Failed T cell-based immunotherapies in the presence of genomic alterations in antigen presentations pathways may be overcome by NK cell-based immunotherapy. This approach may still be limited by the ...presence of immunosuppressive myeloid populations. Here, we demonstrate that NK cells (haNKs) engineered to express a PD-L1 chimeric antigen receptor (CAR) haNKs killed a panel of human and murine head and neck cancer cells at low effector-to-target ratios in a PD-L1-dependent fashion. Treatment of syngeneic tumors resulted in CD8 and PD-L1-dependent tumor rejection or growth inhibition and a reduction in myeloid cells endogenously expressing high levels of PD-L1. Treatment of xenograft tumors resulted in PD-L1-dependent tumor growth inhibition. PD-L1 CAR haNKs reduced levels of macrophages and other myeloid cells endogenously expressing high PD-L1 in peripheral blood from patients with head and neck cancer. The clinical study of PD-L1 CAR haNKs is warranted.
BackgroundThymic epithelial tumors are PD-L1–expressing tumors of thymic epithelial origin characterized by varying degrees of lymphocytic infiltration and a predisposition towards development of ...paraneoplastic autoimmunity. PD-1–targeting antibodies have been evaluated, largely in patients with thymic carcinoma. We sought to evaluate the efficacy and safety of the anti-PD-L1 antibody, avelumab (MSB0010718C), in patients with relapsed, advanced thymic epithelial tumors and conduct correlative immunological studies.MethodsSeven patients with thymoma and one patient with thymic carcinoma were enrolled in a phase I, dose-escalation trial of avelumab (MSB0010718C), and treated with avelumab at doses of 10 mg/kg to 20 mg/kg every 2 weeks until disease progression or development of intolerable side effects. Tissue and blood immunological analyses were conducted.ResultsTwo of seven (29%) patients with thymoma had a confirmed Response Evaluation Criteria in Solid Tumors–defined partial response, two (29%) had an unconfirmed partial response and three patients (two thymoma; one thymic carcinoma) had stable disease (43%). Three of four responses were observed after a single dose of avelumab. All responders developed immune-related adverse events that resolved with immunosuppressive therapy. Only one of four patients without a clinical response developed immune-related adverse events. Responders had a higher absolute lymphocyte count, lower frequencies of B cells, regulatory T cells, conventional dendritic cells, and natural killer cells prior to therapy.ConclusionThese results demonstrate anti-tumor activity of PD-L1 inhibition in patients with relapsed thymoma accompanied by a high frequency of immune-related adverse events. Pre-treatment immune cell subset populations differ between responders and non-responders.Trial registrationClinicalTrials.gov - NCT01772004. Date of registration – January 21, 2013.
BackgroundThere are highly effective treatment strategies for estrogen receptor (ER)+, progesterone receptor (PR)+, and HER2+ breast cancers; however, there are limited targeted therapeutic ...strategies for the 10%–15% of women who are diagnosed with triple-negative breast cancer. Here, we hypothesize that ER targeting drugs induce phenotypic changes to sensitize breast tumor cells to immune-mediated killing regardless of their ER status.MethodsReal-time cell analysis, flow cytometry, qRT-PCR, western blotting, and multiplexed RNA profiling were performed to characterize ER+ and ER− breast cancer cells and to interrogate the phenotypic effects of ER targeting drugs. Sensitization of breast cancer cells to immune cell killing by the tamoxifen metabolite 4-hydroxytamoxifen (4-OHT) and fulvestrant was determined through in vitro health-donor natural killer cell 111IN-release killing assays. A syngeneic tumor study was performed to validate these findings in vivo.ResultsPretreatment with tamoxifen metabolite 4-OHT or fulvestrant resulted in increased natural killer (NK)–mediated cell lysis of both ER+ and ER− breast cancer cells. Through multiplexed RNA profiling analysis of 4-OHT-treated ER+ and ER− cells, we identified increased activation of apoptotic and death receptor signaling pathways and identified G protein-coupled receptor for estrogen (GPR30) engagement as a putative mechanism for immunogenic modulation. Using the specific GPR30 agonist G-1, we demonstrate that targeted activation of GPR30 signaling resulted in increased NK cell killing. Furthermore, we show that knockdown of GPR30 inhibited 4-OHT and fulvestrant mediated increases to NK cell killing, demonstrating this is dependent on GPR30 expression. Moreover, we demonstrate that this mechanism remains active in a 4-OHT-resistant MCF7 cell line, showing that even in patient populations with ER+ tumors that are resistant to the cytotoxic effects of tamoxifen, 4-OHT treatment sensitizes them to immune-mediated killing. Moreover, we find that fulvestrant pretreatment of tumor cells synergizes with the IL-15 superagonist N-803 treatment of NK cells and sensitizes tumor cells to killing by programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK) cells. Finally, we demonstrate that the combination of fulvestrant and N-803 is effective in triple-negative breast cancer in vivo.ConclusionTogether, these findings demonstrate a novel effect of ER targeting drugs on the interaction of ER+ and, surprisingly, ER− tumors cells with the immune system. This study is the first to demonstrate the potential use of ER targeting drugs as immunomodulatory agents in an ER agnostic manner and may inform novel immunotherapy strategies in breast cancer.
Therapeutic cancer vaccines can be effective for treating patients, but clinical responses vary considerably from patient to patient. Early indicators of a favorable response are crucial for making ...individualized treatment decisions and advancing vaccine design, but no validated biomarkers are currently available. In this study, we used glycan microarrays to profile antiglycan antibody responses induced by PROSTVAC-VF, a poxvirus-based cancer vaccine currently in phase III clinical trials. Although the vaccine is designed to induce T-cell responses to prostate-specific antigen, we demonstrate that this vaccine also induces humoral responses to a carbohydrate on the poxvirus, the Forssman disaccharide (GalNAcα1–3GalNAcβ). These responses had a statistically significant correlation with overall survival in two independent sample sets (P = 0.015 and 0.008) comprising more than 100 patients. Additionally, anti-Forssman humoral responses correlated with clinical outcome in a separate study of PROSTVAC-VF combined with a radiopharmaceutical (Quadramet). Studies on control subjects demonstrated that the survival correlation was specific to the vaccine. The results provide evidence that antiglycan antibody responses may serve as early biomarkers of a favorable response to PROSTVAC-VF and offer unique insights for improving vaccine design.
We recently reported the clinical results of a Phase I trial combining ipilimumab with a vaccine containing transgenes for prostate-specific antigen (PSA) and for a triad of costimulatory molecules ...(PROSTVAC) in patients with metastatic castration-resistant prostate cancer. Thirty patients were treated with escalating ipilimumab and a fixed dose of vaccine. Of 24 chemotherapy-naïve patients, 58 % had a PSA decline. Combination therapy did not exacerbate the immune-related adverse events associated with ipilimumab. Here, we present updated survival data and an evaluation of 36 immune cell subsets pre- and post-therapy. Peripheral blood mononuclear cells were collected before therapy, at 13 days and at 70 days post-initiation of therapy, and phenotyped by flow cytometry for the subsets of T cells, regulatory T cells, natural killer cells, and myeloid-derived suppressor cells. Associations between overall survival (OS) and immune cell subsets prior to treatment, and the change in a given immune cell subset 70 days post-initiation of therapy, were evaluated. The median OS was 2.63 years (1.77–3.45). There were trends toward associations for longer OS and certain immune cell subsets before immunotherapy: lower PD-1
+
Tim-3
NEG
CD4
EM
(
P
= 0.005, adjusted
P
= 0.010), higher PD-1
NEG
Tim-3
+
CD8 (
P
= 0.002, adjusted
P
= 0.004), and a higher number of CTLA-4
NEG
Tregs (
P
= 0.005, adjusted
P
= 0.010). We also found that an increase in Tim-3
+
natural killer cells post- versus pre-vaccination associated with longer OS (
P
= 0.0074, adjusted
P
= 0.015). These results should be considered as hypothesis generating and should be further evaluated in larger immunotherapy trials.
The nuclear transcription factor brachyury has previously been shown to be a strong mediator of the epithelial-to-mesenchymal transition (EMT) in human carcinoma cells and a strong negative ...prognostic factor in several tumor types. Brachyury is overexpressed in a range of human carcinomas as well as in chordoma, a rare tumor for which there is no standard systemic therapy. Preclinical studies have shown that a recombinant Saccharomyces cerevisiae (yeast) vaccine encoding brachyury (GI-6301) can activate human T cells in vitro. A phase I dose-escalation (3+3 design) trial enrolled 34 patients at 4 dose levels 3, 3, 16, and 11 patients, respectively, at 4, 16, 40, and 80 yeast units (YU). Expansion cohorts were enrolled at 40- and 80-YU dose levels for analysis of immune response and clinical activity. We observed brachyury-specific T-cell immune responses in the majority of evaluable patients despite most having been heavily pretreated. No evidence of autoimmunity or other serious adverse events was observed. Two chordoma patients showed evidence of disease control (one mixed response and one partial response). A patient with colorectal carcinoma, who enrolled on study with a large progressing pelvic mass and rising carcinoembryonic antigen (CEA), remains on study for greater than 1 year with stable disease, evidence of decreased tumor density, and decreased serum CEA. This is the first-in-human study to demonstrate the safety and immunogenicity of this therapeutic cancer vaccine and provides the rationale for exploration in phase II studies. A randomized phase II chordoma study is now enrolling patients.
Checkpoint inhibitors targeting the PD-1/PD-L1 axis are promising immunotherapies shown to elicit objective responses against multiple tumor types, yet these agents fail to benefit most patients with ...carcinomas. This highlights the need to develop effective therapeutic strategies to increase responses to PD-1/PD-L1 blockade. Histone deacetylase (HDAC) inhibitors in combination with immunotherapies have provided preliminary evidence of anti-tumor effects. We investigated here whether exposure of either natural killer (NK) cells and/or tumor cells to two different classes of HDAC inhibitors would augment (a) NK cell‒mediated direct tumor cell killing and/or (b) antibody-dependent cellular cytotoxicity (ADCC) using avelumab, a fully human IgG1 monoclonal antibody targeting PD-L1. Treatment of a diverse array of human carcinoma cells with a clinically relevant dose of either the pan-HDAC inhibitor vorinostat or the class I HDAC inhibitor entinostat significantly enhanced the expression of multiple NK ligands and death receptors resulting in enhanced NK cell‒mediated lysis. Moreover, HDAC inhibition enhanced tumor cell PD-L1 expression both in vitro and in carcinoma xenografts. These data demonstrate that treatment of a diverse array of carcinoma cells with two different classes of HDAC inhibitors results in enhanced NK cell tumor cell lysis and avelumab-mediated ADCC. Furthermore, entinostat treatment of NK cells from healthy donors and PBMCs from cancer patients induced an activated NK cell phenotype, and heightened direct and ADCC-mediated healthy donor NK lysis of multiple carcinoma types. This study thus extends the mechanism and provides a rationale for combining HDAC inhibitors with PD-1/PD-L1 checkpoint blockade to increase patient responses to anti-PD-1/PD-L1 therapies.
Lessons Learned
Modified vaccinia Ankara‐Bavarian Nordic (MVA‐BN)‐Brachyury followed by fowlpox virus‐BN‐Brachyury was well tolerated upon administration to patients with advanced cancer.
Sixty‐three ...percent of patients developed CD4+ and/or CD8+ T‐cell responses to brachyury after vaccination.
BN‐Brachyury vaccine also induced T‐cell responses against CEA and MUC1, which are cascade antigens, that is, antigens not encoded in the vaccines.
Background
Brachyury, a transcription factor, plays an integral role in the epithelial–mesenchymal transition, metastasis, and tumor resistance to chemotherapy. It is expressed in many tumor types, and rarely in normal tissues, making it an ideal immunologic target. Bavarian Nordic (BN)‐Brachyury consists of vaccination with modified vaccinia Ankara (MVA) priming followed by fowlpox virus (FPV) boosting, each encoding transgenes for brachyury and costimulatory molecules.
Methods
Patients with metastatic solid tumors were treated with two monthly doses of MVA‐brachyury s.c., 8 × 108 infectious units (IU), followed by FPV‐brachyury s.c., 1 × 109 IU, for six monthly doses and then every 3 months for up to 2 years. The primary objective was to determine safety and tolerability.
Results
Eleven patients were enrolled from March 2018 to July 2018 (one patient was nonevaluable). No dose‐limiting toxicities were observed. The most common treatment‐related adverse event was grade 1/2 injection‐site reaction observed in all patients. Best overall response was stable disease in six patients, and the 6‐month progression‐free survival rate was 50%. T cells against brachyury and cascade antigens CEA and MUC1 were detected in the majority of patients.
Conclusion
BN‐Brachyury vaccine is well tolerated and induces immune responses to brachyury and cascade antigens and demonstrates some evidence of clinical benefit.
BackgroundImmune checkpoint blockade (ICB) has achieved unprecedented success in treating multiple cancer types. However, clinical benefit remains modest for most patients with solid malignancies ...with the majority having primary resistance to these therapies. Additionally, many patients that initially respond often acquire resistance. Tumor-intrinsic loss of MHC class I and aberrations in the interferon gamma (IFNγ) pathway have been shown to play an important role in ICB resistance. Entinostat, a class I HDAC inhibitor, has previously been shown to upregulate MHC class I molecules and antigen processing machinery (APM). NHS-IL12 (M9140), an IL12 fusion protein targeting necrotic tissue, increases anti-tumor effector functions through an influx of interferon gamma into the tumor microenvironment (TME). Here, we investigated the combination of Entinostat and NHS-IL12 in three different murine ICB-refractory tumor models (TC-1/a9, CMT.64, or RVP3) harboring varying MHC-class I and APM deficiencies.MethodsEntinostat and murine NHS-IL12 were administered to mice bearing lung TC-1/a9 (HPV16 E6/E7+), CMT.64 (lung), or RVP3 (sarcoma) tumors. Anti-tumor efficacy and survival were monitored. Comprehensive tumor and spleen immune profile analyses were carried out using flow cytometry. Tumor supernantants and sera were analyzed for cytokine and chemokine profiles. Additionally, whole transcriptomic analysis was carried out on TC-1/a9 tumors. TCGA datasets were analyzed for translational relevance.ResultsWe demonstrated that combination therapy elicits potent anti-tumor activity in ICB-resistant MHC-I deficient tumors through prolonged activation of cytotoxic CD8+ T cells. TC-1/a9 tumor-bearing athymic nude mice and depletion studies confirmed CD8 T cells were required for tumor growth control and survival. Importantly, we show for the first time that combination therapy synergizes to promote antigen presentation, MHC-I and APM upregulation and enrichment of IFNγ and antigen processing pathways increasingly described as hallmarks of ICB resistance. Moreover, combination therapy promoted M1-macrophages and activated antigen presenting cells while suppressing M2-macrophages and Tregs in a tumor-dependent manner. Combination therapy induced high levels of IFNγ, IL-12, CXCL9 and CXCL13 in the TME, resulting in significant anti-tumor activity including in the IFNγ signaling-impaired RVP3 tumor model. A biomarker signature of the mechanism involved in these studies is associated with patients‘ overall survival across multiple tumor types.ConclusionsOur findings provide a rationale for combining NHS-IL12 with Entinostat in the clinical setting for patients unresponsive to ICB.AcknowledgementsThe authors thank Curtis Randolph for his excellent technical assistance.
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