune checkpoint receptor PD-1, which is reported to mark "exhausted" Treg with lower suppressive function. T-cell immunoglobulin mucin (Tim)-3, a negative regulator of Th1 immunity, is expressed by a sizeable fraction of TIL Tregs, but the functional status of Tim-3 Tregs remains unclear. CD4CTLA-4CD25 Treg cells were sorted from freshly excised head and neck squamous cell carcinoma (HNSCC) TIL based on Tim-3 expression. Functional and phenotypic features of these Tim-3 and Tim-3 TIL Tregs were tested by o suppression assays and multi-color flow cytometry. Gene-expression profiling and NanoString analysis of Tim-3 TIL Treg were performed. A murine HNSCC tumor model was used to test the effect of anti-PD-1 immunotherapy on Tim-3 Treg. Despite high PD-1 expression, Tim-3 TIL Treg displayed a greater capacity to inhibit naïve T-cell proliferation than Tim-3 Treg. Tim-3 Treg from human HNSCC TIL also displayed an effector-like phenotype, with more robust expression of CTLA-4, PD-1, CD39, and IFN-γ receptor. Exogenous IFN-γ treatment could partially reverse the suppressive function of Tim-3 TIL Treg. Anti-PD-1 immunotherapy downregulated Tim-3 expression on Tregs isolated from murine HNSCC tumors, and this treatment reversed the suppressive function of HNSCC TIL Tregs. Tim-3 Treg are functionally and phenotypically distinct in HNSCC TIL, and are highly effective at inhibiting T-cell proliferation despite high PD-1 expression. IFN-γ induced by anti-PD-1 immunotherapy may be beneficial by reversing Tim-3 Treg suppression. .

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. .