Tumor-free Animals Research Articles

Abstract B057: The CD122-biased immunostimulatory cytokine NKTR-214 combined with checkpoint blockade leads to mobilization of antitumor immunity and synergistic activity

Abstract Background: NKTR-214 is a novel immunostimulatory cytokine which provides a sustained and biased activation signal through the heterodimeric IL-2R receptor (IL2Rbg or CD122), resulting in significant expansion of CD8+ T and NK cells relative to regulatory T cells in the tumor microenvironment. As a single agent in preclinical tumor models, NKTR-214 significantly increases the intratumoral CD8T/Treg ratio (>400). Here we examine activity and pharmacodynamic effects of NKTR-214 alone and combined with immune checkpoint blockade in both a murine tumor model and in a human in vitro co-culture system. Methods: Mice bearing subcutaneous CT26 colon tumors were treated with single agent NKTR-214 (q9d), murine anti-CTLA-4 or anti-PD-1 (twice-weekly), or combinations of these agents. Serum cytokines were assessed by multiplex ELISA (Quansys). T cell clonality and infiltration were assessed in tumors 7 days after treatment initiation, with TCR V and J beta usage determined by ImmunoSEQ (Adaptive Biotechnologies). Effects on human cytokine and chemokine expression were examined by the BioMAP Combo ELECT (DiscoverX) in vitro system utilizing human fibroblasts or endothelial cells, PBMCs and either HT29 colon adenocarcinoma or HT1299 NSCLC cell lines. 1-PEG-IL2, the most active species of the NKTR-214 prodrug, or Keytruda (anti-PD-1) was added over a range of concentrations alone or in combination, with biomarkers assessed 48 hours later. Results: In tumor-bearing mice, NKTR-214 led to increases in serum IFNg as well as increases in the chemokine MCP-1 (CCL2), a chemoattractant of CD4 T, CD8 T and NK cells. Single-agent NKTR-214 led to significant tumor growth inhibition and 10% tumor free animals, while single-agents anti-CTLA-4 or anti-PD-1 lacked efficacy. Combination of NKTR-214 with checkpoint blockade was synergistic and led to greater tumor-free animals (67% in combination with anti-CTLA-4, 90% with anti-PD-1). NKTR-214 combined with anti-PD-1 was also more efficacious than the combination of anti-CTLA-4 and anti-PD-1. TCR repertoire analysis demonstrated superior increases in TIL clonality and infiltration with NKTR-214 compared to either anti-CTLA-4 or anti-PD-1 alone. NKTR-214 combined with either mode of checkpoint inhibition led to significant increases in both parameters, with the greatest effect occurring when combined with anti-PD-1. In the human BioMAP system, NKTR-214 active species led to significant increases in granzyme B, IFNg, IL-6, IL-17A and TNFa, while these markers were only modestly induced by Keytruda alone. When NKTR-214 was combined with Keytruda, additive and synergistic increases in these factors were found. In addition, the combination significantly decreased uPA in the fibroblast-HT29 system and MDC in the endothelial-HT1299 system, both factors associated with tumor progression. Conclusions: NKTR-214 delivers sustained signaling of the IL-2 pathway. Analysis of multiple immune markers in mice and human tumor-immune cell cultures provide mechanistic rationale for the increased T and NK cell activation in the tumor microenvironment after treatment with NKTR-214. When combined with anti-PD-1, significant increases in T cell receptor clonality, tumor infiltration and immune activation biomarkers are achieved - a significant finding, given that concomitant increase in clonality and infiltration has been correlated with favorable responses in the clinic. In vitro studies with human cells demonstrate the potentiation of immune activation markers associated with anti-tumor effects in combination with anti-PD-1, further suggesting these findings may be translatable from mouse to human. NKTR-214 is currently in a Phase 1 clinical trial to evaluate the pharmacokinetics, pharmacodynamics and activity in an outpatient setting. Citation Format: John L. Langowski, Murali Addepalli, Laurie VanderVeen, Rhoneil Pena, Ravikumar Nutakki, Yolanda Kirksey, Ute Hoch, Jonathan Zalevsky, Stephen K. Doberstein, Deborah H. Charych. The CD122-biased immunostimulatory cytokine NKTR-214 combined with checkpoint blockade leads to mobilization of antitumor immunity and synergistic activity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B057.

Abstract B091: A LAG-3/PD-L1 bispecific antibody inhibits tumor growth in two syngeneic colon carcinoma models

Abstract Combining immunotherapeutic antibodies for treatment of cancer patients has shown benefits over single agent treatment. For example, the addition of anti-PD-1 antibodies to anti-CTLA-4 treatment in advanced melanoma patients has increased objective response rates from 19% to 57.6% (Larkin,J et al. 2015. NEJM 373:23-34). An alternative to combining two antibodies is the development of bispecific antibodies that not only bring two biologies together but may result in novel mechanisms of action that are impossible to attain with combinations. Lymphocyte Activation Gene-3 (LAG-3) is a member of the Ig superfamily expressed on activated T cells, NK cells, pDCs, B cells, γδ T cells and participates in immune suppression, particularly through persistent strong expression in a percentage of regulatory T cells (Tregs). Programmed Cell Death receptor (PD-1) binds to its ligand PD-L1, expressed not only on activated immune cells to inhibit cellular immune responses but also on tumor cells. Expression of LAG-3 and PD-1 leads to T cell exhaustion, allowing tumor escape from immune surveillance. Combining inhibitory antibodies to PD-1 and LAG-3 reactivates T cells leading to efficacy in murine models over single treatment (Woo,S-R et al. 2012. Cancer Res. 15: 917-27) which prompted interest in the development of a LAG-3 and PD-L1 bispecific antibody for inhibiting tumor growth. A murine-specific anti-LAG-3 and PD-L1 bispecific antibody was engineered which binds both antigens simultaneously and with nanomolar affinities. The anti-LAG-3/PD-L1 bispecific antibody inhibits LAG-3 binding to MHCII and PD-L1 binding to PD-1 and CD80, resulting in T cell activation in an in vitro assay. This potency translates into in vivo efficacy, where the anti-LAG-3/PD-L1 bispecific antibody decreased tumor burden in the MC38 colon carcinoma tumor model, both in early-established or well-established tumors. At the end of the study tumor-free animals were more numerous in the LAG-3/PD-L1 bispecific group than in the group given a combination of individual anti-LAG-3 and PD-L1 antibodies. The results were recapitulated in the CT26 murine colon cancer model, where the LAG-3/PD-L1 bispecific showed an increase of anti-tumor activity as compared to the combination of anti-LAG-3 and anti-PD-L1 antibodies. Thus, the preclinical data supports developing an anti-human LAG-3/PD-L1 bispecific for the treatment of cancer patients. Citation Format: Jacqueline Doody, Matthew Kraman, Katy Everett, Carlo Zimarino, Katarzyna Kmiecik, Mustapha Faroudi, Paul Thompson, Mateusz Wydro, Francisca Wollerton, Carlos Gaspar, Mihriban Tuna. A LAG-3/PD-L1 bispecific antibody inhibits tumor growth in two syngeneic colon carcinoma models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B091.

1078P - Combining complementary mechanisms of immune activation: NKTR-214, a biased IL-2 pathway agonist and immune checkpoint antagonists

NKTR-214 is an agonist of the IL-2 pathway that provides a biased, sustained signal to the IL-2R&bgr;γ complex resulting in expansion of CD8+ T cells compared to regulatory T cells in the tumor. (1) NKTR-214 is currently in an outpatient Phase 1 clinical trial to evaluate MTD, pharmacokinetics, pharmacodynamics and mechanism in solid tumors. Here we describe T-cell clonality and formation of tumor-reactive immune memory after administering NKTR-214 and checkpoint inhibitor antibodies in murine tumor models. Mice bearing subcutaneous established EMT6 breast or CT26 colon tumors were treated with single agent NKTR-214 (q9d), murine anti-CTLA4 or anti-PD1 (twice weekly), or their combinations. Anti-tumor memory was assessed by 1) rechallenging tumor-free mice and 2) transferring splenocytes from tumor-free animals into tumor-bearing recipients. Immune cell enumeration used flow cytometry; T-cell clonality used the ImmunoSEQ platform (Adaptive Biotechnologies). While EMT6 and CT26 were refractory to single agent regimens, NKTR-214 achieved synergistic anti-tumor activity with antiPD1 or antiCTLA4 superior to both antibodies combined. Tumor rechallenge demonstrated anti-tumor memory was durable, specific, and marked by vigorous proliferative memory T cells. The two antibodies produced significant increases in T cell density but modest increases in T cell clonality. In contrast, when NKTR-214 combined with either checkpoint antibody, a greater increase in both T cell density and clonality was observed. NKTR-214 delivers a long-lived, biased activation of the potent IL-2 pathway, favorable pharmacokinetics and mechanistic complementarity to checkpoint inhibition. The combination increases T cell clonality paralleling improved efficacy. The data support the concept of enhancing anti-tumor immunologic memory by combining agonist and antagonist mechanisms, providing increased T cell density and clonality in the tumor. 1. Charych et al., NKTR-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models. Clinical Cancer Research, 22, 680-690, 2016

Abstract B077: Evaluating oncolytic vaccines targeting mutated epitopes in a murine fibrosarcoma model CMS-5

Abstract Oncolytic viruses have traditionally been used as stand alone therapy delivered to animals and patients to lytically destroy tumor tissue. However, it has recently become clear that involvement of the immune system is an extremely important aspect of these therapies. To further enhance anti-tumor immune responses we have utilized an oncolytic vaccine approach. This approach involves priming the immune system to a specific tumor associated antigen (TAA) with an Adenovirus and then subsequently delivering an oncolytic Rhabdovirus expressing the same tumor antigen leading to robust anti-tumor immune responses and improved efficacy. For the studies presented herein we have developed oncolytic vaccines targeting mutated epitopes expressed in CMS-5. Immune responses generated towards these epitopes through oncolytic vaccination will be characterized and their ability to control tumor growth will be evaluated. We hypothesize that the foreign nature of these mutated epitopes will allow for the generation of substantial immune responses following oncolytic vaccination in tumor bearing mice. In preliminary studies, oncolytic vaccination has been able to generate robust immune responses leading to as many as 60% of circulating CD8+ T cells responding to a single mutated epitope in tumor free animals. Therapeutic oncolytic vaccination of CMS-5 tumor bearing animals was still able to generate large anti-tumor immune responses, however these responses were cut in half compared to tumor free animals. The resultant anti-tumor immune response was able to significantly control tumor growth, and the magnitude of these responses correlated with survival. Even though there was a delay in tumor growth following therapy, the responses were not durable and all mice ultimately failed the therapy. We are currently engaged in studies to further characterize the immune responses generated and determine what, if any, immunosuppression led to failure of our therapy; even in the presence of anti-tumor immune responses exceeding 20% of peripheral CD8+ T cells. Nanostring analysis of the tumor microenvironment are being conducted focusing on gene signatures for immune cells and inflammation and these data will be confirmed by histology and flow cytometry. These studies will help shed light on potential mechanisms of immune evasion/suppression following oncolytic vaccination and point to potential targets for modulation of the oncolytic vaccine induced immune response. Citation Format: Kyle Stephenson, Matthew Atherton, John Bell, Brian Lichty. Evaluating oncolytic vaccines targeting mutated epitopes in a murine fibrosarcoma model CMS-5. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B077.

Establishment of an in vitro equine papillomavirus type 2 (EcPV2) neutralization assay and a VLP-based vaccine for protection of equids against EcPV2-associated genital tumors

The consistent and specific presence of Equus caballus papillomavirus type 2 (EcPV2) DNA and mRNA in equine genital squamous cell carcinoma (gSCC) is suggestive of an etiological role in tumor development.To further validate this concept, EcPV2-neutralizing serum antibody titers were determined by an EcPV2 pseudovirion (PsV) neutralization assay. Furthermore, an EcPV2 L1 virus-like particle (VLP)-based vaccine was generated and its prophylactic efficacy evaluated in vivo.All 6/6 gSCC-affected, but only 3/20 tumor-free age-matched animals revealed EcPV2-neutralizing serum antibody titers by PsV assay. Vaccination of NZW rabbits and BalbC mice with EcPV2 L1 VLP using Freund׳s or alum respectively as adjuvant induced high-titer neutralizing serum antibodies (1600–12,800). Passive transfer with rabbit EcPV2–VLP immune sera completely protected mice from experimental vaginal EcPV2 PsV infection.These findings support the impact of EcPV2 in equine gSCC development and recommend EcPV2 L1 VLP as prophylactic vaccine against EcPV2 infection and associated disease in equids.

ONO-4059—a Potent and Selective Reversible Bruton’s Tyrosine Kinase (Btk) Inhibitor: Single Agent, Twice Daily (BD) Dosing and Dosing with Food Results in Sustained, High Trough Levels of ONO-4059, Translating into 100% Tumour Remission in a TMD-8 Xenograft Model

Purpose: Bruton’s tyrosine kinase (Btk) is a key regulator of the BCR signaling pathway and abberant BCR signaling has been implicated in the survival of malignant B-cells. The activated B-cell-like (ABC) sub-type of Diffuse Large B-Cell Lymphoma (DLBCL) correlates with poor prognosis. There is still a high unmet, medical need for new therapies, preferably chemo-sparing, to help treat patients with ABC-DLBCL and CLL as well as other B-cell malignancies. ONO-4059 is a highly selective, orally bioavailable inhibitor of Btk kinase activity with a potency (IC50) of 2.2 nM. ONO-4059 reversibly blocks BCR signaling and B-cell proliferation and activation. Data from the ongoing Phase 1 study (ONO-4059POE001), where ONO-4059 is administered as monotherapy (QD) demonstrated a best overall response rate of 47% (7/15) in non-GCB DLBCL patients (Walter et al, ASCO 2014; Rule et al, EHA 2014). We hypothesized that the efficacy of ONO-4059 could be further enhanced by increasing the drug trough concentration. To address this, we examined different dosing regimens of ONO-4059 in an ABC-DLBCL xenograft model.Methods: TMD-8 tumour cells (ABC-DLBCL cell line) were implanted sub-cutaneously into female SCID mice. Randomization of mice occurred when mean tumour volume was 100-200 or 400-450 mm3. ONO-4059 was administered orally or mixed in food at doses up to 20 mg/kg/day and animals were dosed QD or BD. Tumour volumes were measured twice a week after initiation of treatment, and tumour volumes were determined using the formula volume (= width2 x length)/2. Animals were euthanized when the tumours reached a maximum volume of 3,000 mm3.Results: For the100-200 mm3 tumour groups, tumour growth inhibition at the final treatment day was 23% in QD, 72.9% in BD and 100% in dose mixed in food, groups respectively. For the 400-450 mm3 tumour groups, no growth inhibition was observed in the QD group and, growth inhibitions of 27.5% in BD and 100% in dose mixed in food were observed. Interestingly, the treatment with ONO-4059-containing diets resulted in tumour remission in 10/10 animals, in both 100-200 and 400-450 mm3 treatment groups, whereas no tumour free animals were observed in the other treatment groups. The PK concentration and phosphorylated Btk (pBtk) inhibition levels of those animals whose dose was mixed in with food were higher than that of other treatment groups.Conclusion: Our previous study demonstrated that 100% tumour remission can be achieved partially with an ONO-4059 and GA101 or rituximab combination (Yoshizawa et al, ASH 2013). However, to date, no orally bioavailable targeted-agent administered as monotherapy has demonstrated 100% tumour remission in an advanced ABC-DLBCL xenograft model. Although the clinical data for ONO-4059 given as monotherapy (QD) is very encouraging in both relapsed and refractory CLL/NHL patients, this data indicates that a more frequent dosing regimen such as BD may be a more effective treatment, especially for non-GCB DLBCL and warrants further investigation in the clinical setting, along with food effect studies. DisclosuresYoshizawa:Ono Pharmaceutical Co., Ltd.: Employment. Yasuhiro:Ono Pharmaceutical Co., Ltd.: Employment. Honda:Ono Pharmaceutical Co., Ltd.: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment.

Immune consequences of decreasing tumor vasculature with antiangiogenic tyrosine kinase inhibitors in combination with therapeutic vaccines.

This study investigated the effects on the tumor microenvironment (TME) of combining antiangiogenic tyrosine kinase inhibitors (TKI) with therapeutic vaccines, and in particular, how vascular changes affect tumor-infiltrating immune cells. We conducted studies using a TKI (sunitinib or sorafenib) in combination with recombinant vaccines in two murine tumor models: colon carcinoma (MC38-CEA) and breast cancer (4T1). Tumor vasculature was measured by immunohistochemistry using three endothelial cell markers: CD31 (mature), CD105 (immature/proliferating), and CD11b (monocytic). We assessed oxygenation, tight junctions, compactness, and pressure within tumors, along with the frequency and phenotype of tumor-infiltrating lymphocytes (TIL), myeloid-derived suppressor cells (MDSC), and tumor-associated macrophages (TAM) following treatment with antiangiogenic TKIs alone, vaccine alone, or the combination of a TKI with vaccine. The combined regimen decreased tumor vasculature, compactness, tight junctions, and pressure, leading to vascular normalization and increased tumor oxygenation. This combination therapy also increased TILs, including tumor antigen-specific CD8 T cells, and elevated the expression of activation markers FAS-L, CXCL-9, CD31, and CD105 in MDSCs and TAMs, leading to reduced tumor volumes and an increase in the number of tumor-free animals. The improved antitumor activity induced by combining antiangiogenic TKIs with vaccine may be the result of activated lymphoid and myeloid cells in the TME, resulting from vascular normalization, decreased tumor-cell density, and the consequent improvement in vascular perfusion and oxygenation. Therapies that alter tumor architecture can, thus, have a dramatic impact on the effectiveness of cancer immunotherapy.

Breaking immune tolerance by targeting CD25+ regulatory T cells is essential for the anti-tumor effect of the CTLA-4 blockade in an HLA-DR transgenic mouse model of prostate cancer.

Recent studies suggest that the cancer immunotherapy based on the blockade of the CTLA-4-mediated inhibitory pathway is efficacious only in select populations, predominantly for immunogenic tumors or when delivered in combination with modalities that can break immunologic tolerance to tumor antigens. We studied the effect of CD25+ cell depletion and CTLA-4 blockade on the growth of Transgenic Mouse Adenocarcinoma of Prostate (TRAMP)-PSA tumor cells in DR2bxPSA F1 mice. In these mice, immunological tolerance to PSA was established in a context of the HLA-DRB1*1501(DR2b) allele. In our model, single administration of anti-CD25 antibody prior to tumor inoculation significantly increased IFN-γ production in response to the CD8 T cell epitope PSA65-73 , and delayed TRAMP-PSA tumor growth compared to mice treated with isotype control antibodies. In contrast, the anti-tumor effect of the anti-CTLA-4 antibody as a monotherapy was marginal. The combinatory treatment with anti-CD25/anti-CTLA-4 antibodies significantly enhanced anti-tumor immunity and caused more profound delay in tumor growth compared to each treatment alone. The proportion of tumor-free animals was higher in the group that received combination treatment (21%) compared to other groups (2-7%). The enhanced anti-tumor immunity in response to the CD25 depletion or CTLA-4 blockade was only seen in the immunogenic TRAMP-PSA tumor model, whereas the effect was completely absent in mice bearing poorly immunogenic TRAMP-C1 tumors. Our data suggest that breaking immunological tolerance to "self" antigens is essential for the therapeutic effect of CTLA-4 blockade. Such combinatory treatment may be a promising approach for prostate cancer immunotherapy.

Anti-Tumour Efficacy Study Of The Bruton's Tyrosine Kinase (Btk) Inhibitor, ONO-4059, In Combination With The Glycoengineered Type II Anti-CD20 Monoclonal Antibody, Obinutuzumab (GA101) Demonstrates Superior In Vivo Efficacy Compared To ONO-4059 In Combination With Rituximab

PurposeThe activated B-cell-like (ABC) sub-type of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. There is still a high medical need for new therapies, preferably chemo-sparing, to help treat patients with ABC-DLBCL and CLL as well as other B-cell malignancies. The B-cell receptor signaling pathway is known to be dysregulated in NHL/CLL and given Btk is a downstream mediator of BCR signalling, Btk constitutes an interesting and obvious therapeutic target. Given the high potency and selectivity of the novel Btk inhibitor ONO-4059, it was hypothesized that, the anti-tumour activity of ONO-4059 could be further enhanced by combining it with the anti-CD20 Abs, Obinutuzumab (GA101) or Rituximab (RTX), as a novel therapeutic strategy. MethodsTMD-8 tumour cells, a human ABC-DLBCL cell line, were implanted sub-cutaneously into female SCID mice. Randomization of mice occurred when mean tumour volume was 400-450 mm3. ONO-4059 was administered orally at a dose of 10 mg/kg bid, and GA101 or RTX were administered intravenously both at a sub-optimal dose of 3 mg/kg once weekly. Tumour volumes were measured twice a week after initiation of treatment, and tumour volumes were determined using the formula volume (= width2 x length)/2. Animals were euthanized when the tumours reached a maximum volume of 3,000 mm3. ResultsTreatment with ONO-4059 combined with either GA101 or RTX resulted in a significant inhibition of tumour growth in the TMD-8 xenograft model. GA101 monotherapy showed superior anti-tumour activity compared with RTX monotherapy in terms of tumor growth inhibition (TGI 78% vs 54% at day 21). ONO-4059 combined with GA101 or RTX at sub-optimal doses was significantly better than the respective Abs or ONO-4059 given as monotherapy with TGI of 90% for the GA101 combination and 86% for the rituximab combination. ONO-4059 as single agent mediated a TGI of 63%. In addition combination treatment with GA101 resulted in tumour remission in 3/10 animals, whereas combination treatment with rituximab resulted in tumor remission in only1/10 animals. GA101 monotherapy resulted in 1/10 tumor-free animals, whereas no tumor free animals were observed in the rituximab and ONO-4059 monotherapy groups. ConclusionRecent studies indicate that targeting Btk is effective in the treatment of B-cell malignancies. Our results demonstrate that treatment with ONO-4059 in combination with GA101 or rituximab results in a combined effect in vivo and is superior to the respective monotherapies. Furthermore, ONO-4059 in combination with the novel glycoengineered Type II CD20 antibody GA101 is more effective than the combination of ONO-4059 with rituximab in this DLBCL xenograft model. When interpreting these data it should be noted that both, obinutuzumab and rituximab were dosed at sub-optimal doses as they can induce tumor remission when dosed at 10 mg/kg. Taken together these data indicate that the combination of ONO-4059 with rituximab, and particularily obinutuzumab may be an effective treatment for ABC-DLBCL. Disclosures:Yoshizawa:Ono Pharmaceutical CO., Ltd: Employment. Yasuhiro:Ono Pharmaceutical Co., Ltd.: Employment. Birkett:Ono Pharma UK: Employment. Klein:Roche Glycart AG: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment.TableTumour Growth inhibition (%TGI) in treatment groupGroupDay 21 (TGI, %)pNumber of tumour-free animals (n=10)GA101, 3mg/kg77.9%< 0.0011RTX, 3mg/kg54.3%< 0.0010ONO-4059, 10mg/kg62.6%< 0.0010GA101, 3mg/kg +ONO-4059, 10mg/kg90.2%< 0.0013RTX, 3mg/kg +ONO-4059, 10mg/kg86.4%< 0.0011

Improving immunotherapy of hepatocellular carcinoma (HCC) using dendritic cells (DC) engineered to express IL‐12 in vivo

Interleukin 12 (IL-12), one of the most potent Th1-cytokines, has been used to improve dendritic cells (DC)-based immunotherapy of cancer. However, it failed to achieve clinical response in patients with hepatocellular carcinoma (HCC). In this study, improved conditions of immunotherapy with DC engineered to express IL-12 were studied in murine subcutaneous HCC. Tumour-lysate pulsed DC were transduced with IL-12-encoding adenoviruses or cultivated with recombinant (r)IL-12. DC were injected intratumourally, subcutaneously or intravenously at different stages of tumour-development. Dendritic cell overexpressing IL-12 by adenoviruses showed enhanced expression of costimulatory molecules and stronger priming of HCC-specific effector cells than DC cultured with rIL-12. Intratumoural but not systemic injections of IL-12-DC induced the strongest antitumoural effects reaching complete regressions in 75% of early-staged tumours and in 33% of advanced tumours. Importantly, antitumoural effects could be further enhanced through combination with sorafenib. Analysing the tumour-environment, IL-12-DC increased the levels of Th1-cytokines/chemokines and of CD4(+) -, CD8(+) -T- and NK-cells. Induced immunity was tumour-specific and sustained since all tumour-free animals were protected towards hepatic tumour-cell rechallenge. However, IL-12-DC also enhanced immunosuppressive cytokines, regulatory T cells and even myeloid-derived suppressor cells within the tumours. Induced IL-12-overexpression by adenoviral vectors can effectively immunostimulate DC. Intratumoural but not systemic injection of activated IL-12-DC was crucial for effective tumour regression. The mechanism of this approach seems to be the induction of a sufficient Th1 tumour-environment allowing the recruitment of effector cells rather than the inhibition of tumour immunosuppression. Thus, improved immunotherapy with IL-12-DC represents a promising approach towards HCC.

Abstract 4489: 5’-Sulfamoyl purines are highly active in many xenografts of human solid tumors via oral, intravenous, and intraperitoneal routes of delivery.

Abstract The anticancer activity of 5’-sulfamoyl-purines in cell culture has been previously reported, but there are no published investigations of activity in vivo. One of the earliest members of this class, 5’-O-aminosulfonyl-adenosine, NSC133114, was reported to have 200nM IC50 in L1210 cells (A.Bloch, Biochemistry 10:4394, 1971). We found the average GI50 for NSC133114 was 10 nM across the full NCI-60 cell line panel. When tested in vivo, NSC 133114 was active in the NCI hollow fiber assay, with an overall score of 28 (maximum possible score is 96). NSC750854, the 6-desamino derivative of NSC133114, also had an average GI50 of 10 nM in the NCI-60 panel, but it was nearly twice as active in vivo as NSC133114, with a hollow fiber score of 52. Thus, NSC750854 was chosen for more extensive testing in a set of 8 distinct xenografts. The A498 human renal tumor was the most sensitive xenograft, responding to NSC 750854 via the PO, IP and IV routes. Complete regressions of tumors and tumor-free animals at the end of the study were produced by NSC750854 when administered IP once daily at 5 mg/kg for 2 cycles of 5 days or 3.75 mg/kg twice daily (BID) for 2 cycles of 5 days. At an oral dose of 5 mg/kg given BID for 2 cycles of 6-10 doses each, NSC 750854 produced A498 tumor stasis in the absence of body weight loss. Administered IV at 6.4 mg/kg every other day for 2 cycles of 5 doses or 3.2 mg/kg BID every other day for 2 cycles of 10 doses, NSC 750854 also produced tumor regressions, but no tumor free animals at the end of the study. RPMI-8226 myeloma was the xenograft with the next most sensitive response to NSC750854, with durable tumor stasis following administration by all 3 routes. NSC750854 produced tumor stasis and some regressions in the U251 CNS tumor xenograft, but they were more transient. The PC-3 prostate and LOX melanoma xenografts were responsive to NSC750854 with tumor growth stasis of varying durations. NSC 750854 was effective by all 3 routes of administration but the most dramatic activity was noted with the IP dose route. Among the 8 models tested, the least responsive tumors under the conditions evaluated were Colo 205 colon, HCT-15 colon and UACC-62 melanoma. NSC750854 produced tumor growth suppression but no durable tumor stasis in these 3 models. These patterns of activity in vitro and in vivo for NSC750854 are substantially different than the patterns observed for the anticancer purines that are approved for clinical use. Ongoing studies with NSC 750854 include determinations of activity in advanced tumors, pediatric human tumor models, pharmacologic behavior, toxicologic profile in additional species, and mechanisms of action. This work was conducted under an approved IACUC protocol in AAALACi accredited facilities and supported by federal funds from the National Cancer Institute, NIH, under Contract No. HHSN261200800001E. Citation Format: Melinda G. Hollingshead, Michelle Gottholm-Ahalt, Howard Stotler, John Carter, Cheryl Domitrovich, Adrienne Horner, Jerry M. Collins. 5’-Sulfamoyl purines are highly active in many xenografts of human solid tumors via oral, intravenous, and intraperitoneal routes of delivery. [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 4489. doi:10.1158/1538-7445.AM2013-4489

Abstract 5508: Antitumor activity of cabazitaxel in pediatric patient-derived tumor xenografts (PDX).

Abstract Background: Cabazitaxel (Jevtana®), a semi-synthetic taxane, was approved for patients with metastatic hormone-refractory prostate cancer previously treated with docetaxel-based therapy. Cabazitaxel, which stabilizes microtubules as efficiently as docetaxel, has a better antiproliferative activity than docetaxel against chemotherapy resistant tumor cell lines, a broad spectrum of in vivo antitumor activity in docetaxel-sensitive tumors and in tumor models in which docetaxel was either poorly active or inactive. Unlike docetaxel, cabazitaxel is able to cross the blood-brain barrier and is active in intracranial human glioblastomas (AACR 2000). The preclinical combination of cabazitaxel and cisplatin exhibits therapeutic synergism (AACR 2011). Finally, cabazitaxel demonstrates greater activity than docetaxel in 3 pediatric tumor xenografts, 1 rhabdomyosarcoma and 2 Ewing's sarcomas (AACR 2012). Methods: The anti-tumor activity of cabazitaxel was further evaluated in comparison to docetaxel in 3 pediatric patient-derived tumor xenografts (PDX): 2 osteosarcomas derived from lung metastases of a 19 year old male (DM77) and a 3 year old female (DM113) patient; 1 Ewing's sarcoma taken from the bone of a 17 year old male patient (DM101). Cabazitaxel and docetaxel were dosed IV at 5.8, 9.3, 15 or 24.2 mg/kg, every 4 days for a total of 3 doses (initial tumor burden = 125 - 250 mm3). Results: Both cabazitaxel and docetaxel demonstrated antitumor activity in these 3 PDX. In DM113, cabazitaxel is significantly more active than docetaxel at 9.3, 15 and 24.2 mg/kg. Additionally, when comparing the numbers of partial regressions (PR ≥ 50 % initial tumor size for at least 7days), cabazitaxel is more potent than docetaxel at 15 (4/10 PR versus 0/10 PR, respectively) and at 24.2 mg/kg (5/10 PR versus 1/10 PR, respectively). Using body weight loss as a gross indicator of toxicity, docetaxel appears to more toxic than cabazitaxel in the 2 other PDX studies. In DM101, cabazitaxel at 15 or 24.2 mg/kg doses was significantly more active than docetaxel at the same dose or at the equitoxic dose (9.3 or 15 mg/kg, respectively). In addition, cabazitaxel induces more complete regressions (CR = no palpable tumors for at least 7days) and tumor free animals on day 60 (TF) than docetaxel (9/9 CR and 7/9 TF for cabazitaxel versus 4/9 CR and 1/9 TF for docetaxel at 15 mg/kg; 9/9 CR and 8/9 TF for cabazitaxel versus 3/9 CR and 2/9 TFS for docetaxel at 24.2 mg/kg). Finally, in DM77, cabazitaxel at 15 and 9.3 mg/kg is also more active than docetaxel at an equivalent dose or a toxicity adjusted dose (9.3 or 5.8 mg/kg, respectively). Conclusion: Both cabazitaxel and docetaxel are active in these 3 human pediatric PDX; however, cabazitaxel demonstrates greater activity than docetaxel. Taken together, preclinical data with cabazitaxel including activity in brain tumors and therapeutic synergism with cisplatin, support its development in pediatric indications. Citation Format: Patricia Vrignaud. Antitumor activity of cabazitaxel in pediatric patient-derived tumor xenografts (PDX). [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 5508. doi:10.1158/1538-7445.AM2013-5508

Systemic TNFα Gene Therapy Synergizes With Liposomal Doxorubicine in the Treatment of Metastatic Cancer

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

Cidofovir treatment improves the pathology caused by the growth of human papillomavirus-positive cervical carcinoma xenografts in athymic nude mice

Cidofovir has shown antiproliferative effects against human papillomavirus (HPV)-positive cells and successfully suppressed the growth of HPV-positive xenografts in athymic nude mice. The present study evaluated the effect of cidofovir on several disease parameters in this animal model.Intratumoral administration of cidofovir resulted in a beneficial effect on body weight gain, a reduction in splenomegaly, a partial restoration of tryptophan catabolism, and diminished the inflammatory state induced by the xenografts. Administration of cidofovir to tumor-free animals did not have a direct effect on these parameters.Beyond suppressing tumor growth, intratumoral treatment with cidofovir ameliorated the pathology associated with HPV-tumor growth.

Gender Bias in Skin Cancer: Role of Catalase Revealed

Sullivan et al. describe their finding that lower skin catalase activity in male as compared with female mice may be responsible for the increased skin carcinogenesis observed in UVB radiation-exposed male mice. This adds to the growing literature that points toward a gender bias in the pathogenesis of skin cancer. Delineating the events between UV exposure and carcinogenesis in relation to skin's antioxidant activity will provide insight into disease progression, prognosis, and responses to therapy. If substantiated with further studies in humans, this information may aid in designing gender-specific preventive measures aimed at reversing immune suppression through targeting the altered redox status in UV-exposed individuals.

The vitamin E analog, alpha-tocopheryloxyacetic acid enhances the anti-tumor activity of trastuzumab against HER2/neu-expressing breast cancer

BackgroundHER2/neu is an oncogene that facilitates neoplastic transformation due to its ability to transduce growth signals in a ligand-independent manner, is over-expressed in 20-30% of human breast cancers correlating with aggressive disease and has been successfully targeted with trastuzumab (Herceptin®). Because trastuzumab alone achieves only a 15-30% response rate, it is now commonly combined with conventional chemotherapeutic drugs. While the combination of trastuzumab plus chemotherapy has greatly improved response rates and increased survival, these conventional chemotherapy drugs are frequently associated with gastrointestinal and cardiac toxicity, bone marrow and immune suppression. These drawbacks necessitate the development of new, less toxic drugs that can be combined with trastuzumab. Recently, we reported that orally administered alpha-tocopheryloxyacetic acid (α-TEA), a novel ether derivative of alpha-tocopherol, dramatically suppressed primary tumor growth and reduced the incidence of lung metastases both in a transplanted and a spontaneous mouse model of breast cancer without discernable toxicity.MethodsIn this study we examined the effect of α-TEA plus HER2/neu-specific antibody treatment on HER2/neu-expressing breast cancer cells in vitro and in a HER2/neu positive human xenograft tumor model in vivo.ResultsWe show in vitro that α-TEA plus anti-HER2/neu antibody has an increased cytotoxic effect against murine mammary tumor cells and human breast cancer cells and that the anti-tumor effect of α-TEA is independent of HER2/neu status. More importantly, in a human breast cancer xenograft model, the combination of α-TEA plus trastuzumab resulted in faster tumor regression and more tumor-free animals than trastuzumab alone.ConclusionDue to the cancer cell selectivity of α-TEA, and because α-TEA kills both HER2/neu positive and HER2/neu negative breast cancer cells, it has the potential to be effective and less toxic than existing chemotherapeutic drugs when used in combination with HER2/neu antibody.

2A Peptide-based, Lentivirus-mediated Anti-death Receptor 5 Chimeric Antibody Expression Prevents Tumor Growth in Nude Mice

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces tumor cell death via death receptors on target cells, without adverse effects on most normal cells. Its receptors are therefore an attractive target for antibody-mediated tumor therapy. Here, we report the creation of a lentivirus vector constructed by linking the heavy chain and the light chain of the antibody with a 2A/furin self-processing peptide in a single open reading frame that expresses a novel chimeric antibody (named as zaptuximab) with tumoricidal activity, which is consisted of the variable region of a mouse anti-human DR5 monoclonal antibody, AD5-10, and the constant region of human immunoglobulin G1. Lentivirus-expressed zaptuximab bound specifically to its antigen, DR5, and exhibited significant apoptosis-inducing activity in various tumor cell lines. The packaged recombinant virus lenti-HF2AL showed strong apoptosis-inducing activity in vitro. Meanwhile, inoculated subcutaneous human colon HCT116 tumor formation in nude mice were inhibited significantly. Moreover, there was a synergistic effect of mitomycin C (MMC) on the observed tumoricidal efficacy, prolonging the life span of nude mice with orthotopic human lung tumor cancers. These data suggest that lentivirus-mediated, 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility as an anticancer treatment and may represent a rational adjuvant therapy in combination with chemotherapy.

Investigation of fatty acid profile for diagnostic purposes in mouse melanoma

The aim of this study was to investigate whether fatty acid profile is a suitable marker for diagnostic purposes in mouse melanoma. Twelve C57Bl/6 male mice were implanted with B16 mouse melanoma cells (106 cells/animal) orthotopically (subcutaneously). After the implantation 4-4 animals were bled by cardiac puncture following narcosis, at days 7, 14, and 21. In order to investigate fatty acid profiles a method based on extraction and HPLC-MS was developed. Signal intensities of 14 fatty acids were determined by mass spectrometry in tumor-free animals as well as tumor bearing animals at the three time points. Mathematical analysis showed non-significant profile changes when control (tumor-free) animals were compared to tumor-implanted ones as well as during tumor progression on week 1, 2 and 3. In case of three fatty acids (myristic acid, palmitoleic acid and eicosadienoic acid) a trend was observed during tumor progression but its statistical significance cannot be evaluated without further investigations. The fatty acid profile cannot be used for early diagnoses in mouse melanoma.

Abstract LB-232: APG350, a dimerized single chain TRAIL receptor agonist with enhanced functional properties

Abstract TRAIL (Apo2L) is a member of the Tumor Necrosis Factor Superfamily (TNF-SF) that induces apoptosis through binding to two closely related receptors, TRAIL-R1 and TRAIL-R2. Its unique ability of selective apoptosis induction in tumor cells makes TRAIL an attractive molecule for tumor therapy. APG350 is a newly designed TRAIL-receptor agonist comprising two single-chain TRAIL-receptor binding domains (each consisting of three covalently linked TRAIL protomers) that are dimerized via the Fc-part of human IgG1. APG350 has six receptor binding sites per molecule and binds to both death inducing TRAIL receptors. In vitro, APG350 shows potent apoptosis induction on a wide subset of human tumor cell-lines, on cancer stem cells and on primary tumor cells. Mechanistically its improved ability to form clusters on target cells composed of six TRAIL-receptors each, distinguishes APG350 from current clinical development candidates. Agonistic antibodies like Conatumumab or Apomab are capable to bind two receptor chains per molecule, while recombinant TRAIL (e.g. Dulanermin) binds three receptor chains per molecule. Direct in vitro comparison of APG350 with recombinant TRAIL (APG400) and a TRAIL-R2 specific agonistic monoclonal antibody revealed superior apoptosis induction capacity for APG350. Analysis of the PK-parameters in mice showed a half life of 23.1h for APG350 (1.04h for APG400) indicating a significantly improved PK in comparison to APG400. The half life of APG350 in a Cynomolgus monkey was 26.7h. Comparative treatment of mice bearing Colo205 xenograft tumors with APG350 and APG400 employing one treatment cycle with five consecutive daily intravenous injections showed a superior efficacy of APG350 with respect to tumor volume reduction and number of tumor free animals. Even mice with initially large tumors (∼600 mm3) were treated effectively with APG350. Remarkably, APG350 also showed pronounced dose dependent anti tumor efficacy on xenograft-tumors derived from colon cancer stem cells (CSC). Furthermore large CSC-derived tumors could be treated effectively and successful re-treatment of previously responding tumors demonstrated that tumors did not gain drug resistance. In a pilot experiment APG350 treatment of mice bearing a slowly growing primary rectum tumor xenograft also showed a significant tumor volume reduction. In all in vivo studies APG350 was well tolerated at doses between 0.3–100mg/kg bw in mice and doses up to 10mg/kg bw in a Cynomolgus monkey. General tolerability and potential effects on liver toxicity were assessed by co-application of APG350 together with a crosslinking antibody in mice. This treatment evoked only minor clinical signs at high doses with no relevant increase in liver enzymes. Currently two non-GLP toxicology studies, a 4-week study in mice and a dose escalation study in Cynomolgus monkeys are ongoing to confirm tolerability in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-232. doi:10.1158/1538-7445.AM2011-LB-232

Abstract 2838: Brief exposure to gefitinib/Iressa during premalignant risk window provides lifelong protection from mammary tumor development in MMTV-erbB-2 transgenic mice

Abstract Although chemopreventive agents targeting estrogen/estrogen receptor (ER) pathway have been effective for ER+ breast cancer, hormonal resistance associated with increased receptor tyrosine kinase activities, such as erbB-2 and/or EGFR, remains a significant issue in beast cancer prevention. Previous studies demonstrated that administration of gefitinib, an orally active EGFR tyrosine kinase inhibitor, to MMTV-erbB-2 transgenic mice inhibited mammary tumor development in these mice, suggesting that gefitinib is a promising agent for the prevention of ER-negative breast cancer. However, the prevention was achieved by high dose (100 mg/kg) and prolonged drug exposure (from 12 to 50 weeks). The tolerance to high dose/long-term of drug administration for the prevention regimens raised concerns in clinical application. In this study, we tested the efficacy of brief exposure to gefitinib during the high risk window in the prevention of mammary tumor development in the MMTV-erbB-2 transgenic mice and studied the underlying mechanisms. We found that brief exposure of gefitinib to these mice at a dosage of 100 mg/kg/day from 16 to 23 weeks (total 8 wks) resulted in significant chemopreventive effects. In contrast to no tumor-free animals in the control group by 52 weeks, mice in gefitinib treated group remained 65% tumor-free at the same age, which was comparable to the results from a previous report that mice with lifelong gefitinib were 75% tumor-free by 45 weeks. We further demonstrated that delayed tumor development in the treated mice was preceded with decreased mammary epithelial density. Molecular analysis indicated that gefitinib inhibited phosphorylation and expression of EGFR, erbB-3 and endogenous erbB-2 in premalignant mammary tissues. Phosphorylation of Akt1 and Erk1/2 was also significantly downregulated. Importantly, although tumors developed from this model were ER-, mammary tissues in the premalignant stage were ERα+; and gefitinib treatment drastically inhibited the phosphorylation and expression of ERα and the transcription of ER target genes, including EGF, EGFR, ESR1, Bcl-2, cyclin D1 and c-myc. Moreover, BrdU incorporation analysis demonstrates that cell proliferation in the mammary glands in gefitinib treated mice was significantly inhibited. Taken together, these data demonstrate that brief treatment with EGFR/erbB-2 targeting agents at the appropriate risk window may provide lifelong protection from mammary tumors, and inhibition of ER-erbB-2 crosstalk may play a critical role in this long lasting preventive process. Given the concerns associated with high dose/long-term gefitinib treatment, our findings are of great significance in directing clinical application of EGFR/erbB-2 targeting agents as chemopreventive agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2838. doi:10.1158/1538-7445.AM2011-2838