Identifying effective immunotherapies for solid tumors remains challenging despite the significant clinical responses observed in subsets of patients treated with immune checkpoint inhibitors. ...Interleukin-15 (IL-15) is a promising cytokine for the treatment of cancer as it stimulates NK and CD8
lymphocytes. However, unfavorable pharmacokinetics and safety concerns render recombinant IL-15 (rIL-15) a less attractive modality. These shortcomings were addressed by the clinical development of heterodimeric IL-15 agonists, including N803. In preclinical tumor models, N803 elicited significant Th1 immune activation and tumor suppressive effects, primarily mediated by NK and CD8
T lymphocytes. In addition, multiple clinical studies have demonstrated N803 to be safe for the treatment of cancer patients. The combination of N803 with the immune checkpoint inhibitor nivolumab demonstrated encouraging clinical responses in nivolumab-naïve and nivolumab-refractory patients with non-small cell lung cancer. In a recent Phase II/III clinical study, most Bacillus Calmette-Guerin (BCG)-refractory bladder cancer patients treated with N803 plus BCG experienced durable complete responses. Currently, N803 is being evaluated preclinically and clinically in combination with various agents, including chemotherapeutics, immune checkpoint inhibitors, vaccines, and other immuno-oncology agents. This report will review the mechanism(s) of action of N803 and how it relates to the preclinical and clinical studies of N803.
BackgroundImmune checkpoint blockade (ICB) has achieved unprecedented success in treating multiple cancer types. However, clinical benefit remains modest for most patients with solid malignancies due ...to primary or acquired resistance. Tumor-intrinsic loss of major histocompatibility complex class I (MHC-I) and aberrations in antigen processing machinery (APM) and interferon gamma (IFN-γ) pathways have been shown to play an important role in ICB resistance. While a plethora of combination treatments are being investigated to overcome ICB resistance, there are few identified preclinical models of solid tumors harboring these deficiencies to explore therapeutic interventions that can bypass ICB resistance. Here, we investigated the combination of the epigenetic modulator entinostat and the tumor-targeted immunocytokine NHS-IL12 in three different murine tumor models resistant to αPD-1/αPD-L1 (anti-programmed cell death protein 1/anti-programmed death ligand 1) and harboring MHC-I, APM, and IFN-γ response deficiencies and differing tumor mutational burden (TMB).MethodsEntinostat and NHS-IL12 were administered to mice bearing TC-1/a9 (lung, HPV16 E6/E7+), CMT.64 lung, or RVP3 sarcoma tumors. Antitumor efficacy and survival were monitored. Comprehensive tumor microenvironment (TME) and spleen analysis of immune cells, cytokines, and chemokines was performed. Additionally, whole transcriptomic analysis was carried out on TC-1/a9 tumors. Cancer Genome Atlas (TCGA) datasets were analyzed for translational relevance.ResultsWe demonstrate that the combination of entinostat and NHS-IL12 therapy elicits potent antitumor activity and survival benefit through prolonged activation and tumor infiltration of cytotoxic CD8+ T cells, across αPD-1/αPD-L1 refractory tumors irrespective of TMB, including in the IFN-γ signaling-impaired RVP3 tumor model. The combination therapy promoted M1-like macrophages and activated antigen-presenting cells while decreasing M2-like macrophages and regulatory T cells in a tumor-dependent manner. This was associated with increased levels of IFN-γ, IL-12, chemokine (C-X-C motif) ligand 9 (CXCL9), and CXCL13 in the TME. Further, the combination therapy synergized to promote MHC-I and APM upregulation, and enrichment of JAK/STAT (janus kinase/signal transducers and activators of transcription), IFN-γ-response and antigen processing-associated pathways. 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 the tumor-targeting NHS-IL12 with the histone deacetylase inhibitor entinostat in the clinical setting for patients unresponsive to αPD-1/αPD-L1 and/or with innate deficiencies in tumor MHC-I, APM expression, and IFN-γ signaling.
Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and ...hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Here we show for the first time that both enzalutamide and abiraterone render prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT.
Natural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate ...early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined.
Tumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed.
Cultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab.
Tumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.
The development of vaccines, especially RNA-based, directed against patient-specific tumor neoepitopes is an active and productive area of cancer immunotherapy. Promising clinical results in melanoma ...and other solid tumor types are emerging. As with all cancer therapy modalities, neoepitope vaccine development and delivery also has some drawbacks, including the level of effort to develop a patient-specific product, accuracy of algorithms to predict neoepitopes, and with the exception of melanoma and some other tumor types, biopsies of metastatic lesions of solid tumors are often not available. We hypothesize that in some circumstances the use of rationally designed combinations of “off-the-shelf” agents may prove an additional path to enable the patient to produce his/her own “neoepitope vaccine” in situ. These combination therapies may consist of agents to activate a tumor-associated T-cell response, potentiate that response, reduce or eliminate immunosuppressive entities in the tumor microenvironment, and/or alter the phenotype of tumor cells to render them more susceptible to immune-mediated lysis. Examples are provided in both preclinical and clinical studies in which combinations of “off-the-shelf” agents lead to the generation of T cells directed against tumor-derived neoepitopes with consequent antitumor activity.
There is strong evidence that chemotherapy can induce tumor necrosis which can be exploited for the targeted delivery of immuno-oncology agents into the tumor microenvironment (TME). We hypothesized ...that docetaxel, a chemotherapeutic agent that induces necrosis, in combination with the bifunctional molecule NHS-IL-12 (M9241), which delivers recombinant IL-12 through specific targeting of necrotic regions in the tumor, would provide a significant antitumor benefit in the poorly inflamed murine tumor model, EMT6 (breast), and in the moderately immune-infiltrated tumor model, MC38 (colorectal). Docetaxel, as monotherapy or in combination with NHS-IL-12, promoted tumor necrosis, leading to the improved accumulation and retention of NHS-IL-12 in the TME. Significant antitumor activity and prolonged survival were observed in cohorts receiving docetaxel and NHS-IL-12 combination therapy in both the MC38 and EMT6 murine models. The therapeutic effects were associated with increased tumor infiltrating lymphocytes and were dependent on CD8
+
T cells. Transcriptomics of the TME of mice receiving the combination therapy revealed the upregulation of genes involving crosstalk between innate and adaptive immunity factors, as well as the downregulation of signatures of myeloid cells. In addition, docetaxel and NHS-IL-12 combination therapy effectively controlled tumor growth of PD-L1 wild-type and PD-L1 knockout MC38 in vivo, implying this combination could be applied in immune checkpoint refractory tumors, and/or tumors regardless of PD-L1 status. The data presented herein provide the rationale for the design of clinical studies employing this combination or similar combinations of agents.
Here we describe a novel bifunctional fusion protein, designated N-809. This molecule comprises the IL-15/IL15Rα superagonist complex containing the Fc-domain of IgG1 (N-803, formerly designated as ...ALT-803) fused to two single chain anti-PD-L1 domains. The fully human IgG1 portion of the N-809 molecule was designed to potentially mediate antibody dependent cellular cytotoxicity (ADCC). The studies reported here show that N-809 has the same ability to bind PD-L1 as an anti-PD-L1 monoclonal antibody. RNAseq studies show the ability of N-809 to alter the expression of an array of genes of both CD4
+
and CD8
+
human T cells, and to enhance their proliferation; CD8
+
T cells exposed to N-809 also have enhanced ability to lyse human tumor cells. An array of genes was differentially expressed in human natural killer (NK) cells following N-809 treatment, and there was increased expression of several surface activating receptors; there was, however, no increase in the expression of inhibitory receptors known to be upregulated in exhausted NK cells. N-809 also increased the cytotoxic potential of NK cells, as shown by increased expression of granzyme B and perforin. The lysis of several tumor cell types was increased when either NK cells or tumor cells were exposed to N-809. Similarly, the highest level of ADCC was seen when both NK cells (from donors or cancer patients) and tumor cells were exposed to N-809. These studies thus demonstrate the multi-functionality of this novel agent.
Numerous reports have now demonstrated that the epithelial-to-mesenchymal transition (EMT) process is involved in solid tumor progression, metastasis, and drug resistance. Several transcription ...factors have been implicated as drivers of EMT and metastatic progression, including Twist. Overexpression of Twist has been shown to be associated with poor prognosis and drug resistance for many carcinomas and other tumor types. The role of Twist in experimental cancer metastases has been principally studied in the 4T1 mammary tumor model, where silencing of Twist in vitro has been shown to greatly reduce in vivo metastatic spread. Transcription factors such as Twist are generally believed to be "undruggable" because of their nuclear location and lack of a specific groove for tight binding of a small molecule inhibitor. An alternative approach to drug therapy targeting transcription factors driving the metastatic process is T-cell-mediated immunotherapy. A therapeutic vaccine platform that has been previously characterized consists of heat-killed recombinant Saccharomyces cerevisiae (yeast) capable of expressing tumor-associated antigen protein. We report here the construction and characterization of a recombinant yeast expressing the entire Twist protein, which is capable of inducing both CD8(+) and CD4(+) Twist-specific T-cell responses in vivo. Vaccination of mice reduced the size of primary transplanted 4T1 tumors and had an even greater antitumor effect on lung metastases of the same mice, which was dependent on Twist-specific CD8(+) T cells. These studies provide the rationale for vaccine-induced T-cell-mediated therapy of transcription factors involved in driving the metastatic process.
To understand the impact of clinically relevant radiation therapy (RT) on tumor immune gene expression and to utilize the changes that occur during treatment to improve cancer treatment outcome, we ...examined how immune response genes are modulated in prostate cancer cells of varying p53 status. LNCaP (p53 wild-type), PC3 (p53 null) and DU145 (p53 mutant) cells received a 10 Gy single dose or 1 Gy × 10 multifractionated radiation dose to simulate hypofractionated and conventionally fractionated prostate radiotherapy. Total RNA was isolated 24 h after multifractionated radiation treatment and single-dose treatments and subjected to microarray analysis and later validated by RT-PCR. RT-PCR was utilized to identify total-dose inflection points for significantly upregulated genes in response to multifractionated radiation therapy. Radiation-induced damage-associated molecular pattern molecules (DAMPs) and cytokine analyses were performed using bioluminescence and ELISA. Multifractionated doses activated immune response genes more robustly than single-dose treatment, with a relatively larger number of immune genes upregulated in PC3 compared to DU145 and LNCaP cells. The inflection point of multifractionated radiation-induced immune genes in PC3 cells was observed in the range of 8–10 Gy total radiation dose. Although both multifractionated and single-dose radiation-induced proinflammatory DAMPs and positively modulated the cytokine environment, the changes were of higher magnitude with multifractionated therapy. The findings of this study together with the gene expression data suggest that cells subjected to multifractionated radiation treatment would promote productive immune cell–tumor cell interactions.
Breast tumors commonly harbor low mutational burden, low PD-L1 expression, defective antigen processing/presentation, and an immunosuppressive tumor microenvironment (TME). In a malignancy mostly ...refractory to checkpoint blockade, there is an unmet clinical need for novel combination approaches that increase tumor immune infiltration and tumor control. Preclinical data have guided the development of this clinical trial combining 1) BN-Brachyury (a poxvirus vaccine platform encoding the tumor associated antigen brachyury), 2) bintrafusp alfa (a bifunctional protein composed of the extracellular domain of the TGF-βRII receptor (TGFβ "trap") fused to a human IgG1 anti-PD-L1), 3), entinostat (a class I histone deacetylase inhibitor), and 4) T-DM1 (ado-trastuzumab emtansine, a standard of care antibody-drug conjugate targeting HER2). We hypothesize that this tetratherapy will induce a robust immune response against HER2
breast cancer with improved response rates through 1) expanding tumor antigen-specific effector T cells, natural killer cells, and immunostimulatory dendritic cells, 2) improving antigen presentation, and 3) decreasing inhibitory cytokines, regulatory T cells, and myeloid-derived suppressor cells. In an orthotopic HER2
murine breast cancer model, tetratherapy induced high levels of antigen-specific T cell responses, tumor CD8
T cell/Treg ratio, and augmented the presence of IFNγ- or TNFα-producing CD8
T cells and IFNγ/TNFα bifunctional CD8
T cells with increased cytokine production. Similar effects were observed in tumor CD4
effector T cells. Based on this data, a phase 1b clinical trial evaluating the stepwise addition of BN-Brachyury, bintrafusp alfa, T-DM1 and entinostat in advanced breast cancer was designed. Arm 1 (TNBC) receives BN-Brachyury + bintrafusp alfa. Arm 2 (HER2
) receives T-DM1 + BN-Brachyury + bintrafusp alfa. After safety is established in Arm 2, Arm 3 (HER2
) will receive T-DM1 + BN-Brachyury + bintrafusp alfa + entinostat. Reimaging will occur every 2 cycles (1 cycle = 21 days). Arms 2 and 3 undergo research biopsies at baseline and after 2 cycles to evaluate changes within the TME. Peripheral immune responses will be evaluated. Co-primary objectives are response rate and safety. All arms employ a safety assessment in the initial six patients and a 2-stage Simon design for clinical efficacy (Arm 1 if ≥ three responses of eight then expand to 13 patients; Arms 2 and 3 if ≥ four responses of 14 then expand to 19 patients per arm). Secondary objectives include progression-free survival and changes in tumor infiltrating lymphocytes. Exploratory analyses include changes in peripheral immune cells and cytokines. To our knowledge, the combination of a vaccine, an anti-PD-L1 antibody, entinostat, and T-DM1 has not been previously evaluated in the preclinical or clinical setting. This trial (NCT04296942) is open at the National Cancer Institute (Bethesda, MD).
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