Cell-mediated Antitumor Responses Research Articles

Abstract 2363: Combinational therapy with specific immunization plus checkpoint inhibition results in enhanced tumor regression and survival benefit

Abstract Effective immunotherapeutic treatment of various cancers may require combinational approaches to induce specific immunity to the tumor as well as inhibition of immune check-points. We have developed a viral gene delivery platform to immunize against numerous tumor associated antigens (TAA) such as carcinoembryonic antigen (CEA), HER2/neu and HPV 16. We investigated the therapeutic benefit of the immunotherapeutics alone and in combination with check-point inhibitors such as programmed death-ligand 1 (PD-1) blockade and LAG3 blockade in murine tumor models. As single agents, immunization with the viral delivery platform encoding the TAA induced target-specific cell-mediated immunity and anti-tumor responses in the respective model. When the immunotherapies were combined with immune checkpoint blockade, an increased level of anti-tumor activity against was observed in addition to an improvement in survival. Tumor microenvironment analysis immunized mice revealed an increase in CD8+ tumor-infiltrating lymphocytes (TILs). In addition, we observed induction of suppressive mechanisms such as programmed death-ligand 1 (PD-L1) expression on tumor cells and an increase in PD-1+ TILs. When immunotherapy was combined with anti-PD-1 antibody, we observed CD8+ TILs at the same increased level but found that a smaller fraction of these were PD-1+. Furthermore, we observed a reduction in PD-L1 expression on tumor cells, providing a mechanism by which combination therapy favors tumor clearance and a rationale for pairing antigen-specific vaccines with checkpoint inhibitors in future clinical trials. Citation Format: Elizabeth Gabitzsch, Adrian Rice, Yvette Latchman, Joseph P. Balint, Frank Jones. Combinational therapy with specific immunization plus checkpoint inhibition results in enhanced tumor regression and survival benefit. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2363.

Administration of Gemcitabine After Pancreatic Tumor Resection in Mice Induces an Antitumor Immune Response Mediated by Natural Killer Cells

Even after potentially curative R0 resection, patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis owing to high rates of local recurrence and metastasis to distant organs. However, we have no suitable transgenic animal models for surgical interventions. To induce formation of pancreatic tumor foci, we electroporated oncogenic plasmids into pancreata of LSL-KrasG12D× p53fl/fl mice; mutant Kras was expressed in p53fl/fl mice using a sleeping beauty transposon. We co-delivered a transposon encoding a constitutively active form of Akt2 (myrAkt2). Carcinogenesis and histopathologic features of tumors were examined. Metastasis was monitored by bioluminescence imaging. Tumors were resected and mice were given gemcitabine, and tumor recurrence patterns and survival were determined. Immune cells were collected from resection sites and analyzed by flow cytometry and in depletion experiments. After electroporation of oncogenic plasmids, mice developed a single pancreatic tumor nodule with histopathologic features of human PDAC. Pancreatic tumors that expressed myrAkt2 infiltrated the surrounding pancreatic tissue and neurons and became widely metastatic, reflecting the aggressive clinical features of PDAC in patients. Despite early tumor resection, mice died from locally recurring and distant tumors, but adjuvant administration of gemcitabine after tumor resection prolonged survival. In mice given adjuvant gemcitabine or vehicle, gemcitabine significantly inhibited local recurrence of tumors, but not metastasis to distant organs, similar to observations in clinical trials. Gemcitabine inhibited accumulation of CD11b+Gr1intF4/80int myeloid-derived suppressor cells at the resection margin and increased the number of natural killer (NK) cells at this location. NK cells but not Tcells were required for gemcitabine-mediated antitumor responses. Gemcitabine administration after resection of pancreatic tumors in mice activates NK cell-mediated antitumor responses and inhibits local recurrence of tumors, consistent with observations from patients with PDAC. Transgenic mice with resectable pancreatic tumors might be promising tools to study adjuvant therapy strategies for patients.

Mutations in CD58 and Other Immune System Related Genes in Hodgkin Lymphoma

Hodgkin Lymphoma (HL) is a B cell derived malignancy characterized by a minority of tumor cells, known as Hodgkin Reed-Sternberg (HRS) cells. The background is composed of a wide variety of inflammatory cells with T cells representing the largest population. Chemokines and cytokines produced by HRS cells and by the infiltrating cells shape the environment and provide proliferative and survival signals to the HRS cells. Despite this critical dependence on the microenvironment, HRS cells also need to apply mechanisms to escape from both antigen-dependent and innate immune responses. HRS cells have evolved multiple mechanisms to evade cytotoxic T cell (CTL) and natural killer (NK) cell mediated anti-tumor responses. These mechanisms include secretion of immune-suppressive factors (IL10, TGFβ and others), recruitment of regulatory and helper T cells, expression of PDL1 and CD95 and loss of HLA expression. Recent publications show that mutations in immune system related genes might represent a mechanism of HRS cells to evade detection by immune cells. The aim of this study was to validate whole exome sequencing results of seven HL cell lines focusing on immune system associated genes. We previously showed that B2M mutations affect the ATG start codon in L428 (heterozygous) and DEV (homozygous) cells. B2M mRNA levels were reduced in both cell lines as compared to L1236, whereas HLA-A, HLA-B and HLA-C mRNA levels were in the same range. Consistent with these findings we observed no membranous B2M and HLA class I expression by flow cytometry in the two cell lines with mutated B2M genes. In primary diagnostic HL tissue we showed lack of membranous B2M in 51% of the cases. We now studied two additional genes in more detail. CD58 gene mutations were observed in KMH2 and DEV cells. By manual inspection of the alignments using the Integrative Genomics Viewer (IGV), we also noticed a lack of reads of exons 1, 2 and 3 in SUPHD1. Heterozygous mutations and homozygous loss of exons 1-3 were confirmed for all three cell lines. CD58 mRNA levels were low or absent in SUPHD1 and KMH2 cells and normal in DEV. CD58 protein expression as determined by flow, western blot and IHC was low or absent in all 3 mutated HL cell lines in comparison to four cell lines with wild type CD58. Tumor cells of 36 primary HL cases with good treatment outcome showed a strong CD58 expression in all cases. As HL cell lines are derived from end stage HL patients, we next studied CD58 expression in relapsed HL patients. No or weak CD58 staining was observed in HRS cells in 6 out of 45 patients who experienced a relapse. Our results indicate that mutations in CD58 and loss of CD58 expression are common in HL derived cell lines and that loss of CD58 expression in tumor cells is restricted to relapsed HL patients. Heterozygous CSF2RB mutations in KMH2, SUPHD1, DEV and L1236 were validated by RNA-seq and Sanger-seq. As CSF2RB encodes the common β chain (CD131) shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, we also measured the expression of these 3 α chain receptors. We observed the same expression pattern between CD131 and CD116 (GM-CSF α receptor chain) in HL cell lines by flow cytometry suggesting that these mutations mainly affect the GM-CSF receptor. In conclusion, we show that mutations of immune system genes are common in HL. Deleterious mutations in B2M explain the lack of HLA class I expression, indicating that this genetic alteration is responsible for defective antigen presentation. Deleterious mutations or deletions of CD58 exons result in loss of CD58 protein expression. This will lead to loss of binding to CD2 expressed on T cells and will result in a defect in T cell adhesion and activation. Overall these results indicate that mutations are likely to contribute to the immune escape mechanisms applied by the HRS cells. DisclosuresNo relevant conflicts of interest to declare.

Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells.

CD8+ T cell-mediated immune response plays an important role in inhibiting progression of hepatocellular carcinoma (HCC). For strategic immunotherapy, it is critical to understand why some of the tumor cells escape from this immune attack. In this study, we investigated how HCC cells alter endogenous anti-tumor immunity and their related signaling pathways. We found that HCC cells, both in vitro and in vivo, substantially secret and express amphiregulin (AR). AR in turn activates immunosuppressive function of intratumoral CD4+Foxp3+ regulatory T cells (Tregs), a major inhibitor of CD8+ T cells. Using either lentiviral siRNA, or AR neutralizing antibody, we blocked the expression and function of AR to test the specificity of AR mediated activation of Tregs, Biochemical and cell biology studies were followed and confirmed that blocking of AR inhibited Tregs activation. In addition, we found that AR can trigger the activation of rapamycin complex 1(mTORC1) signaling in Tregs. The mTORC1 inhibitor rapamycin treatment led to compromise Treg function and resulted in enhancing anti-tumor function of CD8+ T cells. Blocking AR/EGFR signaling in Tregs with Gefitinib also enhanced anti-tumor immunity and decreased tumor size in a mouse xenograft tumor model. Taken together, our study suggested a novel mechanism of functional interaction between HCC and Tregs for regulating anti-tumor function of CD8+ T cells.

Nuclear FAK Controls Chemokine Transcription, Tregs, and Evasion of Anti-tumor Immunity

SummaryFocal adhesion kinase (FAK) promotes anti-tumor immune evasion. Specifically, the kinase activity of nuclear-targeted FAK in squamous cell carcinoma (SCC) cells drives exhaustion of CD8+ T cells and recruitment of regulatory T cells (Tregs) in the tumor microenvironment by regulating chemokine/cytokine and ligand-receptor networks, including via transcription of Ccl5, which is crucial. These changes inhibit antigen-primed cytotoxic CD8+ T cell activity, permitting growth of FAK-expressing tumors. Mechanistically, nuclear FAK is associated with chromatin and exists in complex with transcription factors and their upstream regulators that control Ccl5 expression. Furthermore, FAK’s immuno-modulatory nuclear activities may be specific to cancerous squamous epithelial cells, as normal keratinocytes do not have nuclear FAK. Finally, we show that a small-molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, also drives depletion of Tregs and promotes a CD8+ T cell-mediated anti-tumor response. Therefore, FAK inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic opportunities.

Reconstitution models to evaluate natural killer T cell function in tumor control.

Natural killer T (NKT) cells are glycolipid-reactive T lymphocytes that function in immunosurveillance and immune regulation. However, reduced tumor control in NKT cell-deficient Jα18(-/-) mice may be confounded by an overall reduction in T-cell receptor (TCR) repertoire diversity in these animals. Mechanistic studies are also hindered by a lack of tools to target molecules specifically in NKT cells. To address these issues, we developed protocols to expand functional NKT cells and stably reconstitute them in Jα18(-/-) mice. In vivo delivery of α-galactosylceramide (α-GalCer)-loaded dendritic cells expanded NKT cells in wild-type mice without skewing CD4 or TCR Vβ expression profiles. Expanded NKT cells exhibited enhanced cytokine responses upon re-stimulation with glycolipid or CD3 ligation. Adoptive transfer of recently expanded wild-type or interferon (IFN)-γ(-/-) NKT cells protected recipient Jα18(-/-) mice from B16 melanoma metastasis without the need for additional glycolipid stimulation. However, NKT cell reconstitution in recipient Jα18(-/-) mice was short lived. Long-term reconstitution was only achieved when expanded NKT cells were transferred into sublethally irradiated recipients. Thirty days after transfer, NKT cell numbers, phenotype and α-GalCer-induced cytokine responses were equivalent to naive wild-type mice. Jα18(-/-) recipients reconstituted with wild-type or IFN-γ(-/-) NKT cells were both protected from B16 melanoma metastasis following α-GalCer treatment, and NK cell transactivation was intact in mice reconstituted with IFN-γ(-/-) NKT cells. These studies validate the use of reconstitution protocols to investigate the mechanisms of NKT cell immune function, demonstrating that NKT cell-derived IFN-γ and the altered TCR repertoire in Jα18(-/-) mice do not impact NKT cell-mediated antitumor responses.

6-Thioguanine-loaded polymeric micelles deplete myeloid-derived suppressor cells and enhance the efficacy of T cell immunotherapy in tumor-bearing mice.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6chi Ly6g− monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 days post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6clo Ly6g+ granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1int Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6chi macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8+ T cells in melanoma cells expressing OVA. These findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1702-8) contains supplementary material, which is available to authorized users.

Maraba MG1 Virus Enhances Natural Killer Cell Function via Conventional Dendritic Cells to Reduce Postoperative Metastatic Disease

This study characterizes the ability of novel oncolytic rhabdoviruses (Maraba MG1) to boost natural killer (NK) cell activity. Our results demonstrate that MG1 activates NK cells via direct infection and maturation of conventional dendritic cells. Using NK depletion and conventional dendritic cells ablation studies in vivo, we established that both are required for MG1 efficacy. We further explored the efficacy of attenuated MG1 (nonreplicating MG1-UV(2min) and single-cycle replicating MG1-Gless) and demonstrated that these viruses activate conventional dendritic cells, although to a lesser extent than live MG1. This translates to equivalent abilities to remove tumor metastases only at the highest viral doses of attenuated MG1. In tandem, we characterized the antitumor ability of NK cells following preoperative administration of live and attenuated MG1. Our results demonstrates that a similar level of NK activation and reduction in postoperative tumor metastases was achieved with equivalent high viral doses concluding that viral replication is important, but not necessary for NK activation. Biochemical characterization of a panel of UV-inactivated MG1 (2-120 minutes) revealed that intact viral particle and target cell recognition are essential for NK cell-mediated antitumor responses. These findings provide mechanistic insight and preclinical rationale for safe perioperative virotherapy to effectively reduce metastatic disease following cancer surgery.

Fludarabine downregulates indoleamine 2,3-dioxygenase in tumors via a proteasome-mediated degradation mechanism.

Indoleamine 2,3-dioxygenase (IDO) is found in multiple malignancies and exerts immunosuppressive effects that are central in protecting tumors from host T lymphocyte rejection. IDO is an enzyme involved in the catabolism of tryptophan resulting in inhibition of T lymphocyte function. While inhibition of IDO enzymatic activity results in tumor rejection, it is still unknown how we can directly target IDO expression within tumors using drugs. We have chosen to interfere with IDO expression by targeting the key-signaling event signal transducer and activator of transcription 1 (STAT1). We evaluated the efficacy of fludarabine, previously described to inhibit STAT1 phosphorylation. Interestingly, fludarabine was efficient in suppressing protein expression and consequently IDO activity in two different cell lines derived from breast cancer and melanoma when IDO was activated with interferon-gamma (IFN-γ) or supernatants prepared from activated T lymphocytes. However, fludarabine had no inhibitory effect on STAT1 phosphorylation. Other IFN-γ-responsive genes were only marginally inhibited by fludarabine. The level of IDO transcript was unaffected by this inhibitor, suggesting the involvement of post-transcriptional control. Strikingly, we have found that the inhibition of proteasome partially protected IDO from fludarabine-induced degradation, indicating that fludarabine induces IDO degradation through a proteasome-dependent pathway. Currently used in the clinic to treat some malignancies, fludarabine has the potential for use in the treatment of human tumors through induction of IDO degradation and consequently, for the promotion of T cell-mediated anti-tumor response.

TNF signaling drives myeloid-derived suppressor cell accumulation (P2051)

Abstract TNF, an inflammatory cytokine that is enriched in the tumor microenvironment, promotes tumor growth and subverts innate immune responses to cancer cells. We previously reported that tumors implanted in TNF receptor-deficient (Tnfr-/-) mice are spontaneously rejected; however, the molecular mechanisms underlying this rejection are unclear. Here we report that TNF signaling drives the peripheral accumulation of myeloid-derived suppressor cells (MDSCs). MDSCs expand extensively during inflammation and tumor progression in mice and humans and can enhance tumor growth by repressing T cell-mediated antitumor responses. Peripheral accumulation of MDSCs was drastically impaired in Tnfr-/- mice. Signaling of TNFR-2, but not TNFR-1, promoted MDSC survival through upregulation of cellular FLICE-inhibitory protein (c-FLIP) and inhibition of caspase-8 activity. Loss of TNFRs impaired the induction of MDSCs from bone marrow cells, but this could be reversed by treatment with caspase inhibitors. These results demonstrate that TNFR-2 signaling promotes MDSC survival and accumulation and helps tumor cells evade the immune system.

Lung tumor NF-κB signaling promotes T cell–mediated immune surveillance

NF-κB is constitutively activated in many cancer types and is a potential key mediator of tumor-associated inflammation, tumor growth, and metastasis. We investigated the role of cancer cell NF-κB activity in T cell-mediated antitumor responses. In tumors rendered immunogenic by model antigen expression or following administration of antitumor vaccines, we found that high NF-κB activity leads to tumor rejection and/or growth suppression in mice. Using a global RNA expression microarray, we demonstrated that NF-κB enhanced expression of several T cell chemokines, including Ccl2, and decreased CCL2 expression was associated with enhanced tumor growth in a mouse lung cancer model. To investigate NF-κB function in human lung tumors, we identified a gene expression signature in human lung adenocarcinoma cell lines that was associated with NF-κB activity level. In patient tumor samples, overall lung tumor NF-κB activity was strongly associated with T cell infiltration but not with cancer cell proliferation. These results therefore indicate that NF-κB activity mediates immune surveillance and promotes antitumor T cell responses in both murine and human lung cancer.

Abstract 1237: Mechanism of action of L-BLP25 in preclinical pancreatic tumor models.

Abstract Background. L-BLP25 is an investigational antigen-specific cancer immunotherapeutic agent targeting human mucin 1 (MUC1). A single low dose of cyclophosphamide (CPA) is known to be immunostimulatory and may enhance the anti-tumor effects of L-BLP25 when given 3 days before treatment initiation. Here we report the immune and anti-tumor efficacy of L-BLP25 with CPA pretreatment in subcutaneous (s.c.) and orthotopic models of murine pancreatic cancer. These models allow for reproduction of the clinical scenario, where 8 weekly doses of L-BLP25 are administered. Methods. Murine pancreatic tumor cells Panc02 transfected with human MUC1 were injected for comparison s.c. or orthotopically into human MUC1 transgenic mice. Mice received either vehicle control (PBS), CPA (100 mg/kg), L-BLP25 (100 μg), or L-BLP25 (100 μg) + CPA (100 mg/kg). CPA was administered intravenously (i.v.) 3 days prior to initiation of 8 weekly intradermal (i.d.) or s.c. L-BLP25 injections. Tumor volume was monitored and splenic cell populations were quantified by FACS. In the s.c. tumor model, the contribution of CD4+ and CD8+ T cell subsets to the inhibition of tumor growth was studied. In the orthotopic model, antigen-specific IFN-γ secretion was assessed via ex vivo T cell re-stimulation with antigen alone. Results. In the s.c. model of pancreatic cancer, tumor volume was reduced compared to vehicle at day 49 after i.d. administration of L-BLP25 (41.4%, p<0.02) and L-BLP25 + CPA (55.5%, p<0.02). In multiple studies, tumor volume was consistently lower in mice treated with L-BLP25 + CPA vs. L-BLP25 alone. L-BLP25 + CPA s.c. treatment was effective at day 49 with 44% greater inhibition of tumor growth and more pronounced extended survival compared to i.d. treatment, supporting the route of administration in the clinic. Various splenic immune cell populations were increased by s.c. L-BLP25 + CPA treatment compared to vehicle and L-BLP25 alone, including the CD8+ effector memory/Treg ratio. Mice treated with s.c. L-BLP25 + CPA after CD8+ T cell depletion had a greater tumor volume compared to T cell-sufficient mice, while CD4+ T cell depletion had no effect. In the orthotopic model of pancreatic cancer, median survival was improved with s.c. L-BLP25 + CPA treatment vs. vehicle (p<0.05) and vs. L-BLP25 alone (non-significant). When stimulated ex vivo with the BP25 peptide, the frequency of IFN-γ producing CD4+ T cells isolated from the spleens of mice treated with L-BLP25 + CPA was significantly higher than in all other treatment groups (p<0.001). CD8+ T cells were found to produce no IFN-γ after in vitro re-stimulation. Conclusions. When administered after a single, low-dose pre-treatment of CPA, L-BLP25 immunotherapy exhibits anti-tumor activity, immune activation and prolonged survival in murine pancreatic cancer models. T cell depletion data indicate a CD8+ T cell-mediated anti-tumor response, whilst IFN-γ production by CD4+ helper T cells may contribute to prolonged survival. Citation Format: Kenneth Hance, Bo Marelli, Jin Qi, Guozhong Qin, Huakui Yu, Hong Wang, Xiaomei Xu, Robert Tighe, Beatrice Brunkhorst, Yan Lan, Robert Hofmeister, Helen Sabzevari, Michael Wolf. Mechanism of action of L-BLP25 in preclinical pancreatic tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1237. doi:10.1158/1538-7445.AM2013-1237

Abstract LB-290: Targeting MUC16 with the antibody-drug conjugate (ADC) DMUC5754A in patients with platinum-resistant ovarian cancer: A phase I study of safety and pharmacokinetics.

Abstract Background: MUC16 is a large transmembrane protein overexpressed by the majority of ovarian cancers (OC), compared with normal tissues. The role of MUC16 in the pathogenesis of ovarian cancer is unknown; however, MUC16 may facilitate the binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity, and may inhibit natural killer cell-mediated anti-tumor cytotoxic responses. Conjugation of a highly potent cytotoxic drug to a MUC16-specific monoclonal antibody represents a novel approach to treatment of MUC16-expressing tumors. DMUC5754A is an ADC containing the humanized IgG1 anti-MUC16 monoclonal antibody linked to the potent anti-mitotic agent MMAE and demonstrates anti-tumor activity in MUC16-expressing tumor xenograft models. Methods: This phase I study evaluated safety, pharmacokinetics (PK), and pharmacodynamic (PD) activity of DMUC5754A (0.3-3.2 mg/kg) given every 3 weeks (q3w) to patients with advanced recurrent platinum-resistant OC. A standard 3+3 design was used to determine the maximum-tolerated dose, followed by cohort expansion. Tumor tissue was assessed for expression of MUC16 and other relevant markers. Clinical activity was evaluated per RECIST. Results: Forty-four patients (22 escalation, 22 expansion at 2.4 mg/kg), median age 62 (44-79), ECOG PS 0-1, received a median of 4 doses (range 1-20) of DMUC5754A. Two DLTs, 1 Grade 4 neutropenia and 1 Grade 4 uric acid increase, occurred at the maximally administered dose of 3.2 mg/kg. Grade ≥ 3 related adverse events (AE) occurring in ≥ 5% of patients included fatigue (4 at 2.4 mg/kg; 9% total) and neutropenia (1 at 3.2 mg/kg, 3 at 2.4 mg/kg; 9% total). The most common related AEs over all dose levels were fatigue (57%), nausea (34%), vomiting (27%), decreased appetite (25%), peripheral neuropathy (25%), and diarrhea (23%). Serious drug-related AEs (SAE) were small intestine obstruction (2 patients), hypocalcemia (1 patient), and neutropenia (1 patient). Total antibody and conjugated MMAE were not impacted by circulating CA125 and displayed dose-dependent PK with clearance decreasing as dose increased. Accumulation of total antibody, conjugated MMAE and free MMAE was not observed due to their short half-lives (≤5 days). Tumor MUC16 expression was evaluable in 42 patients, showing 20% IHC 0, 16% IHC 2+, and 64% IHC 3+. All confirmed responses (1 CR and 4 PRs) occurred in patients treated at 2.4 mg/kg and whose tumors were MUC16 IHC 2+ or 3+. Six additional patients had minor responses (3 at 2.4 mg/kg). Sixteen of the twenty-nine patients at 2.4 mg/kg were on study ≥ 105 days. Human epididymis protein 4 was a potential surrogate marker for serologic response measures in the presence of anti-MUC16/CA125-binding therapy. Conclusions: DMUC5754A at 2.4 mg/kg q3w has an encouraging safety profile and evidence of anti-tumor activity in MUC16-expressing OC. Further studies are planned. Citation Format: Joyce Liu, Kathleen Moore, Michael Birrer, Suzanne Berlin, Ursula Matulonis, Jeffrey Infante, Jian Xi, Robert Kahn, Yulei Wang, Katie Wood, Daniel Coleman, Daniel Maslyar, Eric Humke, Howard Burris. Targeting MUC16 with the antibody-drug conjugate (ADC) DMUC5754A in patients with platinum-resistant ovarian cancer: A phase I study of safety and pharmacokinetics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-290. doi:10.1158/1538-7445.AM2013-LB-290

TNF signaling drives myeloid-derived suppressor cell accumulation

TNF, an inflammatory cytokine that is enriched in the tumor microenvironment, promotes tumor growth and subverts innate immune responses to cancer cells. We previously reported that tumors implanted in TNF receptor-deficient (Tnfr-/-) mice are spontaneously rejected; however, the molecular mechanisms underlying this rejection are unclear. Here we report that TNF signaling drives the peripheral accumulation of myeloid-derived suppressor cells (MDSCs). MDSCs expand extensively during inflammation and tumor progression in mice and humans and can enhance tumor growth by repressing T cell-mediated antitumor responses. Peripheral accumulation of MDSCs was drastically impaired in Tnfr-/- mice. Signaling of TNFR-2, but not TNFR-1, promoted MDSC survival through upregulation of cellular FLICE-inhibitory protein (c-FLIP) and inhibition of caspase-8 activity. Loss of TNFRs impaired the induction of MDSCs from bone marrow cells, but this could be reversed by treatment with caspase inhibitors. These results demonstrate that TNFR-2 signaling promotes MDSC survival and accumulation and helps tumor cells evade the immune system.

Th9 cells promote antitumor immune responses in vivo

Th9 cells are a subset of CD4+ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8+ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8+ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell-mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8+ CTL-mediated antitumor immunity and indicate that Th9 cell-based cancer immunotherapy may be a promising therapeutic approach.

Immunization with E1A expressing tumor cell line enhances antigen specific tumor immunity (74.5)

Abstract The stable expression of Adenovirus (Ad) E1A in tumor cells reduces tumorigenicity by 1,000 fold, an effect dependent on the elicitation of an NK cell and T cell mediated antitumor response. We generated B6 fibrosarcoma cell lines (MCA tumor cells) that stably expressed ovalbumin (MCA-OVA) or full-length E1A fused to ovalbumin (MCA-E1A-OVA) to test the hypotheses that E1A could function as a molecular adjuvant to induce a robust anti-tumor immune response to other weakly or non-immunogenic tumor antigens. MCA E1A-OVA was more susceptible to killing by NK in vitro and 1000 fold less tumorigenic than MCA or MCA-OVA in vivo. Immunization with 1X10^5 live MCA-OVA or MCA- E1A-OVA tumor cells elicited comparable numbers of IFNγ- and TNFα -producing OVA specific CD8 T cells that had similar cytolytic activity against OVA-expressing target cells in in vivo CTL killing assays. Immunization of MCA-OVA or MCA-E1A-OVA cells also increased the number of MCA-OVA cells required to form tumors by 1,000 fold in an anatomically distant site. However, primary tumors developed at the site of immunization with MCA-OVA but not MCA-E1A-OVA cells. These data demonstrate that immunization of live MCA-E1A or MCA-OVA tumor cells can elicit a robust, systemic anti-tumor immune response. However, primary tumor progression occurs at the site of inoculation following immunization with MCA-OVA cells despite the induction of an anti-tumor immune response.

Abstract 1536: A novel immunocytokine generates a CD8+ T cell-mediated anti-tumor response in vivo

Abstract Interleukin-12 (IL-12) is a potent TH1 cytokine produced by dendritic cells and macrophages to activate NK cells and T cells. Data from preclinical studies in mice indicate that this cytokine may be useful for the treatment of cancer. However, cases of severe toxicity in human trials have impeded its successful clinical development. Targeting delivery of IL-12 to a tumor while reducing systemic IL-12 exposure could make this a safer, more effective cancer therapeutic agent. One strategy is to conjugate IL-12 to a tumor-binding antibody, as antibody conjugates have previously been used to deliver radioisotopes, other cytokines, and small molecule therapeutics to tumors in vivo. NHS-IL12 is a novel immunocytokine consisting of two molecules of the IL-12 heterodimer covalently fused to a fully humanized antibody (NHS76). This antibody targets necrotic portions of tumors because of its high affinity for single- and double-stranded DNA, which are often exposed as tumors outgrow their blood supply. NHS-IL12 may therefore be useful against a wide range of solid tumors, and mouse models can be used to study its mechanisms of action. Initial imaging studies clearly showed superior in vivo localization of IL-12 to mouse tumors following subcutaneous NHS-IL12 injection as compared to administration of recombinant IL-12 (rIL-12) or IL-12 fused to a control antibody (BC1). In the MC38/CEA+ colorectal carcinoma model in CEA.Tg mice, treatment with NHS-IL12 significantly delayed subcutaneous tumor growth more effectively than treatment with rIL-12. NHS-IL12 did not inhibit MC38/CEA+ tumor cell growth in vitro, suggesting that NHS-IL12 acts indirectly on tumors in vivo most probably via an immune-mediated mechanism. Subsequent findings revealed that tumor-bearing mice treated with NHS-IL12 developed a strong CD8+ T cell response directed against the endogenous p15E tumor antigen. In one study, a subset of mice bearing MC38/CEA+ tumors had complete tumor regression following NHS-IL12 treatment. Each of these mice still had a strong p15E-specific CTL response more than two months after tumor rejection, indicating that p15E may be the major tumor rejection antigen for this in vivo tumor model. Subsequent in vivo depletion studies are planned to investigate which immune cell subsets contribute to the anti-tumor efficacy of this agent. Taken together, these data show that targeted delivery of IL-12 to tumors can overcome the immunosuppressive tumor microenvironment and prime a robust CD8+ T cell response. These findings suggest that NHS-IL12 may be a promising cancer therapeutic as a single agent and/or as a potent vaccine adjuvant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1536. doi:1538-7445.AM2012-1536

Tumor-Induced CD8+ T-Cell Dysfunction in Lung Cancer Patients

Lung cancer is the leading cause of cancer deaths worldwide and one of the most common types of cancers. The limited success of chemotherapy and radiotherapy regimes have highlighted the need to develop new therapies like antitumor immunotherapy. CD8+ T-cells represent a major arm of the cell-mediated anti-tumor response and a promising target for developing T-cell-based immunotherapies against lung cancer. Lung tumors, however, have been considered to possess poor immunogenicity; even so, lung tumor-specific CD8+ T-cell clones can be established that possess cytotoxicity against autologous tumor cells. This paper will focus on the alterations induced in CD8+ T-cells by lung cancer. Although memory CD8+ T-cells infiltrate lung tumors, in both tumor-infiltrating lymphocytes (TILs) and malignant pleural effusions, these cells are dysfunctional and the effector subset is reduced. We propose that chronic presence of lung tumors induces dysfunctions in CD8+ T-cells and sensitizes them to activation-induced cell death, which may be associated with the poor clinical responses observed in immunotherapeutic trials. Getting a deeper knowledge of the evasion mechanisms lung cancer induce in CD8+ T-cells should lead to further understanding of lung cancer biology, overcome tumor evasion mechanisms, and design improved immunotherapeutic treatments for lung cancer.

Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn’s neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.

Maximal T Cell–Mediated Antitumor Responses Rely upon CCR5 Expression in Both CD4+ and CD8+ T Cells

Immune responses against cancer rely upon leukocyte trafficking patterns that are coordinated by chemokines. CCR5, the receptor for chemotactic chemokines MIP1alpha, MIP1beta, and RANTES (CCL3, CCL4, CCL5), exerts major regulatory effects on CD4(+)- and CD8(+) T cell-mediated immunity. Although CCR5 and its ligands participate in the response to various pathogens, its relevance to tumoral immune control has been debated. Here, we report that CCR5 has a specific, ligand-dependent role in optimizing antitumor responses. In adoptive transfer studies, efficient tumor rejection required CCR5 expression by both CD4(+) and CD8(+) T cells. CCR5 activation in CD4(+) cells resulted in CD40L upregulation, leading to full maturation of antigen-presenting cells and enhanced CD8(+) T-cell crosspriming and tumor infiltration. CCR5 reduced chemical-induced fibrosarcoma incidence and growth, but did not affect the onset or progression of spontaneous breast cancers in tolerogenic Tg(MMTV-neu) mice. However, CCR5 was required for TLR9-mediated reactivation of antineu responses in these mice. Our results indicate that CCR5 boosts T-cell responses to tumors by modulating helper-dependent CD8(+) T-cell activation.