BackgroundHPV-associated cancers continue to present a major health concern despite the development of prophylactic vaccines. Currently checkpoint inhibition is the only FDA-approved immunotherapy ...available for HPV-associated cancers, and the response rate remains low. PDS0101 is a liposomal nanoparticle HPV16-specific therapeutic vaccine that has previously been shown to generate strong HPV-specific responses in pre-clinical and clinical studies. We assessed the efficacy of PDS0101 in combination with two other immune-modulating agents. PDS0301 (previously designated NHS-IL12, M94241) is a tumor-targeting immunocytokine composed of the histone binding NHS76 antibody fused to two molecules of IL-12. The epigenetic modifier Entinostat (ENT), a class I histone deacetylase (HDAC) inhibitor, has been shown to increase tumor cell susceptibility to cytotoxic T cell mediated killing and increase necrosis.MethodsThe TC-1 murine carcinoma, which expresses HPV16 E6 and E7 but lacks PD-L1, was used in a syngeneic mouse model to evaluate the anti-tumor effects, survival, and changes to peripheral immune cells and the tumor microenvironment induced by combination treatment with PDS0101, PDS0301, and ENT. Tumor infiltrating lymphocytes (TILs) were assessed via flow cytometry, immunohistochemistry, and single cell RNA sequencing. Serum and tumor supernatant analytes were evaluated via ELISA and cytometric bead array.ResultsSuperior anti-tumor activity and prolonged survival were observed when all three drugs were used in combination, and elevated levels of HPV-specific T cells in the tumor correlated strongly with decreased tumor size. PDS0101 was the primary driver of HPV-specific T cells. PDS0301 reduced M2 macrophages in the tumor and increased effector memory CD8 T cells, and ENT decreased CD4 T cells in the tumor. All three agents were necessary for optimal tumor infiltration of granzyme B+ T and NK cells.ConclusionsThese data provide rationale for the combination of therapeutic cancer vaccines, immune-activating tumor targeting cytokines, and HDAC inhibitors in the clinical setting, especially in checkpoint refractory HPV-associated cancers.Ethics ApprovalAll animal procedures reported in this study that were performed by NCI-CCR affiliated staff were approved by the NCI Animal Care and Use Committee (ACUC) and in accordance with federal regulatory requirements and standards.
PURPOSE: Docetaxel has activity against androgen-independent prostate cancer and preclinical studies have shown that taxane-based
chemotherapy can enhance antitumor response of vaccines. The primary ...objective of this study was to determine if concurrent
docetaxel (with dexamethasone) had any effect on generating an immune response to the vaccine. Secondary end points were whether
vaccine could be given safely with docetaxel and the clinical outcome of the treatment regimen. EXPERIMENTAL DESIGN: The vaccination
regimen was composed of (a) recombinant vaccinia virus (rV) that expresses the prostate-specific antigen gene (rV-PSA) admixed
with (b) rV that expresses the B7.1 costimulatory gene (rV-B7.1), and (c) sequential booster vaccinations with recombinant
fowlpox virus (rF-) containing the PSA gene (rF-PSA). Patients received granulocyte macrophage colony-stimulating factor with
each vaccination. Twenty-eight patients with metastatic androgen-independent prostate cancer were randomized to receive either
vaccine and weekly docetaxel or vaccine alone. Patients on the vaccine alone arm were allowed to cross over to receive docetaxel
alone at time of disease progression. The ELISPOT assay was used to monitor immune responses for PSA-specific T cells. RESULTS:
The median increase in these T-cell precursors to PSA was 3.33-fold in both arms following 3 months of therapy. In addition,
immune responses to other prostate cancer-associated tumor antigens were also detected postvaccination. Eleven patients who
progressed on vaccine alone crossed over to receive docetaxel at time of progression. Median progression-free survival on
docetaxel was 6.1 months after receiving vaccine compared with 3.7 months with the same regimen in a historical control. CONCLUSION:
This is the first clinical trial to show that docetaxel can be administered safely with immunotherapy without inhibiting vaccine
specific T-cell responses. Furthermore, patients previously vaccinated with an anticancer vaccine may respond longer to docetaxel
compared with a historical control of patients receiving docetaxel alone. Larger prospective clinical studies will be required
to validate these findings.
Purpose: We reported previously the first randomized study of any kind in patients with nonmetastatic, castrate-resistant prostate
cancer. The study employed vaccine, the hormone nilutamide, and the ...combined therapy (crossover for each arm) with an endpoint
of time to progression. We now report survival analyses at 6.5 years from the initiation of therapy with a median potential
follow-up of 4.4 years.
Experimental Design: Forty-two patients were randomized to receive either a poxvirus-based prostate-specific antigen (PSA) vaccine or nilutamide.
Patients in either arm who developed increasing PSA without radiographic evidence of metastasis could cross over to receive
the combined therapies.
Results: Median survival among all patients was 4.4 years from date of enrollment. Median survival exhibited a trend toward improvement
for patients initially randomized to the vaccine arm (median, 5.1 versus 3.4 years; P = 0.13). Starting from the on-study date, the retrospectively determined subset of 12 patients who initially received vaccine
and then later received nilutamide suggested improved survival compared with the 8 patients who began with nilutamide and
subsequently were treated with vaccine (median, 6.2 versus 3.7 years; P = 0.045). A subgroup analysis of patients randomized to the vaccine arm versus the nilutamide arm showed substantial improvements
in survival if at baseline patients had a Gleason score <7 ( P = 0.033) and PSA <20 ng/dL ( P = 0.013) or who had prior radiation therapy ( P = 0.018).
Conclusions: These data indicate that patients with nonmetastatic castration-resistant prostate cancer (D0.5) who receive vaccine before
second-line hormone therapy may potentially result in improved survival compared with patients who received hormone therapy
and then vaccine. These data also suggest that patients with more indolent disease may derive greater clinical benefit from
vaccine alone or vaccine before second-line hormone therapy compared with hormone therapy alone or hormone therapy followed
by vaccine. These findings have potential implications for both the design and endpoint analysis of larger vaccine combination
therapy trials.
Purpose: Many patients with clinically localized prostate cancer develop biochemical failure despite excellent local therapy perhaps
due to occult metastatic disease. One potential solution is the ...utilization of a well-tolerated systemic therapy (e.g., vaccine)
in concert with local therapy.
Experimental Design: We present a randomized phase II clinical trial designed to determine if a poxviral vaccine encoding prostate-specific antigen
(PSA) can induce a PSA-specific T-cell response when combined with radiotherapy in patients with clinically localized prostate
cancer. Thirty patients were randomized in a 2:1 ratio into vaccine plus radiotherapy or radiotherapy-only arms. Those patients
in the combination arm received a “priming” vaccine with recombinant vaccinia (rV) PSA plus r V containing the T-cell costimulatory
molecule B7.1 (rV-B7.1) followed by monthly booster vaccines with recombinant fowlpox PSA. The vaccines were given with local
granulocyte-macrophage colony-stimulating factor and low-dose systemic interleukin-2. Standard external beam radiation therapy
was given between the fourth and the sixth vaccinations.
Results: Seventeen of 19 patients in the combination arm completed all eight vaccinations and 13 of these 17 patients had increases
in PSA-specific T cells of at least 3-fold versus no detectable increases in the radiotherapy-only arm ( P < 0.0005). There was also evidence of de novo generation of T cells to well-described prostate-associated antigens not found in the vaccine, providing indirect evidence
of immune-mediated tumor killing. The vaccine was well tolerated.
Conclusion: This vaccine regimen can be safely given in patients undergoing radiation therapy for localized prostate cancer, with the
majority of patients generating a PSA-specific cellular immune response to vaccine.
Purpose: Poxviral vectors have a proven safety record and can be used to incorporate multiple transgenes. Prior clinical trials with
poxviral vaccines have shown that immunologic tolerance to ...self-antigens can be broken. Carcinoembryonic antigen (CEA) and
MUC-1 are overexpressed in a substantial proportion of common solid carcinomas. The primary end point of this study was vaccine
safety, with immunologic and clinical responses as secondary end points.
Experimental Design: We report here a pilot study of 25 patients treated with a poxviral vaccine regimen consisting of the genes for CEA and MUC-1,
along with a triad of costimulatory molecules (TRICOM; composed of B7.1, intercellular adhesion molecule 1, and lymphocyte
function–associated antigen 3) engineered into vaccinia (PANVAC-V) as a prime vaccination and into fowlpox (PANVAC-F) as a
booster vaccination.
Results: The vaccine was well tolerated. Apart from injection-site reaction, no grade ≥2 toxicity was seen in more than 2% of the
cycles. Immune responses to MUC-1 and/or CEA were seen following vaccination in 9 of 16 patients tested. A patient with clear
cell ovarian cancer and symptomatic ascites had a durable (18-month) clinical response radiographically and biochemically,
and one breast cancer patient had a confirmed decrease of >20% in the size of large liver metastasis.
Conclusions: This vaccine strategy seems to be safe, is associated with both CD8 and CD4 immune responses, and has shown evidence of clinical
activity. Further trials with this agent, either alone or in combination with immunopotentiating and other therapeutic agents,
are warranted.
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.
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.
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.
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.
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.
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