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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.

Activation of antigen-specific T-lymphocyte responses may be needed to cure disorders caused by chronic infection with low-risk human papillomavirus (lrHPV). Safe and effective adjuvant therapies for such disorders are needed. The safety and efficacy of a novel gorilla adenovirus vaccine expressing a protein designed to elicit immune responses directed against HPV6 and HPV11, PRGN-2012, was studied using in vitro stimulation of T lymphocytes from patients with recurrent respiratory papillomatosis, in vivo vaccination studies, and therapeutic studies in mice bearing tumors expressing lrHPV antigen. PRGN-2012 treatment induces lrHPV antigen-specific responses in patient T lymphocytes. Vaccination of wild-type mice induces E6-specific T-lymphocyte responses without toxicity. In vivo therapeutic vaccination of mice bearing established HPV6 E6 expressing tumors results in HPV6 E6-specific CD8+ T-lymphocyte immunity of sufficient magnitude to induce tumor growth delay. The clinical study of PRGN-2012 in patients with disorders caused by chronic infection with lrHPV is warranted.

NHS-IL12 is a novel immunocytokine designed for delivery of IL-12 to the tumor microenvironment (TME). NHS-IL12 consists of two molecules of IL-12 fused to a human IgG1 (NHS76) recognizing DNA/histone complexes, which are often exposed in the necrotic portions of tumors. Preclinical studies demonstrated the tumor-targeting ability and longer plasma half-life for NHS-IL12 when compared with recombinant IL-12 (rIL-12). NHS-IL12 outperformed rIL-12 in enhancing the proliferation and activation of immune as well as antigen-presenting cells, resulting in a more robust primary immune response. NHS-IL12 also reduced the number and function of suppressive myeloid cells (myeloid derived suppressor cells/macrophages) within the TME. In a murine bladder tumor model, NHS-IL12 administration led to a coordinated increase in host immunity with a reduction of immunosuppressive myeloid cells in the TME resulting in substantial reduction in tumor growth. Several preclinical studies have demonstrated increased overall anti-tumor efficacy when NHS-IL12 was combined with either immune-based therapeutics or chemotherapeutic approaches.

BackgroundIn preclinical studies, combining M9241 (a novel immunocytokine containing interleukin (IL)-12 heterodimers) with avelumab (anti-programmed death ligand 1 antibody) resulted in additive or synergistic antitumor effects. We report dose-escalation and dose-expansion results from the phase Ib JAVELIN IL-12 trial investigating M9241 plus avelumab.MethodsIn the dose-escalation part of JAVELIN IL-12 (NCT02994953), eligible patients had locally advanced or metastatic solid tumors; in the dose-expansion part, eligible patients had locally advanced or metastatic urothelial carcinoma (UC) that had progressed with first-line therapy. Patients received M9241 at 4, 8, 12, or 16.8 µg/kg every 4 weeks (Q4W) plus avelumab 10 mg/kg every 2 weeks (Q2W, dose levels (DLs) 1–4) or M9241 16.8 µg/kg Q4W plus avelumab 800 mg once a week for 12 weeks followed by Q2W (DL5/dose expansion). Primary endpoints for the dose-escalation part were adverse events (AEs) and dose-limiting toxicities (DLTs), and those for the dose-expansion part were confirmed best overall response (BOR) per investigator (Response Evaluation Criteria in Solid Tumors V.1.1) and safety. The dose-expansion part followed a two-stage design; 16 patients were enrolled and treated in stage 1 (single-arm part). A futility analysis based on BOR was planned to determine whether stage 2 (randomized controlled part) would be initiated.ResultsAt data cut-off, 36 patients had received M9241 plus avelumab in the dose-escalation part. All DLs were well tolerated; one DLT occurred at DL3 (grade 3 autoimmune hepatitis). The maximum-tolerated dose was not reached, and DL5 was declared the recommended phase II dose, considering an observed drug–drug interaction at DL4. Two patients with advanced bladder cancer (DL2 and DL4) had prolonged complete responses. In the dose-expansion part, no objective responses were recorded in the 16 patients with advanced UC; the study failed to meet the criterion (≥3 confirmed objective responses) to initiate stage 2. Any-grade treatment-related AEs occurred in 15 patients (93.8%), including grade ≥3 in 8 (50.0%); no treatment-related deaths occurred. Exposures for avelumab and M9241 concentrations were within expected ranges.ConclusionsM9241 plus avelumab was well tolerated at all DLs, including the dose-expansion part, with no new safety signals. However, the dose-expansion part did not meet the predefined efficacy criterion to proceed to stage 2.

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.

Clinical successes have been achieved with checkpoint blockade therapy, which facilitates the function of T cells recognizing tumor-specific mutations known as neoepitopes. It is a reasonable hypothesis that therapeutic cancer vaccines targeting neoepitopes uniquely expressed by a patient’s tumor would prove to be an effective therapeutic strategy. With the advent of high-throughput next generation sequencing, it is now possible to rapidly identify these tumor-specific mutations and produce therapeutic vaccines targeting these patient-specific neoepitopes. However, initial reports suggest that when used as a monotherapy, neoepitope-targeted vaccines are not always sufficient to induce clinical responses in some patients. Therefore, research has now turned to investigating neoepitope vaccines in combination with other cancer therapies, both immune and non-immune, to improve their clinical efficacies.

Investigation of the efficacy and mechanisms of human immuno-oncology agents has been hampered due to species-specific differences when utilizing preclinical mouse models. Peripheral blood mononuclear cell (PBMC) humanized mice provide a platform for investigating the modulation of the human immune-mediated antitumor response while circumventing the limitations of syngeneic model systems. Use of humanized mice has been stymied by model-specific limitations, some of which include the development of graft versus host disease, technical difficulty and cost associated with each humanized animal, and insufficient engraftment of some human immune subsets. Recent advances have addressed many of these limitations from which have emerged humanized models that are more clinically relevant. This review characterizes the expanded usage, advantages and limitations of humanized mice and provides insights into the development of the next generation of murine humanized models to further inform clinical applications of cancer immunotherapeutic agents.

BackgroundImmune checkpoint inhibitors (ICIs) have changed the cancer treatment landscape, but immune-related adverse events (irAEs) can affect a wide range of tissues in patients receiving ICIs. Severe irAEs can be life-threatening or fatal and prohibit patients from receiving further ICI treatment. While the clinical features of irAEs are well documented, the pathological mechanisms and predictive biomarkers are largely unknown. In addition, there is a critical need to preserve ICI-induced anti-tumor immunity while controlling for irAEs, which requires deciphering molecular and cellular signatures associated specifically with irAEs beyond those more generally linked to anti-tumor immunity.MethodsTo unbiasedly identify immune cells and states associated with irAEs, we applied CITE-seq to measure transcripts and surface proteins (83 protein markers) from PBMCs collected from patients with thymic epithelial tumors before and after treatment with an anti-PD-L1 antibody (avelumab, NCT01772004, NCT03076554).ResultsSamples from 9 patients were analyzed. No patient had a history of pre-existing paraneoplastic autoimmune disease. Anti-tumor activity was observed in all cases, and 5 patients had clinical and/or biochemical evidence of immune-related muscle inflammation (myositis with or without myocarditis). Multilevel models applied within highly resolved cell clusters revealed transcriptional states associated with ICI response and more uniquely with irAEs. A total of 190,000 cells were included in the analysis after quality control. Most notably, CD45RA+ effector memory CD8 T cells with an mTOR transcriptional signature were highly enriched at baseline and post treatment in patients with irAEs.ConclusionsOur findings suggest the potential therapeutic avenues by using mTOR inhibitors to dampen autoimmune responses while potentially sparing anti-tumor activity, to prevent treatment discontinuation and improve clinical outcomes for cancer patients treated with ICIs.AcknowledgementsThis research was supported in part by the Intramural Research Program of the NCI (the Center for Cancer Research), NIAID and NIAMS, and through a Cooperative Research and Development Agreement between the National Cancer Institute and EMD Serono.Trial RegistrationNCT01772004, NCT03076554Ethics ApprovalThis study is approved by NCI institutional review board.

BackgroundThe safety and efficacy of bintrafusp alfa, a first-in-class bifunctional fusion protein targeting TGFβ and PD-L1 pathways, have been demonstrated in patients with HPV-related cancers in an open-label, multicenter phase 1 trial (NCT02517398), and an open-label, single-center phase 2 trial (NCT03427411). The current study aimed to identify immune related biomarkers prior to and following 1 cycle of bintrafusp alfa that associate with clinical benefit.MethodsImmune parameters were compared in patients (n=65) deriving clinical benefit from bintrafusp alfa (defined as BOR of stable disease (SD) or better, which included SD, mixed, partial, and complete responses) versus patients with a BOR of progressive disease (PD). Peripheral blood was obtained from patients before and after 1 cycle of therapy, and evaluated for complete blood counts, plasma cytokines/soluble factors, 158 immune subsets, and T cells specific for HPV-16 E6 and E7.ResultsPrior to therapy, patients who developed a BOR of SD or better had lower counts of neutrophils, monocytes, and platelets, lower levels of TGF-β1 and sCD73, and higher levels of sCD27:sCD40L than patients with a BOR of PD. Lower baseline frequencies of MDSCs, monocytes, naïve CD4+ and CD8+ T cells, and CD8+ T cells that express CD73, an immune checkpoint associated with adenosine metabolism, were detected in patients with a BOR of SD or better than those with PD. Following 1 cycle of treatment, lymphocyte counts were reduced, while neutrophil counts and the NLR were increased, in patients with PD compared to those with a BOR of SD or better. IL-8, a cytokine involved in tumor progression and associated with reduced clinical benefit to immune checkpoint inhibitors, was increased in patients with PD compared to those with a BOR of SD or better, while conventional dendritic cells and CD8+ T cells expressing the proliferative marker ki67 were increased in patients with a BOR of SD or better compared to those with PD. Greater increases in the frequency and magnitude of HPV-16 specific CD8+ T-cells were also detected in individuals with a BOR of SD or better compared to PD.ConclusionsImmune profiling identified specific measures prior to therapy, as well as changes induced early after therapy (preceding restaging), that may serve as predictive biomarkers to identify patients with HPV-related cancers most likely to benefit from bintrafusp alfa. These findings also provide the rationale to combine bintrafusp alfa with other therapies including HPV-targeted therapeutic vaccines and agents that block IL-8 signaling.AcknowledgementsThis research was supported by the Intramural Research Program of the Center for Cancer Research, NCI, National Institutes of Health, and through a Cooperative Research and Development Agreement with EMD Serono Research & Development Institute and GSK.Ethics ApprovalThe study protocol was approved by ethics committees at all participating institutions, and each patient provided written informed consent before study enrollment.

BackgroundSmall cell lung cancer (SCLC) is a highly aggressive tumor with a 5-year survival rate of less than 5%. Traditionally characterized as a neuroendocrine (NE) cancer, several subtypes have now been identified which vary in their phenotypic and transcriptional profiles. Classical NE tumors are molecularly defined as ASCL1+ or NEUROD1+ and exhibit an epithelial phenotype, expressing cytokeratin and E-cadherin (E-Cad). In contrast, non-classical variants express POU2F3 or YAP1 and are enriched in mesenchymal features, such as high levels of vimentin (Vim). Prior studies describe that non-NE variants of SCLC are less susceptible to chemotherapy and may arise via therapeutic selection. With the addition of immune checkpoint blockade to first-line chemotherapy for the treatment of advanced SCLC, understanding whether SCLC variants respond differently to immunotherapy is crucial.MethodsWe utilized a range of pre-clinical models to investigate whether molecular and phenotypic variants of SCLC differ in their susceptibility to immune-mediated lysis. Following extensive characterization at the RNA and protein levels for expression of ASCL1, NEUROD1, POU2F3, YAP1, epithelial E-Cad, mesenchymal Vim, and other markers of cell phenotype, a panel of cells including each variant subtype were selected for further study.ResultsUpon exposure to healthy donor effector NK cells, the more epithelial cells were highly susceptible to NK-mediated cytotoxicity while all mesenchymal SCLC cells remained highly refractory to NK-mediated lysis. This prompted us to investigate immunotherapy approaches such as the addition of N803, a mutant IL-15 superagonist, to improve the activation and proliferation of NK cells. In a xenograft model utilizing the mesenchymal YAP1+ H841 cell line subcutaneously implanted into nude mice devoid of all immune cells except for NK cells, we observed that the weekly administration of N803 resulted in a significant increase in the number of activated NK cells within the spleens of treated mice. Additionally, NK cells from treated mice produced significantly higher levels of IFN-gamma and granzyme B, resulting in a significant decrease in overall tumor burden.ConclusionsOur data indicates that N803-activated NK cells effectively mediate lysis of SCLC across all variant types, including those previously completely refractory to traditional NK cell lysis. These results highlight the potential of N803 as a novel immune-based intervention for the treatment of all variants of SCLC.Ethics ApprovalPBMCs were obtained from healthy donors at the NIH Clinical Center Blood Bank (NCT00001846). All animal studies were approved and conducted in accordance with an IACUC-approved animal protocol (LTIB-57) with the approval the NIH/NCI Institutional Animal Care and Use Committee.

BackgroundBackground: Treatment of patients with castration-resistant prostate cancer (CRPC) includes the use of next-generation hormonal therapies such as abiraterone or enzalutamide. Although these agents extend survival, a significant proportion of patients exhibit primary or acquired resistance to treatment. In recent years, immune checkpoint blockade has led to remarkable responses in patients with several tumor types, however, CRPC has remained resistant to immunotherapy. Previous studies have demonstrated that different tumor variants could emerge along the progression of prostate cancer, including tumors undergoing phenotypic plasticity in the context of an epithelial-mesenchymal transition. Our laboratory and others have shown that phenotypic plasticity is a driver of resistance to immunotherapy. Based on this knowledge, we investigated whether changes in tumor phenotype could affect the response of CRPC to immune-based therapies, and ways this can be mitigated.MethodsThe androgen sensitive LNCAP prostate cancer cell line was used to derive LNCAP cells resistant to enzalutamide (LNCAP-EnzaR) or abiraterone (LNCAP-AbiR). Resistant cell lines and parental LNCAP cells were comparatively evaluated for features of EMT and neuroendocrine phenotype via RT-PCR, ELISA, western blot, immunofluorescence, and RNAseq. Changes in the susceptibility to NK-cell mediated cytotoxicity were evaluated with NK cells isolated from peripheral blood from healthy donors. LNCAP-EnzaR cells were also grown in vivo in NSG MHC-deficient mice, and tumors were characterized for phenotypic markers and potential therapeutic targets.ResultsAcquisition of resistance to both enzalutamide and abiraterone was associated with a significant increase in mesenchymal tumor features, including high levels of vimentin and fibronectin, and the loss of epithelial features and cell-to-cell attachments. LNCAP-Enza-R and LNCAP-AbiR cells showed a significant reduction (up to 90%) in susceptibility to NK-cell mediated cytotoxicity and antibody-dependent cell cytotoxicity (ADCC), compared with parental cells. These results prompted us to investigate approaches to improve immune-mediated lysis, including inhibition of estrogen receptor 1 (ESR1), which was identified as highly upregulated in LNCAP-EnzaR cells via RNAseq analysis. In a xenograft model of LNCAP-EnzaR cells, we corroborated the maintenance of tumor phenotypic plasticity and the expression of actionable targets.ConclusionsOur data indicates that acquisition of resistance to androgen receptor inhibition is associated with marked reduction of susceptibility to immune attack, and the acquisition of tumor phenotypic plasticity. Future studies will investigate approaches that revert tumor plasticity, including blockade of ESR1, TGF-beta or IL-8, for potential improvement of tumor susceptibility to immune attack in CRPC.Ethics ApprovalPBMCs were obtained from healthy donors at the NIH Clinical Center Blood Bank (NCT00001846). All animal studies were approved and conducted in accordance with an IACUC-approved animal protocol (LTIB-57) with the approval the NIH/NCI Institutional Animal Care and Use Committee.

BackgroundMVA-BN-brachyury-TRICOM is a recombinant vector-based therapeutic cancer vaccine designed to induce an immune response against brachyury. Brachyury, a transcription factor overexpressed in advanced cancers, has been associated with treatment resistance, epithelial-to-mesenchymal transition, and metastatic potential. MVA-BN-brachyury-TRICOM has demonstrated immunogenicity and safety in previous clinical trials of subcutaneously administered vaccine. Preclinical studies have suggested that intravenous administration of therapeutic vaccines can induce superior CD8+ T cell responses, higher levels of systemic cytokine release, and stronger natural killer cell activation and proliferation. This is the first-in-human study of the intravenous administration of MVA-BN-brachyury-TRICOM.MethodsBetween January 2020 and March 2021, 13 patients were treated on a phase 1, open-label, 3+3 design, dose-escalation study at the National Institutes of Health Clinical Center. The study population was adults with advanced solid tumors and was enriched for chordoma, a rare sarcoma of the notochord that overexpresses brachyury. Vaccine was administered intravenously at three DLs on days 1, 22, and 43. Blood samples were taken to assess drug pharmacokinetics and immune activation. Imaging was conducted at baseline, 1 month, and 3 months post-treatment. The primary endpoint was safety and tolerability as determined by the frequency of dose-limiting toxicities; a secondary endpoint was determination of the recommended phase 2 dose.ResultsNo dose-limiting toxicities were observed and no serious adverse events were attributed to the vaccine. Vaccine-related toxicities were consistent with class profile (ie, influenza-like symptoms). Cytokine release syndrome up to grade 2 was observed with no adverse outcomes. Dose-effect trend was observed for fever, chills/rigor, and hypotension. Efficacy analysis of objective response rate per RECIST 1.1 at the end of study showed one patient with a partial response, four with stable disease, and eight with progressive disease. Three patients with stable disease experienced clinical benefit in the form of improvement in pain. Immune correlatives showed T cell activation against brachyury and other tumor-associated cascade antigens.ConclusionsIntravenous administration of MVA-BN-brachyury-TRICOM vaccine was safe and tolerable. Maximum tolerated dose was not reached. The maximum administered dose was 109 infectious units every 3 weeks for three doses. This dose was selected as the recommended phase 2 dose.Trial registration numberNCT04134312.

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.

Most monoclonal antibodies (MAbs), including immune checkpoint inhibitor MAbs, are delivered intravenously (i.v.) to patients. Recent clinical studies have demonstrated that some anti-PD1 MAbs may also be delivered subcutaneously (s.c.), with clinical outcomes similar of those obtained with i.v.-delivered agents. Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the human transforming growth factor β receptor II (TGF-βRII or TGF-β “trap”) fused to the heavy chain of an IgG1 antibody blocking programmed death ligand 1 (anti-PDL1), was designed to target two key immunosuppressive pathways in the tumor microenvironment (TME). Bintrafusp alfa is currently being administered i.v. in clinical studies. The studies reported here demonstrate that systemic or s.c. delivery of bintrafusp alfa, each administered at five different doses, induces similar anti-tumor effects in breast and colorectal carcinoma models. An interrogation of the TME for CD8+ and CD4+ T cells, regulatory T cells (Tregs), monocytic myeloid-derived suppressor cells (M-MDSCs) and granulocytic (G) MDSCs showed similar levels and phenotype of each cell subset when bintrafusp alfa was given systemically or s.c. Subcutaneous administration of bintrafusp alfa also sequestered TGFβ in the periphery at similar levels seen with systemic delivery. To our knowledge, this is the most comprehensive preclinical evaluation of any checkpoint inhibitor MAb given s.c. vs systemically, and the first to demonstrate this phenomenon using a bifunctional agent. These studies provide preclinical rationale to explore s.c. approaches for bintrafusp alfa in the clinic.

Purpose of Review Patients with Lynch syndrome have a high probability of developing colorectal and other carcinomas. This review provides a comprehensive assessment of the immunologic aspects of Lynch syndrome pathogenesis and provides an overview of potential immune interventions for patients with Lynch syndrome polyps and Lynch syndrome–associated carcinomas. Recent Findings Immunogenic properties of the majority of Lynch syndrome polyps and associated cancers include microsatellite instability leading to a high mutational burden and the development of novel frameshift peptides, i.e., neoantigens. In addition, patients with Lynch syndrome develop T cell responses in the periphery and in the tumor microenvironment (TME) to tumor-associated antigens, and a proinflammatory cytokine TME has also been identified. However, Lynch syndrome lesions also possess immunosuppressive entities such as alterations in MHC class I antigen presentation, TGFβ receptor mutations, regulatory T cells, and upregulation of PD-L1 on tumor-associated lymphocytes. Summary The rich immune microenvironment of Lynch syndrome polyps and associated carcinomas provides an opportunity to employ the spectrum of immune-mediating agents now available to induce and enhance host immune responses and/or to also reduce immunosuppressive entities. These agents can be employed in the so-called prevention trials for the treatment of patients with Lynch syndrome polyps and for trials in patients with Lynch syndrome–associated cancers.

There are approximately 44,000 cases of human papillomavirus–associated (HPV-associated) cancer each year in the United States, most commonly caused by HPV types 16 and 18. Prophylactic vaccines successfully prevent healthy people from acquiring HPV infections via HPV-specific antibodies. In order to treat established HPV-associated malignancies, however, new therapies are necessary. Multiple recombinant gorilla adenovirus HPV vaccine constructs were evaluated in NSG-β2m–/– peripheral blood mononuclear cell–humanized mice bearing SiHa, a human HPV16+ cervical tumor, and/or in the syngeneic HPV16+ TC-1 model. PRGN-2009 is a therapeutic gorilla adenovirus HPV vaccine containing multiple cytotoxic T cell epitopes of the viral oncoproteins HPV 16/18 E6 and E7, including T cell enhancer agonist epitopes. PRGN-2009 treatment reduced tumor volume and increased CD8+ and CD4+ T cells in the tumor microenvironment of humanized mice bearing the human cervical tumor SiHa. PRGN-2009 monotherapy in the syngeneic TC-1 model also reduced tumor volumes and weights, generated high levels of HPV16 E6–specific T cells, and increased multifunctional CD8+ and CD4+ T cells in the tumor microenvironment. These studies provide the first evaluation to our knowledge of a therapeutic gorilla adenovirus HPV vaccine, PRGN-2009, showing promising preclinical antitumor efficacy and induction of HPV-specific T cells, along with the rationale for its evaluation in clinical trials.

While vaccines directed against the SARS-CoV-2 spike protein will have varying degrees of effectiveness in preventing SARS-CoV-2 infections, the severity of infection will be determined by multiple host factors including the ability of immune cells to lyse virus-infected cells. This review will discuss the complexity of both adaptive and innate immunomes and how a flow-based assay can detect up to 158 distinct cell subsets in the periphery. This assay has been employed to show the effect of age on differences in specific immune cell subsets, and the differences in the immunome between healthy donors and age-matched cancer patients. Also reviewed are the numerous soluble factors, in addition to cytokines, that may vary in the pathogenesis of SARS-CoV-2 infections and may also be employed to help define the effectiveness of a given vaccine or other antiviral agents. Various steroids have been employed in the management of autoimmune adverse events in cancer patients receiving immunotherapeutics and may be employed in the management of SARS-CoV-2 infections. The influence of steroids on multiple immune cells subsets will also be discussed.

BackgroundDifferent types of tumors have varying susceptibility to immunotherapy and hence require different treatment strategies; these cover a spectrum ranging from ‘hot’ tumors or those with high mutational burden and immune infiltrates that are more amenable to targeting to ‘cold’ tumors that are more difficult to treat due to the fewer targetable mutations and checkpoint markers. We hypothesized that an effective anti-tumor response requires multiple agents that would (1) engage the immune response and generate tumor-specific effector cells; (2) expand the number and breadth of the immune effector cells; (3) enable the anti-tumor activity of these immune cells in the tumor microenvironment; and (4) evolve the tumor response to widen immune effector repertoire.MethodsA hexatherapy combination was designed and administered to MC38-CEA (warm) and 4T1 (cool) murine tumor models. The hexatherapy regimen was composed of adenovirus-based vaccine and IL-15 (interleukin-15) superagonist (N-803) to engage the immune response; anti-OX40 and anti-4-1BB to expand effector cells; anti-PD-L1 (anti-programmed death-ligand 1) to enable anti-tumor activity; and docetaxel to promote antigen spread. Primary and metastatic tumor growth inhibition were measured. The generation of anti-tumor immune effector cells was analyzed using flow cytometry, ELISpot (enzyme-linked immunospot), and RNA analysis.ResultsThe MC38-CEA and 4T1 tumor models have differential sensitivities to the combination treatments. In the ‘warm’ MC38-CEA, combinations with two to five agents resulted in moderate therapeutic benefit while the hexatherapy regimen outperformed all these combinations. On the other hand, the hexatherapy regimen was required in order to decrease the primary and metastatic tumor burden in the ‘cool’ 4T1 model. In both models, the hexatherapy regimen promoted CD4+ and CD8+ T cell proliferation and activity. Furthermore, the hexatherapy regimen induced vaccine-specific T cells and stimulated antigen cascade. The hexatherapy regimen also limited the immunosuppressive T cell and myeloid derived suppressor cell populations, and also decreased the expression of exhaustion markers in T cells in the 4T1 model.ConclusionThe hexatherapy regimen is a strategic combination of immuno-oncology agents that can engage, expand, enable, and evolve the immune response and can provide therapeutic benefits in both MC38-CEA (warm) and 4T1 (cool) tumor models.

BackgroundTherapeutic regimens designed to augment the immunological response of a patient with breast cancer (BC) to tumor tissue are critically informed by tumor mutational burden and the antigenicity of expressed neoepitopes. Herein we describe a neoepitope and cognate neoepitope-reactive T-cell identification and validation program that supports the development of next-generation immunotherapies.MethodsUsing GPS Cancer, NantOmics research, and The Cancer Genome Atlas databases, we developed a novel bioinformatic-based approach which assesses mutational load, neoepitope expression, human leukocyte antigen (HLA)-binding prediction, and in vitro confirmation of T-cell recognition to preferentially identify targetable neoepitopes. This program was validated by application to a BC cell line and confirmed using tumor biopsies from two patients with BC enrolled in the Tumor-Infiltrating Lymphocytes and Genomics (TILGen) study.ResultsThe antigenicity and HLA-A2 restriction of the BC cell line predicted neoepitopes were determined by reactivity of T cells from HLA-A2-expressing healthy donors. For the TILGen subjects, tumor-infiltrating lymphocytes (TILs) recognized the predicted neoepitopes both as peptides and on retroviral expression in HLA-matched Epstein-Barr virus–lymphoblastoid cell line and BC cell line MCF-7 cells; PCR clonotyping revealed the presence of T cells in the periphery with T-cell receptors for the predicted neoepitopes. These high-avidity immune responses were polyclonal, mutation-specific and restricted to either HLA class I or II. Interestingly, we observed the persistence and expansion of polyclonal T-cell responses following neoadjuvant chemotherapy.ConclusionsWe demonstrate our neoepitope prediction program allows for the successful identification of neoepitopes targeted by TILs in patients with BC, providing a means to identify tumor-specific immunogenic targets for individualized treatment, including vaccines or adoptively transferred cellular therapies.

Immunotherapy of immunologically cold solid tumors may require multiple agents to engage immune effector cells, expand effector populations and activities, and enable immune responses in the tumor microenvironment (TME). To target these distinct phenomena, we strategically chose five clinical-stage immuno-oncology agents, namely, (i) a tumor antigen–targeting adenovirus-based vaccine (Ad-CEA) and an IL15 superagonist (N-803) to activate tumor-specific T cells, (ii) OX40 and GITR agonists to expand and enhance the activated effector populations, and (iii) an IDO inhibitor (IDOi) to enable effector-cell activity in the TME. Flow cytometry, T-cell receptor (TCR) sequencing, and RNA-sequencing (RNA-seq) analyses showed that in the CEA-transgenic murine colon carcinoma (MC38-CEA) tumor model, Ad-CEA + N-803 combination therapy resulted in immune-mediated antitumor effects and promoted the expression of costimulatory molecules on immune subsets, OX40 and GITR, and the inhibitory molecule IDO. Treatment with Ad-CEA + N-803 + OX40 + GITR + IDOi, termed the pentatherapy regimen, resulted in the greatest inhibition of tumor growth and protection from tumor rechallenge without toxicity. Monotherapy with any of the agents had little to no antitumor activity, whereas combining two, three, or four agents had minimal antitumor effects. Immune analyses demonstrated that the pentatherapy combination induced CD4+ and CD8+ T-cell activity in the periphery and tumor, and antitumor activity associated with decreased regulatory T-cell (Treg) immunosuppression in the TME. The pentatherapy combination also inhibited tumor growth and metastatic formation in 4T1 and LL2-CEA murine tumor models. This study provides the rationale for the combination of multimodal immunotherapy agents to engage, enhance, and enable adaptive antitumor immunity.

BackgroundWhile prophylactic human papillomavirus (HPV) vaccines will certainly reduce the incidence of HPV-associated cancers, these malignancies remain a major health issue. PDS0101 is a liposomal-based HPV therapeutic vaccine consisting of the immune activating cationic lipid R-DOTAP and HLA-unrestricted HPV16 peptides that has shown in vivo CD8+ T cell induction and safety in a phase I study. In this report, we have employed the PDS0101 vaccine with two immune modulators previously characterized in preclinical studies and which are currently in phase II clinical trials. Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domains of the transforming growth factor-β receptor type II (TGFβRII) fused to a human IgG1 monoclonal antibody blocking programmed cell death protein-1 ligand (PDL1), designed both as a checkpoint inhibitor and to bring the TGFβRII ‘trap’ to the tumor microenvironment (TME). NHS-interleukin-12 (NHS-IL12) is a tumor targeting immunocytokine designed to bring IL-12 to the TME and thus enhance the inflammatory Th1 response.MethodsWe employed TC-1 carcinoma (expressing HPV16 E6 and E7 and devoid of PDL1 expression) in a syngeneic mouse model in monotherapy and combination therapy studies to analyze antitumor effects and changes in immune cell types in the spleen and the TME.ResultsAs a monotherapy, the PDS0101 vaccine generated HPV-specific T cells and antitumor activity in mice bearing HPV-expressing mEER oropharyngeal and TC-1 lung carcinomas. When used as a monotherapy in the TC-1 model, NHS-IL12 elicited antitumor effects as well as an increase in CD8+ T cells in the TME. When used as a monotherapy, bintrafusp alfa did not elicit antitumor effects or any increase in T cells in the TME. When all three agents were used in combination, maximum antitumor effects were observed, which correlated with increases in T cells and T-cell clonality in the TME.ConclusionThese studies provide the rationale for the potential clinical use of combinations of agents that can (1) induce tumor-associated T-cell responses, (2) potentiate immune responses in the TME and (3) reduce immunosuppressive entities in the TME.

BackgroundAnti(α)-programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) monotherapy fails to provide durable clinical benefit for most patients with carcinoma. Recent studies suggested that strategies to reduce immunosuppressive cells, promote systemic T-cell responses and lymphocyte trafficking to the tumor microenvironment (TME) may improve efficacy. N-809 is a first-in-class bifunctional agent comprising the interleukin (IL)-15 superagonist N-803 fused to two αPD-L1 domains. Thus, N-809 can potentially stimulate effector immune cells through IL-15 and block immunosuppressive PD-L1. Here, we examined the antitumor efficacy and immunomodulatory effects of N-809 versus N-803+αPD-L1 combination.MethodsThe ability of N-809 to block PD-L1 and induce IL-15-dependent immune effects was examined in vitro and in vivo. Antitumor efficacy of N-809 or N-803+αPD-L1 was evaluated in two murine carcinoma models and an extensive analysis of immune correlates was performed in the tumor and tumor-draining lymph node (dLN).ResultsWe demonstrate that N-809 blocks PD-L1 and induces IL-15-dependent immune effects. N-809 was well-tolerated and reduced 4T1 lung metastasis, decreased MC38 tumor burden and increased survival versus N-803+αPD-L1. Compared with N-803+αPD-L1, N-809 enhanced natural killer (NK) and CD8+T-cell activation and function in the dLN and TME, relating to increased gene expression associated with interferon and cytokine signaling, lymphoid compartment, costimulation and cytotoxicity. The higher number of TME CD8+T cells was attributed to enhanced infiltration, not in situ expansion. Increased TME NK and CD8+T-cell numbers correlated with augmented chemokine ligands and receptors. Moreover, in contrast to N-803+αPD-L1, N-809 reduced immunosuppressive regulatory T cells (Treg), monocytic myeloid-derived suppressor cells (M-MDSC) and M2-like macrophages in the TME.ConclusionsOur results suggest that N-809 functions by a novel immune mechanism to promote antitumor efficacy. Foremost, N-809 enhances intratumoral lymphocyte numbers by increasing trafficking via altered chemokine levels in the TME and chemokine receptor expression on CD8+T cells and NK cells. In addition, N-809 reduces immunosuppressive and pro-tumorigenic immune cells in the TME, including Treg, M2-like macrophages and M-MDSC. Overall, these novel effects of N-809 promote an inflamed TME, leading to lower tumor burden and increased survival. These results provide mechanistic insight and rationale supporting the potential clinical study of N-809 in patients with carcinoma.

BackgroundDespite the success of immune checkpoint blockade therapy in the treatment of certain cancer types, only a small percentage of patients with solid malignancies achieve a durable response. Consequently, there is a need to develop novel approaches that could overcome mechanisms of tumor resistance to checkpoint inhibition. Emerging evidence has implicated the phenomenon of cancer plasticity or acquisition of mesenchymal features by epithelial tumor cells, as an immune resistance mechanism.MethodsTwo soluble factors that mediate tumor cell plasticity in the context of epithelial-mesenchymal transition are interleukin 8 (IL-8) and transforming growth factor beta (TGF-β). In an attempt to overcome escape mechanisms mediated by these cytokines, here we investigated the use of a small molecule inhibitor of the IL-8 receptors CXCR1/2, and a bifunctional agent that simultaneously blocks programmed death ligand 1 (PD-L1) and traps soluble TGF-β.ResultsWe demonstrate that simultaneous inhibition of CXCR1/2, TGF-β, and PD-L1 signaling synergizes to reduce mesenchymal tumor features in murine models of breast and lung cancer, and to markedly increase expression of tumor epithelial E-cadherin while reducing infiltration with suppressive granulocytic myeloid-derived suppressor cells, significantly enhancing T-cell infiltration and activation in tumors, and leading to improved antitumor activity.ConclusionsThis study highlights the potential benefit of combined blockade of CXCR1/2 and TGF-β signaling for modulation of tumor plasticity and potential enhancement of tumor responses to PD-L1 blockade. The data provide rationale for the evaluation of this novel approach in the clinic.

BackgroundClinical trials have shown the ability of therapeutic vaccines to generate immune responses to tumor-associated antigens (TAAs). What is relatively less known is if this translates into immune-cell (IC) infiltration into the tumor microenvironment. This study examined whether neoadjuvant prostate-specific antigen (PSA)-targeted vaccination with PROSTVAC could induce T-cell immunity, particularly at the tumor site.MethodsAn open-label, phase II study of neoadjuvant PROSTVAC vaccine enrolled 27 patients with localized prostate cancer awaiting radical prostatectomy (RP). We evaluated increases in CD4 and CD8 T-cell infiltrates (RP tissue vs baseline biopsies) using a six-color multiplex immunofluorescence Opal method. Antigen-specific responses were assessed by intracellular cytokine staining after in vitro stimulation of peripheral blood mononuclear cells with overlapping 15-mer peptide pools encoding the TAAs PSA, brachyury and MUC-1.ResultsOf 27 vaccinated patients, 26 had matched prevaccination (biopsy) and postvaccination (RP) prostate samples available for non-compartmentalized analysis (NCA) and compartmentalized analysis (CA). Tumor CD4 T-cell infiltrates were significantly increased in postvaccination RP specimens compared with baseline biopsies by NCA (median 176/mm² vs 152/mm²; IQR 136–317/mm² vs 69–284/mm²; p=0.0249; median ratio 1.20; IQR 0.64–2.25). By CA, an increase in both CD4 T-cell infiltrates at the tumor infiltrative margin (median 198/mm² vs 151/mm²; IQR 123–500/mm² vs 85–256/mm²; p=0.042; median ratio 1.44; IQR 0.59–4.17) and in CD8 T-cell infiltrates at the tumor core (median 140/mm² vs 105/mm²; IQR 91–175/mm² vs 83–163/mm²; p=0.036; median ratio 1.25; IQR 0.88–2.09) were noted in postvaccination RP specimens compared with baseline biopsies. A total of 13/25 patients (52%) developed peripheral T-cell responses to any of the three tested TAAs (non-neoantigens); five of these had responses to more than one antigen of the three evaluated.ConclusionNeoadjuvant PROSTVAC can induce both tumor immune response and peripheral immune response.Trial registration numberNCT02153918.

BackgroundAlthough immune checkpoint inhibitors have revolutionized cancer treatment, clinical benefit with this class of agents has been limited to a subset of patients. Hence, more effective means to target tumor cells that express immune checkpoint molecules should be developed. For the first time, we report a novel natural killer (NK) cell line, programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK), which was derived from NK-92 and was engineered to express high-affinity CD16, endoplasmic reticulum-retained interleukin (IL)-2, and a PD-L1-specific chimeric antigen receptor (CAR). We show that PD-L1 t-haNK cells also retained the expression of native NK receptors and carried a high content of granzyme and perforin granules.MethodsNanoString, flow cytometry, and immunofluorescence analyses were performed to characterize the phenotype of irradiated PD-L1 t-haNK cells. In vitro PD-L1 t-haNK cell activity against cancer cell lines and human peripheral blood mononuclear cells (PBMCs) was determined via flow-based and111In-release killing assays. The antitumor effect of PD-L1 t-haNK cells in vivo was investigated using MDA-MB-231, H460, and HTB1 xenograft models in NOD-scid IL2Rgammanull(NSG) mice. Additionally, the antitumor effect of PD-L1 t-haNK cells, in combination with anti-PD-1 and N-803, an IL-15 superagonist, was evaluated using mouse oral cancer 1 syngeneic model in C57BL/6 mice.ResultsWe show that PD-L1 t-haNK cells expressed PD-L1-targeting CAR and CD16, retained the expression of native NK receptors, and carried a high content of granzyme and perforin granules. In vitro, we demonstrate the ability of irradiated PD-L1 t-haNK cells to lyse 20 of the 20 human cancer cell lines tested, including triple negative breast cancer (TNBC) and lung, urogenital, and gastric cancer cells. The cytotoxicity of PD-L1 t-haNK cells was correlated to the PD-L1 expression of the tumor targets and can be improved by pretreating the targets with interferon (IFN)-γ. In vivo, irradiated PD-L1 t-haNK cells inhibited the growth of engrafted TNBC and lung and bladder tumors in NSG mice. The combination of PD-L1 t-haNK cells with N-803 and anti-PD-1 antibody resulted in superior tumor growth control of engrafted oral cavity squamous carcinoma tumors in C57BL/6 mice. In addition, when cocultured with human PBMCs, PD-L1 t-haNK cells preferentially lysed the myeloid-derived suppressor cell population but not other immune cell types.ConclusionThese studies demonstrate the antitumor efficacy of PD-L1 t-haNK cells and provide a rationale for the potential use of these cells in clinical studies.

Clinical benefit remains elusive for most patients with poorly inflamed carcinomas treated with immune checkpoint blockade. Converting the tumor microenvironment into a functionally inflamed immune hub would increase clinical benefit. By using comprehensive single-cell transcriptome, proteome, and immune cell analysis, we demonstrate here that Entinostat, a class I histone deacetylase inhibitor, facilitates accumulation of the necrosis-targeted recombinant murine immune-cytokine, NHS-rmIL12, in experimental mouse colon carcinomas and poorly immunogenic breast tumor in which the therapy was curative. Combination therapy reprogrammed the tumor innate and adaptive immune milieu to an inflamed landscape, where the concerted action of highly functional CD8+ T cells and activated neutrophils drove a dramatic macrophage M1-like polarization leading to complete tumor eradication in 41.7%-100% of cases. Biomarker signature of favourable overall survival in multiple human tumor types showed close resemblance to the immune pattern generated by Entinostat/NHS-rmIL12 combination therapy. Collectively, these findings provide a rationale for combining NHS-IL12 with Entinostat in the clinical setting. Citation Format: Kristin C. Hicks, Paul L. Chariou, Yohei Ozawa, Christine M. Minnar, Karin M. Knudson, Thomas J. Meyer, Jing Bian, Margaret Cam, Jeffrey Schlom, Sofia R. Gameiro. Tumor-targeted interleukin-12 and Entinostat combination therapy improves cancer survival by reprogramming the tumor immune cell landscape [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P034.

Background Checkpoint inhibitors have not been effective for prostate cancer as single agents. Durvalumab is a human IgG1-K monoclonal antibody that targets programmed death ligand 1 and is approved by the U.S. Food and Drug Administration for locally advanced or metastatic urothelial cancer and locally advanced, unresectable stage 3 non-small cell lung cancer. Olaparib, a poly (ADP-ribose) polymerase inhibitor, has demonstrated an improvement in median progression-free survival (PFS) in select patients with metastatic castration-resistant prostate cancer (mCRPC). Data from other trials suggest there may be improved activity in men with DNA damage repair (DDR) mutations treated with checkpoint inhibitors. This trial evaluated durvalumab and olaparib in patients with mCRPC with and without somatic or germline DDR mutations. Methods Eligible patients had received prior enzalutamide and/or abiraterone. Patients received durvalumab 1500 mg i.v. every 28 days and olaparib 300 mg tablets p.o. every 12 h until disease progression or unacceptable toxicity. All patients had biopsies of metastatic lesions with an evaluation for both germline and somatic mutations. Results Seventeen patients received durvalumab and olaparib. Nausea was the only nonhematologic grade 3 or 4 toxicity occurring in > 1 patient (2/17). No patients were taken off trial for toxicity. Median radiographic progression-free survival (rPFS) for all patients is 16.1 months (95% CI: 4.5–16.1 months) with a 12-month rPFS of 51.5% (95% CI: 25.7–72.3%). Activity is seen in patients with alterations in DDR genes, with a median rPFS of 16.1 months (95% CI: 7.8–18.1 months). Nine of 17 (53%) patients had a radiographic and/or PSA response. Patients with fewer peripheral myeloid-derived suppressor cells and with alterations in DDR genes were more likely to respond. Early changes in circulating tumor cell counts and in both innate and adaptive immune characteristics were associated with response. Conclusions Durvalumab plus olaparib has acceptable toxicity, and the combination demonstrates efficacy, particularly in men with DDR abnormalities. Trial registration ClinicalTrials.gov identifier: NCT02484404. Electronic supplementary material The online version of this article (10.1186/s40425-018-0463-2) contains supplementary material, which is available to authorized users.

Natural killer (NK) cells are innate cytotoxic lymphocytes that play a fundamental role in the immunosurveillance of cancers. NK cells of cancer patients exhibit impaired function mediated by immunosuppressive factors released from the tumor microenvironment (TME), such as transforming growth factor (TGF)-β1. An interleukin (IL)-15 superagonist/IL-15 receptor α fusion complex (IL-15SA/IL-15RA; ALT-803) activates the IL-15 receptor on CD8 T cells and NK cells, and has shown significant anti-tumor activity in several in vivo studies. This in vitro study investigated the efficacy of IL-15SA/IL-15RA on TGF-β1-induced suppression of NK cell-cytotoxic function. IL-15SA/IL-15RA inhibited TGF-β1 from decreasing NK cell lysis of four of four tumor cell lines (H460, LNCap, MCF7, MDA-MB-231). IL-15SA/IL-15RA rescued healthy donor and cancer patient NK cell-cytotoxicity, which had previously been suppressed by culture with TGF-β1. TGF-β1 downregulated expression of NK cell-activating markers and cytotoxic granules, such as CD226, NKG2D, NKp30, granzyme B, and perforin. Smad2/3 signaling was responsible for this TGF-β1-induced downregulation of NK cell-activating markers and cytotoxic granules. IL-15SA/IL-15RA blocked Smad2/3-induced transcription, resulting in the rescue of NK cell-cytotoxic function from TGF-β1-induced suppression. These findings suggest that in addition to increasing NK cell function via promoting the IL-15 signaling pathway, IL-15SA/IL-15RA can function as an inhibitor of TGF-β1 signaling, providing a potential remedy for NK cell dysfunction in the immunosuppressive tumor microenvironment.

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.

BackgroundLeukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an immune inhibitory receptor that binds collagen-like domains commonly found in extracellular matrix (ECM) collagens and complement component C1q. LAIR-1 is expressed on several immune cell types including activated T cells, B cells, NK cells, dendritic cells, and macrophages. Numerous cancer types including gastric, colon, ovarian, bladder, and others, upregulate collagens which enhances tumor growth, metastases, and invasion while actively suppressing antitumor immunity. While a soluble decoy, LAIR-2, is expressed in humans and competes with LAIR-1 for binding of collagen domains, excess LAIR ligands in the tumor often result in an immune suppressive environment.MethodsHere, we report on a novel immunotherapy approach combining NC410, a novel fusion protein consisting of two LAIR-2 molecules grafted on to an IgG1 antibody backbone, capable of targeting the tumor ECM and blocking LAIR-1 signaling; and bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the human transforming growth factor β receptor II (TGF-βRII or TGF-β ”trap”) fused via a flexible linker to the C-terminus of each heavy chain of an IgG1 antibody blocking programmed death ligand 1 (anti–PD-L1).ResultsWe have demonstrated that the combination of NC410 and bintrafusp alfa more effectively controls in vivo tumor growth of the collagen rich MC38 colon and EMT6 mammary carcinomas compared to either monotherapy. We demonstrate that this potent anti-tumor immune response is propagated through the synergy of activated tumor infiltrating lymphocytes and a repolarization of myeloid cells in the tumor microenvironment. MC38 tumors treated with the combination of NC410 plus bintrafusp alfa contained higher numbers of infiltrating T cells, NK cells, and M1 polarized macrophages.ConclusionsThis study highlights the synergy of reshaping the large suppressive myeloid cell populations often present in tumors with activation of adaptive T-cell immune responses dampened by checkpoint inhibition. The results also provide the rationale for the future evaluation of this combination therapy in the clinic.

BackgroundPRGN-2009 is a novel gorilla adenovirus vaccine containing 35 non-HLA-restricted epitopes of HPV 16 and 18 which is being tested in an open-label, NCI-sponsored, single-center Phase I/II study alone and combined with the bifunctional TGF-β ”trap”/anti-PD-L1 fusion protein bintrafusp alfa (BA) (NCT04432597).MethodsFor the Phase I of the trial, eligible patients are adults with previously treated (checkpoint blockade allowed) recurrent/metastatic HPV-associated cancers. Objectives are to assess the safety and determine the recommended phase 2 dose (RP2D) of PRGN-2009 alone and combined with BA. Treatment followed a single-agent 3+3 dose escalation at two dose levels of PRGN-2009 (dose level 1: 1x1011 viral particle units (VPU), dose level 2: 5x1011 VPU) subcutaneously Q2W for 3 times, then Q4W for up to one year in total. After determination of RP2D, a combination cohort of 10 patients treated with PRGN-2009 at the RP2D combined with BA (1200 mg IV Q2W for 52 weeks) opened. Peripheral blood mononuclear cells collected from patients before and after vaccination with PRGN-2009 were stimulated with overlapping peptide pools and assessed by intracellular cytokine staining to identify HPV-16 and HPV-18 specific T-cells, as well as T-cells targeting cascade antigens not encoded by the vaccine.ResultsSix patients were enrolled in the single-agent PRGN-2009 dose-escalation phase (3 with cervical cancer, 2 with anal cancer, 1 with vaginal cancer). Observed adverse events were Grade 1-2 flu-like syndrome (headache, body aches), injection site reactions (erythema, pruritus, soreness, localized edema), fatigue, and rash. There were no dose limiting toxicities, and 5x1011 VPU was selected as RP2D. Four patients had stable disease as best response, (one ongoing, 10 months on treatment).T-cells targeting HPV-16 and/or HPV-18 were increased after vaccination in 100% of patients, with 3/6 (50%) developing HPV-16 T cells and 5/6 (83%) developing HPV-18 T cells. In some patients, the magnitude and breadth of HPV-16 and HPV-18 specific T cells were notably increased after repeated vaccination. T cells that target the cascade antigens brachyury and MUC1 were also increased in all patients evaluated. Multifunctional T-cell responses against all these antigens were also developed after vaccination in the majority of patients. No differences in immunogenicity were noted between the two dose levels. Enrollment is underway in combination with BA. Updated data will be presented.ConclusionsThe Phase 1 results demonstrate the safety of single-agent PRGN-2009 and induction of anti-HPV T-cell immune responses, supporting the hypothesis that PRGN-2009 could potentially induce anti-tumor effects in HPV-associated cancers.AcknowledgementsThis research was supported in part by the Intramural Research Program of the NIH, NCI.Trial RegistrationNCT04432597Ethics ApprovalApproved by the NIH IRB (Ref No 543876). All participants have given informed consent before taking part in the study.

BackgroundRecurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alfa is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types.MethodsWe conducted a phase II clinical trial evaluating bintrafusp alfa in adults with RRP. Papilloma samples before and after treatment with bintrafusp alfa were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison.ResultsDual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject’s own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas.ConclusionsIntact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP.Trial registration numbersNCT03707587 and NCT02859454.

// Anthony S. Malamas 1 , Scott A. Hammond 2 , Jeffrey Schlom 1 and James W. Hodge 1 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA 2 MedImmune LLC, Gaithersburg, Maryland, USA Correspondence to: James W. Hodge, email: // Keywords : OX40, cancer vaccines, combination immunotherapy, twist Received : July 22, 2017 Accepted : July 23, 2017 Published : August 05, 2017 Abstract OX40 is a costimulatory receptor that potentiates proliferation, survival, memory formation, and effector function of CD4 + and CD8 + T-cells, while overcoming the suppressive activity of regulatory T-cells (Tregs). Here, we explored the combination of an OX40L fusion protein (OX40L-FP) with a poxvirus-based cancer vaccine (MVA-Twist-TRICOM) to inhibit tumor metastasis in the 4T1 murine breast cancer model. Contrary to the single agent treatments, the combination therapy significantly decreased the number of metastatic colonies per lung and prolonged survival. Depletion studies demonstrated that these effects were mediated by both CD4 + and CD8 + T-cells. The combination therapy a) increased the total number of T-cells in the CD4 + Foxp3 - population and the CD4 + central and effector memory subsets within the lung, spleen, and draining lymph node, b) enhanced infiltration of CD4 + T-cells into metastatic areas of the lung, and (c) increased the number of functional CD8 + T-cells that produced IFNγ and TNFα. The combination therapy also promoted the development of KLRG1 - CD127 + memory precursor CD8 + T-cells, while reducing those with a KLRG1 + terminally differentiated phenotype. Moreover, the combination of OX40L-FP and vaccine induced greater CD4 + and CD8 + Twist-specific responses. In addition, Tregs isolated from mice receiving the combination were also less immunosuppressive in ex-vivo proliferation assays than those from the OX40L-FP and MVA-Twist-TRICOM monotherapy groups. Such results provide the rationale to combine co-stimulatory agonists with cancer vaccines for the treatment of tumor metastasis.

// Jonathan K. Fallon 1 , Amanda J. Vandeveer 1 , Jeffrey Schlom 1,* and John W. Greiner 1,* 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA * These authors have contributed equally to this work Correspondence to: Jeffrey Schlom, email: // Keywords : checkpoint inhibitor, PD-L1, immunocytokine, interleukin-12, immunotherapy Received : February 01, 2017 Accepted : March 03, 2017 Published : March 11, 2017 Abstract The combined therapeutic potential of an immunocytokine designed to deliver IL-12 to the necrotic regions of solid tumors with an anti-PD-L1 antibody that disrupts the immunosuppressive PD-1/PD-L1 axis yielded a combinatorial benefit in multiple murine tumor models. The murine version of the immunocytokine, NHS-muIL12, consists of an antibody (NHS76) recognizing DNA/DNA-histone complexes, fused with two molecules of murine IL-12 (NHS-muIL12). By its recognition of exposed DNA, NHS-muIL12 targets IL-12 to the necrotic portions of tumors; it has a longer plasma half-life and better antitumor efficacy against murine tumors than recombinant murine IL-12. It is shown here that NHS-muIL12, in an IFN-γ‒dependent mechanism, upregulates mPD-L1 expression on mouse tumors, which could be construed as an immunosuppressive action. Yet concurrent therapy with NHS-muIL12 and an anti-PD-L1 antibody resulted in additive/synergistic antitumor effects in PD-L1‒expressing subcutaneously transplanted tumors (MC38, MB49) and in an intravesical bladder tumor model (MB49). Antitumor efficacy correlated with (a) with a higher frequency of tumor antigen-specific splenic CD8+ T cells and (b) enhanced T cell activation over a wide range of NHS-muIL12 concentrations. These findings suggest that combining NHS-muIL12 and an anti-PD-L1 antibody enhances T cell activation and T cell effector functions within the tumor microenvironment, significantly improving overall tumor regression. These results should provide the rationale to examine the combination of these agents in clinical studies.

Immunosuppressive entities in the tumor microenvironment (TME) remain a major impediment to immunotherapeutic approaches for a majority of patients with cancer. While the immunosuppressive role of transforming growth factor-β (TGF-β) in the TME is well known, clinical studies to date with anti-TGF-β agents have led to limited success. The bifunctional agent bintrafusp alfa (previously designated M7824) has been developed in an attempt to address this issue. Bintrafusp alfa consists of an IgG1targeting programmed death ligand 1 (PD-L1) moiety fused via peptide linkers to the extracellular domain of two TGF-β receptor II molecules designed to ‘trap’ TGF-β in the TME. This agent is able to bring the TGF-β trap to the TME via its anti-PD-L1 component, thus simultaneously attacking both the immunosuppressive PD-L1 and TGF-β entities. A number of preclinical studies have shown bintrafusp alfa capable of (1) preventing or reverting TGF-β-induced epithelial-mesenchymal transition in human carcinoma cells; this alteration in tumor cell plasticity was shown to render human tumor cells more susceptible to immune-mediated attack as well as to several chemotherapeutic agents; (2) altering the phenotype of natural killer and T cells, thus enhancing their cytolytic ability against tumor cells; (3) mediating enhanced lysis of human tumor cells via the antibody-dependent cell-mediated cytotoxicity mechanism; (4) reducing the suppressive activity of Tregcells; (5) mediating antitumor activity in numerous preclinical models and (6) enhancing antitumor activity in combination with radiation, chemotherapy and several other immunotherapeutic agents. A phase I clinical trial demonstrated a safety profile similar to other programmed cell death protein 1 (PD-1)/PD-L1 checkpoint inhibitors, with objective and durable clinical responses. We summarize here preclinical and emerging clinical data in the use of this bispecific and potentially multifunctional agent.

Background Thymic 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. Methods Seven 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. Results Two 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. Conclusion These 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 registration ClinicalTrials.gov - NCT01772004. Date of registration – January 21, 2013. Electronic supplementary material The online version of this article (10.1186/s40425-019-0723-9) contains supplementary material, which is available to authorized users.

Background HuMax-IL8 (now known as BMS-986253) is a novel, fully human monoclonal antibody that inhibits interleukin-8 (IL-8), a chemokine that promotes tumor progression, immune escape, epithelial-mesenchymal transition, and recruitment of myeloid-derived suppressor cells. Studies have demonstrated that high serum IL-8 levels correlate with poor prognosis in many malignant tumors. Preclinical studies have shown that IL-8 blockade may reduce mesenchymal features in tumor cells, making them less resistant to treatment. Methods Fifteen patients with metastatic or unresectable locally advanced solid tumors were enrolled in this 3 + 3 dose-escalation trial at four dose levels (4, 8, 16, or 32 mg/kg). HuMax-IL8 was given IV every 2 weeks, and patients were followed for safety and immune monitoring at defined intervals up to 52 weeks. Results All enrolled patients (five chordoma, four colorectal, two prostate, and one each of ovarian, papillary thyroid, chondrosarcoma, and esophageal) received at least one dose of HuMax-IL8. Eight patients had received three or more prior lines of therapy and five patients had received prior immunotherapy. Treatment-related adverse events occurred in five patients (33%), mostly grade 1. Two patients receiving the 32 mg/kg dose had grade 2 fatigue, hypophosphatemia, and hypersomnia. No dose-limiting toxicities were observed, and maximum tolerated dose was not reached. Although no objective tumor responses were observed, 11 patients (73%) had stable disease with median treatment duration of 24 weeks (range, 4–54 weeks). Serum IL-8 was significantly reduced on day 3 of HuMax-IL8 treatment compared to baseline (p = 0.0004), with reductions in IL-8 seen at all dose levels. Conclusions HuMax-IL8 is safe and well-tolerated. Ongoing studies are evaluating the combination of IL-8 blockade and other immunotherapies. Trial registration NCTN, NCT02536469. Registered 23 August 2015, https://clinicaltrials.gov/ct2/show/NCT02536469?term=NCT02536469&rank=1. Electronic supplementary material The online version of this article (10.1186/s40425-019-0706-x) contains supplementary material, which is available to authorized users.

Progressive tumor growth is associated with deficits in the immunity generated against tumor antigens. Vaccines targeting tumor neoepitopes have the potential to address qualitative defects; however, additional mechanisms of immune failure may underlie tumor progression. In such cases, patients would benefit from additional immune-oncology agents targeting potential mechanisms of immune failure. This study explores the identification of neoepitopes in the MC38 colon carcinoma model by comparison of tumor to normal DNA and tumor RNA sequencing technology, as well as neoepitope delivery by both peptide- and adenovirus-based vaccination strategies. To improve antitumor efficacies, we combined the vaccine with a group of rationally selected immune-oncology agents. We utilized an IL15 superagonist to enhance the development of antigen-specific immunity initiated by the neoepitope vaccine, PD-L1 blockade to reduce tumor immunosuppression, and a tumor-targeted IL12 molecule to facilitate T-cell function within the tumor microenvironment. Analysis of tumor-infiltrating leukocytes demonstrated this multifaceted treatment regimen was required to promote the influx of CD8+ T cells and enhance the expression of transcripts relating to T-cell activation/effector function. Tumor-targeted IL12 resulted in a marked increase in clonality of T-cell repertoire infiltrating the tumor, which when sculpted with the addition of either a peptide or adenoviral neoepitope vaccine promoted efficient tumor clearance. In addition, the neoepitope vaccine induced the spread of immunity to neoepitopes expressed by the tumor but not contained within the vaccine. These results demonstrate the importance of combining neoepitope-targeting vaccines with a multifaceted treatment regimen to generate effective antitumor immunity.

Background While significant strides in the treatment of metastatic bladder cancer have been made with immune checkpoint inhibitors, the treatment of carcinoma in situ and non-muscle invasive, non-metastatic (superficial) human urothelial carcinoma, also termed non-muscle invasive bladder cancer (NMIBC), remains intractable with bacillus Calmette-Guerin (BCG) employed as the standard of care. In this study, an immunocytokine, NHS-muIL12, which consists of two molecules of murine IL-12 fused to NHS76, a tumor necrosis-targeting human IgG1, was examined as an immunotherapeutic in an orthotopic MB49luc bladder tumor model. Methods The antitumor activity of systemic administration of NHS-muIL12 was investigated on MB49luc tumors, an aggressive, bioluminescent orthotopic bladder cancer model. Temporal studies were carried out on MB49luc bladder tumors harvested during various time points during NHS-muIL12 treatment and cellular changes associated with the reduction in tumor burden following NHS-muIL12 were determined by flow cytometry. Effects of those changes on the proliferation/activation of lymphoid cells were also determined. Results Studies revealed a significant reduction in MB49luc bladder tumor burden occurring between days 3 and 6 after the third and final systemic administration of NHS-muIL12. Temporal analyses of the MB49luc bladder tumor microenvironment (TME) initially revealed a large accumulation of myeloid-derived suppressor cells (MDSCs) and macrophages that elicited potent immunosuppression. Immunosuppression was characterized by the inability of CD4+ and CD8+ T cells to respond to broad-based immune stimulants. NHS-muIL12 administration resulted in temporal-dependent reductions in the number of MDSCs, macrophages and tumor-associated TGF-β, which culminated in a re-ignition of CD4+ and CD8+ T cells to elicit potent antitumor responses against MB49luc bladder tumors. Conclusions These findings provide strong evidence that the systemic administration of an immunocytokine consisting of a tumor-targeting Ig through recognition of DNA and DNA-histone complexes coupled to muIL-12 can effectively target the bladder TME; this significantly reduces the myeloid cellular compartment and reverts an immunosuppressive to an immunopermissive TME, ultimately resulting in antitumor effects. These studies provide further rationale for the employment of NHS-IL12 as an immunomodulator and clinical immunotherapeutic for NMIBC.

Background Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV)-driven disorder that causes substantial morbidity and can lead to fatal distal airway obstruction and post-obstructive pneumonias. Patients require frequent surgical debridement of disease, and no approved systemic adjuvant therapies exist. Methods A phase II study was conducted to investigate the clinical activity and safety of programmed death-ligand 1 (PD-L1) blockade with avelumab in patients with RRP. Results Twelve patients were treated. All patients with laryngeal RRP displayed improvement in disease burden, and 5 of 9 (56%) displayed partial responses. None of 4 patients with pulmonary RRP displayed a response. Using each patient’s surgical history as their own control, patients required fewer surgical interventions after avelumab treatment (p = 0.008). A subset of partial responders developed HPV-specific reactivity in papilloma-infiltrating T-cells that correlated with reduced HPV viral load and an increased Tissue Inflammation Signature. Conclusions Avelumab demonstrated safety and clinical activity in patients with laryngeal RRP. Further study of immune checkpoint blockade for RRP, possibly with longer treatment duration or in combination with other immunotherapies aimed at activating antiviral immunity, is warranted. Trial registration NCT, number NCT02859454, registered August 9, 2016. Electronic supplementary material The online version of this article (10.1186/s40425-019-0603-3) contains supplementary material, which is available to authorized users.

BackgroundAntitumor vaccines targeting tumor-associated antigens (TAAs) can generate antitumor immune response. A novel vaccine platform using adenovirus 5 (Ad5) vectors [E1–, E2b–] targeting three TAAs—prostate-specific antigen (PSA), brachyury, and MUC-1—has been developed. Both brachyury and the C-terminus of MUC-1 are overexpressed in metastatic castration-resistant prostate cancer (mCRPC) and have been shown to play an important role in resistance to chemotherapy, epithelial–mesenchymal transition, and metastasis. The transgenes for PSA, brachyury, and MUC-1 all contain epitope modifications for the expression of CD8+ T-cell enhancer agonist epitopes. We report here the first-in-human trial of this vaccine platform.MethodsPatients with mCRPC were given concurrently three vaccines targeting PSA, brachyury, and MUC-1 at 5×1011 viral particles (VP) each, subcutaneously every 3 weeks for a maximum of three doses (dose de-escalation cohort), followed by a booster vaccine every 8 weeks for 1 year (dose-expansion cohort only). The primary objective was to determine the safety and the recommended phase II dose. Immune assays and clinical responses were evaluated.ResultsEighteen patients with mCRPC were enrolled between July 2018 and September 2019 and received at least one vaccination. Median PSA was 25.58 ng/mL (range, 0.65–1006 ng/mL). The vaccine was tolerable and safe, and no grade >3 treatment-related adverse events or dose-limiting toxicities (DLTs) were observed. One patient had a partial response, while five patients had confirmed PSA decline and five had stable disease for >6 months. Median progression-free survival was 22 weeks (95% CI: 19.1 to 34). Seventeen (100%) of 17 patients mounted T-cell responses to at least one TAA, whereras 8 (47%) of 17 patients mounted immune responses to all three TAAs. Multifunctional T-cell responses to PSA, MUC-1, and brachyury were also detected after vaccination in the majority of the patients.ConclusionsAd5 PSA/MUC-1/brachyury vaccine is well tolerated. The primary end points were met and there were no DLTs. The recommended phase II dose is 5×1011 VP. The vaccine demonstrated clinical activity, including one partial response and confirmed PSA responses in five patients. Three patients with prolonged PSA responses received palliative radiation therapy. Further research is needed to evaluate the clinical benefit and immunogenicity of this vaccine in combination with other immuno-oncology agents and/or palliative radiation therapy.Trial registration numberNCT03481816.

BackgroundAs heterogeneous tumors develop in the face of intact immunity, tumor cells harboring genomic or expression defects that favor evasion from T-cell detection or elimination are selected. For patients with such tumors, T cell-based immunotherapy alone infrequently results in durable tumor control.MethodsHere, we developed experimental models to study mechanisms of T-cell escape and demonstrated that resistance to T-cell killing can be overcome by the addition of natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) targeting programmed death ligand-1 (PD-L1).ResultsIn engineered models of tumor heterogeneity, PD-L1 CAR-engineered NK cells (PD-L1 t-haNKs) prevented the clonal selection of T cell-resistant tumor cells observed with T-cell treatment alone in multiple models. Treatment of heterogenous cancer cell populations with T cells resulted in interferon gamma (IFN-γ) release and subsequent upregulation of PD-L1 on tumor cells that escaped T-cell killing through defects in antigen processing and presentation, priming escape cell populations for PD-L1 dependent killing by PD-L1 t-haNKs in vitro and in vivo.ConclusionsThese results describe the underlying mechanisms governing synergistic antitumor activity between T cell-based immunotherapy that results in IFN-γ production, upregulation of PD-L1 on T-cell escape cells, and the use of PD-L1 CAR-engineered NK cells to target and eliminate resistant tumor cell populations.

BackgroundThe standard treatment for non-metastatic castration sensitive prostate cancer (nmCSPC) is androgen deprivation therapy (ADT) or surveillance. This study evaluated the potential synergy of immunotherapy and enzalutamide (without ADT) in nmCSPC. In addition, the immunologic impact of enzalutamide was also evaluated in men with normal testosterone.MethodsPatients with rising prostate-specific antigen (PSA) after definitive therapy, normal testosterone and no radiographic metastasis were randomized to enzalutamide for 3 months with/without PROSTVAC for 6 months. Thereafter, patients could be retreated with another 3 month course of enzalutamide when PSA returned to baseline. Immune profiles were evaluated in these patients.ResultsThirty-eight patients were randomized with a median PSA=4.38 ng/dL and PSA doubling time=4.1 months. No difference was observed between the two groups for PSA growth kinetics, but PSA responses to enzalutamide were noteworthy regardless of PROSTVAC. The median PSA decline after short-course enzalutamide without ADT/testosterone lowering therapy was 99% in both courses. The median time to PSA recovery to baseline after each 84-day course of enzalutamide was also noteworthy because of the duration of response after enzalutamide was discontinued. After the first and second 3 month cycle of enzalutamide, PSA recovery to baseline took a median 224 (range 84–1246) and 189 days (78–400), respectively. The most common adverse events related to the enzalutamide were grade 1 fatigue (71%) and grade 1 breast pain/nipple tenderness (81%). The only grade 3 toxicity was aspartate aminotransferase (AST)/alanine aminotransferase (ALT) elevation in two patients. Enzalutamide was independently associated with immune changes, increasing natural killer cells, naïve-T cells, and decreasing myeloid-derived suppressor cells.ConclusionsThree months of enzalutamide without ADT induced substantial PSA control beyond the treatment period and was repeatable, perhaps representing an alternative to intermittent ADT in nmCSPC. In addition, enzalutamide was associated with immune changes that could be relevant as future immune combinations are developed.Trail registration numberclinicaltrials.gov (NCT01875250).

Prime-boost vaccination with recombinant (r) vaccinia(V)-CEA(6D)-TRICOM (triad of co-stimulatory molecules B7.1, ICAM-1 and LFA-3) and rFowlpox(F)-CEA(6D)-TRICOM infect antigen-presenting cells and direct expression of co-stimulatory molecules. We hypothesized that co-administration of vaccine with GM-CSF and interferon alpha (IFN-α) would have efficacy in CEA-expressing cancers. Patients with CEA-expressing cancers received the rV-CEA(6D)-TRICOM vaccine subcutaneously (s.c.) on day 1 followed by GM-CSF s.c. to the injection site on days 1-4. In Cycle 1, patients received thrice weekly s.c. injections of IFN-α-2b the week after rV-CEA(6D)-TRICOM. In Cycles 2-4, patients received thrice weekly s.c. injections of IFN-α-2b the same week that rF-CEA(6D)-TRICOM was given. The first cohort received no IFN followed by dose escalation of IFN-α in subsequent cohorts. Thirty-three patients were accrued (mean 59.8 years). Grade 3 toxicities included fatigue and hyperglycemia. Grade 4-5 adverse events (unrelated to treatment) were confusion (1), elevated aspartate transaminase (AST)/alanine transaminase (ALT) (1), and sudden death (1). No patients had a partial response, and eight patients exhibited stable disease of ≥3 months. Median progression-free survival and overall survival (OS) were 1.8 and 6.3 months, respectively. Significantly higher serum CD27 levels were observed after vaccine therapy (p = 0.006 post 1-2 cycles, p = 0.003 post 3 cycles, p = 0.03 post 4-7 cycles) and 42 % of patients assayed developed CEA-specific T cell responses. Pre-treatment levels of myeloid-derived suppressor cells correlated with overall survival (p = 0.04). Administration of IFN-α led to significantly increased OS (p = 0.02) compared to vaccine alone. While the vaccine regimen produced no clinical responses, IFN-α administration was associated with improved survival.