mRNA vaccine trafficking and resulting protein expression after intramuscular administration.

The mRNA vaccine route from injection site to critical immunologic tissues, as well as the localization of protein antigen following intramuscular (i.m.) administration, is crucial to generating an effective immune response. Here, we quantified mRNA at the injection site, lymph nodes, and in select tissues. mRNA was primarily present 24 h after administration and then rapidly degraded from local and systemic tissues. Histological analyses of mRNA and expressed protein at the site of administration and in the lymph nodes following i.m. administration of our vaccine in rodents and nonhuman primates (NHPs) were completed, and mRNA and protein expression were detected in tissue resident and infiltrating immune cells at the injection site. In addition, high levels of protein expression were observed within subcapsular and medullary sinus macrophages in draining lymph nodes. More important, results were similar between rodents and NHPs, indicating cross-species similarities.

Evaluating the efficacy of a priming dose of cyclophosphamide prior to pembrolizumab to treat metastatic triple negative breast cancer.

PURPOSE: Triple negative breast cancer (TNBC) is characterized by the presence of immune cells in the tumor microenvironment, however, the response to single-agent immune checkpoint inhibitor (ICI) therapy is modest. Preclinical models have demonstrated that intratumoral regulatory T cells (Tregs) dampen the antitumor response to ICI. We performed a single-arm phase II trial to evaluate the efficacy of a single low dose of cyclophosphamide (Cy) to deplete Tregs administered before initiating pembrolizumab. PATIENTS AND METHODS: 40 patients with pretreated metastatic TNBC were enrolled. The primary endpoints were progression-free survival (PFS) and change in peripheral blood Tregs after Cy. Secondary endpoints included overall response rate (ORR), duration of response, overall survival, treatment-related adverse events (AEs), and correlative evaluations. RESULTS: Median PFS was 1.8 months, and the ORR was 21%. Tregs were not significantly decreased after Cy prior to ICI (-3.3%, p=0.19), and increased significantly after the first cycle of therapy (+21% between cycles 1 and 2, p=0.005). Immune-related AEs were similar to historical pembrolizumab monotherapy, and were associated with response to therapy (p=0.02). Patients with pretreatment tumors harboring increased expression of B cell metagene signatures and increased circulating B cell receptor repertoire diversity were associated with clinical response and immune-related toxicity (IRT). CONCLUSIONS: Among patients with heavily pretreated TNBC, Cy prior to pembrolizumab did not significantly deplete Tregs, and in those with decreased numbers there was rapid recovery following therapy. Increased B cell gene expression in baseline samples was associated with clinical response and IRT.

Restricted myeloperoxidase epitopes drive the adaptive immune response in MPO-ANCA vasculitis.

BACKGROUND: Treatment of autoimmune diseases has relied on broad immunosuppression. Knowledge of specific interactions between human leukocyte antigen (HLA), the autoantigen, and effector immune cells, provides the foundation for antigen-specific therapies. These studies investigated the role of HLA, specific myeloperoxidase (MPO) epitopes, CD4+ T cells, and ANCA specificity in shaping the immune response in patients with anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. METHODS: HLA sequence-based typing identified enriched alleles in our patient population (HLA-DPB1*04:01 and HLA-DRB4*01:01), while in silico and in vitro binding studies confirmed binding between HLA and specific MPO epitopes. Class II tetramers with MPO peptides were utilized to detect autoreactive CD4+ T cells. TCR sequencing was performed to determine the clonality of T cell populations. Longitudinal peptide ELISAs assessed the temporal nature of anti-MPO447-461 antibodies. Solvent accessibility combined with chemical modification determined the buried regions of MPO. RESULTS: We identified a restricted region of MPO that was recognized by both CD4+ T cells and ANCA. The autoreactive T cell population contained CD4+CD25intermediateCD45RO+ memory T cells and secreted IL-17A. T cell receptor (TCR) sequencing demonstrated that autoreactive CD4+ T cells had significantly less TCR diversity when compared to naïve and memory T cells, indicating clonal expansion. The anti-MPO447-461 autoantibody response was detectable at onset of disease in some patients and correlated with disease activity in others. This region of MPO that is targeted by both T cells and antibodies is not accessible to solvent or chemical modification, indicating these epitopes are buried. CONCLUSIONS: These observations reveal interactions between restricted MPO epitopes and the adaptive immune system within ANCA vasculitis that may inform new antigen-specific therapies in autoimmune disease while providing insight into immunopathogenesis.

Inhibition of Wild-Type p53-Expressing AML by the Novel Small Molecule HDM2 Inhibitor CGM097.

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient-derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML.

Lenalidomide Enhances Immune Checkpoint Blockade-Induced Immune Response in Multiple Myeloma.

PURPOSE: PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell-mediated antitumor immune responses. Here, we assessed the impact of single and dual blockade of PD-1/PD-L1, alone or in combination with lenalidomide, on accessory and immune cell function as well as multiple myeloma cell growth in the bone marrow (BM) milieu. EXPERIMENTAL DESIGN: Surface expression of PD-1 on immune effector cells, and PD-L1 expression on CD138(+) multiple myeloma cells and myeloid-derived suppressor cells (MDSC) were determined in BM from newly diagnosed (ND) multiple myeloma and relapsed/refractory (RR) multiple myeloma versus healthy donor (HD). We defined the impact of single and dual blockade of PD-1/PD-L1, alone and with lenalidomide, on autologous anti-multiple myeloma immune response and tumor cell growth. RESULTS: Both ND and RR patient multiple myeloma cells have increased PD-L1 mRNA and surface expression compared with HD. There is also a significant increase in PD-1 expression on effector cells in multiple myeloma. Importantly, PD-1/PD-L1 blockade abrogates BM stromal cell (BMSC)-induced multiple myeloma growth, and combined blockade of PD-1/PD-L1 with lenalidomide further inhibits BMSC-induced tumor growth. These effects are associated with induction of intracellular expression of IFNγ and granzyme B in effector cells. Importantly, PD-L1 expression in multiple myeloma is higher on MDSC than on antigen-presenting cells, and PD-1/PD-L1 blockade inhibits MDSC-mediated multiple myeloma growth. Finally, lenalidomide with PD-1/PD-L1 blockade inhibits MDSC-mediated immune suppression. CONCLUSIONS: Our data therefore demonstrate that checkpoint signaling plays an important role in providing the tumor-promoting, immune-suppressive microenvironment in multiple myeloma, and that PD-1/PD-L1 blockade induces anti-multiple myeloma immune response that can be enhanced by lenalidomide, providing the framework for clinical evaluation of combination therapy.

Identification of Wee1 as a novel therapeutic target for mutant RAS-driven acute leukemia and other malignancies.

Direct targeting of rat sarcoma (RAS), which is frequently mutated, has proven to be challenging, and inhibition of individual downstream RAS mediators has resulted in limited clinical efficacy. We designed a chemical screen to identify compounds capable of potentiating mammalian target of rapamycin (mTOR) inhibition in mutant RAS-positive leukemia, and identified a Wee1 inhibitor. Synergy was observed in both mutant neuroblastoma RAS viral oncogene homolog (NRAS)- and mutant kirsten RAS viral oncogene homolog (KRAS)-positive acute myelogenous leukemia (AML) cell lines and primary patient samples. The observed synergy enhanced dephosphorylation of AKT, 4E-binding protein 1 and s6 kinase, and correlated with increased apoptosis. The specificity of Wee1 as the target of MK-1775 was validated by Wee1 knockdown, as well as partial reversal of drug combination-induced apoptosis by a cyclin-dependent kinase 1 (CDK1) inhibitor. Importantly, we also extended our findings to other mutant RAS-expressing malignancies, including mutant NRAS-positive melanoma, and mutant KRAS-positive colorectal cancer, pancreatic cancer and lung cancer. We observed favorable responses with combined Wee1/mTOR inhibition in human cancer cell lines from multiple malignancies, and inhibition of tumor growth in in vivo models of mutant KRAS lung cancer and leukemia. The present study introduces for the first time Wee1 inhibition combined with mTOR inhibition as a novel therapeutic strategy for the selective treatment of mutant RAS-positive leukemia and other mutant RAS-expressing malignancies.

Lenalidomide Polarizes Th1-specific Anti-tumor Immune Response and Expands XBP1 Antigen-Specific Central Memory CD3+CD8+ T cells against Various Solid Tumors.

INTRODUCTION: Effective combination immunotherapeutic strategies may be required to enhance effector cells' anti-tumor activities and improve clinical outcomes. METHODS: XBP1 antigen-specific cytotoxic T lymphocytes (XBP1-CTL) generated using immunogenic heteroclitic XBP1 US184-192 (YISPWILAV) and XBP1 SP367-375 (YLFPQLISV) peptides or various solid tumor cells over-expressing XBP1 target antigen were evaluated, either alone or in combination with lenalidomide, for phenotype and immune functional activity. RESULTS: Lenalidomide treatment of XBP1-CTL increased the proportion of CD45RO+ memory CD3+CD8+ T cells, but not the total CD3+CD8+ T cells. Lenalidomide upregulated critical T cell activation markers and costimulatory molecules (CD28, CD38, CD40L, CD69, ICOS), especially within the central memory CTL subset of XBP1-CTL, while decreasing TCRαß and T cell checkpoint blockade (CTLA-4, PD-1). Lenalidomide increased the anti-tumor activities of XBP1-CTL memory subsets, which were associated with expression of Th1 transcriptional regulators (T-bet, Eomes) and Akt activation, thereby resulting in enhanced IFN-γ production, granzyme B upregulation and specific CD28/CD38-positive and CTLA-4/PD-1-negative cell proliferation. CONCLUSIONS: These studies suggest the potential benefit of lenalidomide treatment to boost anti-tumor activities of XBP1-specific CTL against a variety of solid tumors and enhance response to an XBP1-directing cancer vaccine regime.