Regression Of Established Tumors Research Articles

Abstract 1764: Inhibition of R-spondin (RSPO) signaling reduces the growth of multiple human tumors

Abstract Deregulation of the canonical Wnt/beta-catenin signaling pathway has long been associated with cancer. Intracellular components of this pathway, including Axin, APC, and beta-catenin are frequently mutated in a range of human tumors. The identity of specific extracellular ligands that contribute to human cancer development through this signaling axis has remained unclear. Molecular characterization of the secreted beta-catenin signaling activities produced by minimally passaged human tumor xenograft models identified RSPO family members produced by multiple tumor types including ovarian, pancreatic, colon, breast and non-small cell lung cancer. In human tumor xenograft models that had RSPO overexpression, in some instances due to genomic translocation, anti-RSPO treatment markedly inhibited tumor growth. In addition, striking combination activity with standard of care chemotherapy agents resulted in regression of established tumors. These results highlight the potential for therapeutic intervention with this newly appreciated signaling axis. Citation Format: Austin Gurney, Fumiko Axelrod, Chris Bond, Jennifer Cain, Cecile Chartier, Marcus Fischer, May Ji, Chris Murriel, Janak Raval, Jalpa Shah, Min Wang, Wan-Ching Yen, Ann Kapoun, John Lewicki, Timothy Hoey. Inhibition of R-spondin (RSPO) signaling reduces the growth of multiple human tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1764. doi:10.1158/1538-7445.AM2014-1764

Abstract 2724: Development of a sensitive assay for measuring pharmacodynamic modulation of c-Met in biopsies

Abstract Introduction: MSC2156119J, a highly selective, potent, reversible, ATP-competitive c-Met inhibitor currently under clinical testing, efficiently inhibits c-Met phosphorylation and downstream signaling in vivo and induces regression of established tumors in xenograft models in preclinical studies (Bladt et al. Clin Cancer Res. 2013;19:2941-51). One initial event following c-Met activation is Y1234/35 phosphorylation in the activation loop of the kinase domain. This results in kinase activation and triggers phosphorylation of tyrosine residues in the c-Met C-terminal tail (eg, Y1349), creating multifunctional docking sites for intracellular adapters. The development of an assay allowing the measurement of these phospho-c-Met epitopes in tumor biopsies will be crucial for establishing an optimal biologic dose for MSC2156119J. Methods: We describe here the development of a highly sensitive Luminex assay that can reliably and reproducibly measure the phosphorylation state of Y1234/35 and Y1349 residues of the c-Met receptor. The high sensitivity of the assay was confirmed by measuring the phospho-c-Met levels in biopsies from patients before and during MSC2156119J treatment. Results: We first assessed the stability of phosphorylated Y1234/35 and Y1349 epitopes in preclinical tumor samples. Analyses revealed that the total c-Met protein is rather stable over time, while the phospho-c-Met epitopes Y1234/35 and Y1349 are relatively unstable; their detection requires rapid processing of tumor samples derived from preclinical tumors or from patients. In tumor samples derived from human xenografts treated with MSC2156119J, Y1349 phosphorylation inhibition varied in different tumor models and did not show strong dose dependence. In contrast, phosphorylation of Y1234/35 residues was effectively inhibited by MSC2156119J in all tumor models tested. Based on these findings, phosphorylation of c-Met Y1234/35 was also assessed in tumor biopsies from patients treated with MSC2156119J in the first-in-man trial (NCT01014936; Falchook et al. J Clin Oncol. 2013;31(suppl):2506). When compared to pretreatment tumor biopsies, on-treatment samples showed effective, dose-dependent inhibition of >90% of c-Met Y1234/35 phosphorylation. Conclusions: We successfully developed an assay capable of detecting phosphorylation of the c-Met Y1234/35 epitope, which is crucial for c-Met activation. Using this assay on tumor samples from xenograft models and on patient-derived tumor biopsies, we demonstrated that MSC2156119J inhibits phosphorylation of c-Met Y1234/35 in a dose-dependent fashion. Therefore, phosphorylation of c-Met Y1234/35 can be used as a pharmacodynamic biomarker of c-Met inhibition and will be an important and valuable element for the selection of the optimal biologic dose of MSC2156119J. Citation Format: Friedhelm Bladt, Frank Jaehrling, Manja Friese-Hamim, Gerald S. Falchook, Hesham M. Amin, Manfred B. Klevesath, Andree Blaukat. Development of a sensitive assay for measuring pharmacodynamic modulation of c-Met in biopsies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2724. doi:10.1158/1538-7445.AM2014-2724

Histone deacetylase inhibition enhances the lymphomacidal activity of the anti-CD22 monoclonal antibody HB22.7

HB22.7, an anti-CD22 monoclonal antibody has shown consistent preclinical activity against non-Hodgkin lymphoma (NHL). Histone deacetylase inhibitors (HDACi) have demonstrated efficacy in lymphoma and can modulate cell surface receptor expression. To augment the lymphomacidal activity of HB22.7 we examined the combination of AR42 (an HDACi) and HB22.7 in vitro and in vivo. The combination resulted in 10-fold increased potency in 6 NHL cell lines when compared to either drug alone. Both drugs reduced tumor progression in xenografts, but the combination was significantly more efficacious and resulted in regression of established tumors, without toxicity. AR42 inhibited HB22.7-mediated CD22 internalization, suggesting that increased efficacy could be due to higher availability of CD22. Overall, the synergistic effects of HB22.7 and AR42 on in vitro cytotoxicity and in vivo anti-tumor activity make this combination an attractive option for further pre-clinical and clinical evaluation.

Immune combinational therapy targeting OX40 and IDO synergistically enhances efficacy of a cancer vaccine

Enhancing an effector immune response by cancer vaccines has not been clinically successful so far. Although the necessary immune response may be elicited using vaccine-based therapies, this has not been sufficient for a positive clinical outcome; since most cancers can escape immune surveillance via multiple immune suppressive mechanisms induced in the tumor environment. A relatively recent strategy to increase the therapeutic efficacy of tumor vaccination is to combine it with alternative immunotherapeutic approaches. Here we tested if simultaneous targeting of the effector arm of the immune system with an agonist anti-OX40 mAb together with targeting of suppressive mechanisms in the tumor, via inhibition of indoleamine-(2,3)-dioxygenase (IDO), could enhance the anti-tumor activity of vaccine in mouse models of cancer. The OX40 molecule is a co-stimulatory receptor expressed on T-cells that can lead to proliferation and enhancement of T-cell effector function when triggered with an agonistic antibody. However, the tumor secretes a number of suppressive signals as a protective mechanism against its destruction, one of which is IDO. We demonstrate that treatment of TC-1 tumor bearing mice with an anti-OX40 agonist antibody in combination with vaccine leads to the enhancement of antigen-specific T cell responses. The dose of 1 mg/kg anti-OX40 antibody stimulated the effector arm of T cells, which ultimately led to a significant increase in the CD8+/regulatory T-cell (Treg) ratio within tumors. Further, this combination of vaccine and anti-OX40 antibody treatment led to a significant inhibition of tumor growth and to a prolonged survival of tumor bearing mice compared to control, and resulted in complete tumor regression in 20% of treated mice. We found, however, that this anti-tumor activity could be further enhanced through the combination of vaccine, anti-OX40 and 1-methyltryptophan (1-MT), an IDO activity inhibitor. IDO has been shown to be secreted by tumor cells, suppressive dendritic cells and macrophages in tumor environment, and is known to be responsible for suppressing effector cells and inducing Tregs. Our data demonstrate that vaccine/anti-OX40 antibody/1-MT combination leads to a more profound inhibition of tumor growth in mice and complete regression of established tumors in 60% of treated mice. In conclusion, we provide evidence that simultaneous targeting of the effector arm of the immune system, via anti-OX40 antibody, and suppressive mechanisms within the tumor, via 1-MT, in combination with cancer vaccine has a synergistic effect for tumor eradication and is a promising strategy that could potentially enhance the overall efficacy of cancer treatment in patients.

Abstract MS01-1: Using T cell receptor engineered T cells for the treatment of cancer

Abstract Adoptive cellular immunotherapy involves the administration of autologous or allogenic tumor reactive T cells into patients to achieve tumor regression and has been successful in transplant-related malignancies, leukemia, and melanoma. The molecule responsible for the recognition of tumor antigens is the T cell receptor (TCR). Best studied in melanoma, this process involves the identification of lymphocytes with high affinity for tumor antigens that can be selected in vitro and expanded and administered to patients. The administration of naturally occurring T cells has been shown to have an objective response rate ranging from 50-70% in metastatic melanoma patients; including bulky invasive tumors at multiple sites including liver, lung, soft tissue and brain. A major limitation to the widespread application of adoptive cell therapy is the difficulty in generating human T cells with anti-tumor reactivity. To overcome this limitation, we can now insert highly active TCR genes into T cells that can recognize tumor antigens. Genetic engineering of TCR genes into normal T cells is a powerful new strategy to generate large numbers of defined antigen-specific cells for therapeutic application. This approach has evolved beyond experimental stage into a clinical reality. The feasibility of TCR engineered T cells has been shown to be an effective clinical strategy resulting in the regression of established tumors in recent clinical trials. Ultimately it may be possible to establish a collection of defined TCR genes that could be used to treat a diverse set of cancers tailored to each patient depending on the expression pattern of their tumor antigens. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr MS01-1.

VIP-Receptor Antagonist Enhanced Antileukemic Activity In Mice

Background and Objective We have previously published that antagonizing vasoactive intestinal peptide (VIP) receptors dramatically decreases PD-1 expression on activated CD8+ T-cells and increases antiviral immunity (Blood 2013, 121:2347-51; PLoS One 2013, 8: e63381). Herein we tested whether short-term pharmacological antagonism of VIP signaling could induce anti-tumor immune responses in mice. Methods B6 mice were inoculated with 0.5 - 2 x 106 luciferase+ murine acute myeloid leukemia cells (Luc+ C1498) and B10BR mice were injected with 3 x 106 luciferase+ murine acute T-cell lymphoma cells (Luc+ LBRM) through tail vein. Mice were treated with one or more daily s.c. injections of 10 μg VIPhyb. Survival of groups that received 1, 3 or 7 doses of VIPhyb were compared with PBS treated controls. Tumor growth was monitored weekly by bioluminescence imaging (BLI). Cytokine expression and expression of immune markers PD-1, PD-1H (VISTA), and PD-L1 on blood and spleen leukocytes were analyzed by flow cytometry. Results Long-term survival (day 80) of tumor-bearing B6 and B10BR mice that received a single-dose of VIPhyb one day before tumor inoculation was 80% for both mouse strains harboring both leukemia cells (p<0.01 vs. control, B6 n=10 and B10BR n=5, Figure 1). A single injection of VIPhyb was more effective than multiple doses in achieving long-term tumor-free survival, with 60% survival among C1498-tumor bearing mice (p<0.01 vs. controls, n=5) treated with 3 doses, and 46% survival in mice (p<0.01 vs. control, n=13) with C1498 and 40% survival in mice (p=0.06 vs. control, n=5,) with LBRM treated with 7 doses. None of the control mice inoculated with C1498 (n=21) or LBRM (n=10) that received PBS injections survived to day 55. To explore the therapeutic effect of VIPhyb on established tumors, B6 mice and B10BR were treated with 7 daily doses of VIPhyb starting 8 days or 15 days after inoculation with Luc+ C1498 or Luc+ LBRM, respectively. Survival of B6 mice bearing C1498 and B10BR mice bearing LBRM that received delayed administration of VIPhyb was 60% (p<0.001 vs. control, n=10) and 20% (p=0.039 vs. control, n=5), respectively, compared with 0 % survival (and faster tumor growth) among control mice (B6 n=10; B10BR n=5) that received PBS injections. Tumor burdens in VIPhyb treated mice measured by BLI showed slower tumor growth, and regression of established tumors compared with mice that received PBS (Figure 1). To elucidate the mechanisms whereby VIPhyb induced anti-tumor activities, expression of serum cytokines (IFN-γ, TNF-α, IL-10 and IL-13), expression of co-inhibitory molecules PD-1, PD-1H, PD-L1, and effector molecules Fas-L and granzyme B were measured in T-cells from VIPhyb- and PBS-treated tumor-bearing B10BR mice. Blood and splenic activated (CD62L-CD25+CD69+) and memory (CD62L+/-CD44+) CD8+ T-cells from VIPhyb-treated tumor–bearing mice expressed higher levels of IFN-γ, FAS-L and granzyme B, and lower levels of PD-1 (but not VISTA/PD-1H) in activated CD8+ T-cells compared with those from PBS-treated mice (Figure 2). Expression levels of TNF-α, IL-10, IL-13, and PD-L1 in blood and splenic dendritic cells were not different comparing with tumor-bearing VIPhyb-treated with PBS-treated control mice. Conclusion Treatment with a small molecule antagonist of VIP-receptor, VIPhyb, dramatically increased immune/T-cell specific anti-leukemic activity. The mechanism by which administration of a VIP receptor antagonist enhanced anti-tumor immunity includes increasing productions of IFN-γ, and expression of FAS-L and granzyme B in and decreasing expression of PD-1 in activated CD8+ T-cells, leading to enhance anti-tumor cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

Abstract PL07-03: Mechanisms of protective tumor immunity.

Abstract Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma and some advanced ovarian carcinoma patients. The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. The pharmacologic inhibition of MFG-E8 function through genetic or engineering-based approaches blocks Treg induction, which intensifies vaccine-induced responses, leading to the regression of established tumors in mice. The clinical translation of these therapeutic strategies to Phase I testing in humans is underway. The detailed analysis of patients achieving sustained clinical benefits from irradiated, autologous GM-CSF secreting tumor cell vaccines and CTLA-4 antibody blockade also affords a rich opportunity to identify antigens associated with immune-mediated tumor destruction and to delineate mechanisms of therapeutic immunity. We elucidated several of the molecular pathways that underlie these antitumor effects, including the NKG2D system and multiple secreted/or cell surface proteins that contribute to tumor promoting inflammation. The therapy-induced antibodies manifest functional activity in vitro, antagonizing tumor cell survival, invasive potential, and angiogenesis. These findings support a key role for humoral immunity in tumor destruction, and highlight interest in more detailed characterization of the antitumor B cell repertoire in vaccinated patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL07-03. Citation Format: Glenn Dranoff. Mechanisms of protective tumor immunity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL07-03.

Abstract 3975: Regression of established tumors requires timely adoptive T cell transfer following whole body irradiation.

Abstract Adoptive immunotherapy with CD8+ T cells has achieved some success in treating established cancer. Response rates are increased by preconditioning the patient with lymphodepleting whole body irradiation (WBI) prior to T cell transfer, including an increased fraction of complete responses. Using a mouse model of autochthonous brain cancer, we previously demonstrated that adoptive transfer of tumor-specific T cells following WBI promotes complete regression of established tumors and long-term survival. However, the mechanisms required to achieve complete responses remain to be defined. Here we investigate kinetic requirements for adoptive T cell transfer to achieve tumor control following WBI. To test the hypothesis that adoptive transfer early after irradiation is required for maximum long-term tumor control, we delayed adoptive transfer of T cells to tumor-bearing mice at varying intervals following irradiation. We show that delay of adoptive transfer did not affect initial T cell priming or the timing of initial appearance of activated T cells at the tumor site. However, we observed a modest decrease in T cell accumulation at the earliest appearance of cells in the brain (5 days post-transfer). Tumors were completely eliminated within 10 days in mice that received T cell transfer one day following WBI, while delayed transfer abrogated this effect. When adoptive transfer was delayed by one week following WBI, incomplete tumor regression was observed at 10 days post-transfer. Although mice in this group lived significantly longer than control animals, they eventually succumbed to lethal tumor recurrence. Our data suggest that lymphodepleting doses of WBI promote a prolonged window for effective T cell priming against the endogenous tumor antigen, but that optimal therapeutic potential requires T cell transfer early after irradiation conditioning. Supported by research grant CA02500 from the National Cancer Institute/National Institutes of Health. Citation Format: Eugene M. Cozza, Lynn R. Budgeon, Neil D. Christensen, Todd D. Schell. Regression of established tumors requires timely adoptive T cell transfer following whole body irradiation. [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 3975. doi:10.1158/1538-7445.AM2013-3975

Abstract SY27-04: Engineering improved cancer vaccines.

Abstract Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma and some advanced ovarian carcinoma patients. The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. The pharmacologic inhibition of MFG-E8 function through genetic or engineering-based approaches blocks Treg induction, which intensifies vaccine-induced responses, leading to the regression of established tumors in mice. The clinical translation of these therapeutic strategies to Phase I testing in humans is underway. The detailed analysis of patients achieving sustained clinical benefits from irradiated, autologous GM-CSF secreting tumor cell vaccines and CTLA-4 antibody blockade also affords a rich opportunity to identify antigens associated with immune-mediated tumor destruction and to delineate mechanisms of therapeutic immunity. We elucidated several of the molecular pathways that underlie these anti-tumor effects, including the NKG2D system and multiple secreted/or cell surface proteins that contribute to tumor promoting inflammation. The therapy-induced antibodies manifest functional activity in vitro, antagonizing tumor cell survival, invasive potential, and angiogenesis. These findings support a key role for humoral immunity in tumor destruction, and highlight interest in more detailed characterization of the anti-tumor B cell repertoire in vaccinated patients. Citation Format: Glenn Dranoff. Engineering improved cancer vaccines. [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 SY27-04. doi:10.1158/1538-7445.AM2013-SY27-04

Abstract 5322: CD44+ prostate cancer cells are regulated by microRNA-708: A novel role of microRNA with potential prognostic and diagnostic significance in prostate cancer .

Abstract A challenging aspect of prostate cancer (PCa) is the high rates of recurrence associated with the disease. About 40% of men with localized prostate cancer suffer from relapse after initial therapy. In these cases, androgen ablation therapy is used to reduce tumor burden/circulating PSA to low or undetectable limits. However, most patients suffer from disease recurrence, as monitored by rising PSA and tumor progression to hormone refractory/castration resistant prostate cancer that is essentially untreatable. Tumor recurrence has been attributed to the existence of tumor-initiating cells (TIC) within the bulk of the tumor; CD44 is an important marker for these cells. In this study, we demonstrate that miR-708 is a key negative regulator of CD44+ subpopulation of PCa cells. miR-708 was underexpressed in CD44+ cells from PCa xenografts. Reconstitution of miR-708 in PCa cell lines or CD44+ PCa cells led to decreased tumorigenicity in vitro. Intratumoral delivery of synthetic miR-708 oligonucleotides triggered regression of established tumors in a murine xenograft model of human prostate cancer. Conversely, miR-708 silencing in a purified CD44- population of PCa cells promoted tumor growth. Functional studies validated CD44 to be a direct target of miR-708. Also, we identified Androgen Receptor (AR) and the serine/threonine kinase AKT2, a member of the PI3K/AKT signaling pathway as functional targets of miR-708. Clinically, low miR-708 expression was associated significantly with poor survival outcome, tumor progression and recurrence in prostate cancer patients. In view of these results we propose that reduced miR-708 expression leads to prostate cancer initiation, progression and development by regulating expression of CD44, AR, AKT2, which in turn regulate proliferative, invasive and migratory abilities of prostate cancer cells. Therefore, miR-708 may represent a novel therapeutic target for prostate cancer. Citation Format: Sharanjot Saini, Sumit Arora, Shahana Majid, Varahram Shahryari, Soichiro Yamamura, Inik Chang, Takeshi Chiyomaru, Shinichiro Fukuhara, Guoren Deng, Yuichiro Tanaka, Rajvir Dahiya. CD44+ prostate cancer cells are regulated by microRNA-708: A novel role of microRNA with potential prognostic and diagnostic significance in prostate cancer . [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 5322. doi:10.1158/1538-7445.AM2013-5322

HA14-1, but not the BH3 mimetic ABT-737, causes Ca2+ dysregulation in platelets and human cell lines

HA14-1, but not the BH3 mimetic ABT-737, causes Ca2+ dysregulation in platelets and human cell lines

Activation of endogenous CD4 T cells stimulates favorable immunologic changes in the tumor milieu and enhances the antitumor effect of lentivector immunization (48.15)

Abstract CD4 T cells are known to help the induction, migration, and effector functions of CD8 antitumor responses. Here, we reported an additional layer of CD4 helper function, i.e. activation of endogenous CD4 T cells could modulate the tumor milieu. We engineered a lentivector to express a nominal fusion Ag composed of hepatitis B surface protein (HBsAg) and IgG2a Fc domain (HBS-Fc-lv) that induced potent CD8 as well as robust CD4 response. Remarkably, immunization with this vector caused regression of established tumors. Immunological analysis revealed that functional CD8 and CD4 T effector (Teff) was markedly increased while the Treg ratio was substantially decreased in the regressing tumors. HBS-Fc-lv immunization also shifted the suppressive tumor lesion microenvironment to one that is immune stimulating. Mechanistically, the decrease of tumor Treg ratio and the improvement of antitumor effect of HBS-Fc-lv immunization were dependent on CD4 activation and IFNg production. CD4 Teff cells were preferentially expanded in the vaccine draining lymph nodes and selectively recruited into tumor lesions as the chemokines for Teff recruitment were significantly increased in the tumor lesions after HBS-Fc-lv immunization. These data indicate that immunization strategies capable of activating both CD8 and CD4 T cells can improve the tumor milieu and generate an enhanced antitumor effect.

Inhibitory effect of sulindac against chemically-induced primary colonic tumors by N-methyl-N-nitrosourea in mice.

To investigate the chemopreventive effect of sulindac, a nonsteroidal anti-inflammatory drug (NSAID), on the growth of N-methyl-N-nitrosourea (MNU)-induced mouse colonic tumors. The experimental colonic tumor model induced by intrarectal instillation of MNU in mice was used in the present study. In the first experiment, MNU intrarectal was instilled and sulindac administered concurrently to a group of mice for a period of 18 wk, while a control group of animals received MNU only for the same period. In the second experiment, two groups of mice that had already been treated with MNU for 12 wk received sulindac or not for another 18 wk. The tumors induced in mice were all located in the distal part of the large intestine. There were no significant differences in the location and the gross appearance of the tumors in the MNU-induced group and control group in both experiments. In the first experiment, sulindac caused a significant reduction in both the number of mice with colonic tumors and the number of tumors per mouse. Sulindac had a significant inhibitory effect on the growth of the MNU-induced tumors. However, in the second experiment, the inhibitory effect of sulindac was less or disappeared. Sulindac has a protective effect against the chemical induction of colonic tumors by MNU in mice. The chemopreventive effect is more significant in the initial stage of the tumor, while in the promotion stage this effect is less or disappeared. Sulindac can not cause the regression of established tumors.

RECENT PROGRESS AND FUTURE DIRECTIONS USING ENGINEERED T CELLS FOR THE TREATMENT OF CANCER

Adoptive immunotherapy with tumor infiltrating lymphocytes (TILs) is a potent therapy for metastatic melanoma. In this process, naturally occurring tumor reactive TILs that bear T-cell receptors (TCR) targeted against tumor cells are generated ex vivo and administrated into the patients. The generation of tumor-reactive T cells is not always possible in all of the patients. To overcome this limitation, we can now insert highly avid TCRs into T cells that can recognize tumor antigens. Genetic engineering of TCR genes into normal T cells is a powerful new strategy to generate large numbers of defined antigen-specific cells for therapeutic application. This approach has evolved beyond experimental stage into a clinical reality. The feasibility of TCR-engineered T cells has been shown to be an effective clinical strategy resulting in the regression of established tumors in recent clinical trials. In this review, we discuss the progress and prospects of TCR-engineered T cells as a therapeutic strategy for treating patients with melanoma and other cancers.

Linear and branched glyco-lipopeptide vaccines follow distinct cross-presentation pathways and generate different magnitudes of antitumor immunity.

BackgroundGlyco-lipopeptides, a form of lipid-tailed glyco-peptide, are currently under intense investigation as B- and T-cell based vaccine immunotherapy for many cancers. However, the cellular and molecular mechanisms of glyco-lipopeptides (GLPs) immunogenicity and the position of the lipid moiety on immunogenicity and protective efficacy of GLPs remain to be determined.Methods/Principal FindingsWe have constructed two structural analogues of HER-2 glyco-lipopeptide (HER-GLP) by synthesizing a chimeric peptide made of one universal CD4+ epitope (PADRE) and one HER-2 CD8+ T-cell epitope (HER420–429). The C-terminal end of the resulting CD4–CD8 chimeric peptide was coupled to a tumor carbohydrate B-cell epitope, based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules. The resulting HER glyco-peptide (HER-GP) was then linked to a palmitic acid moiety, attached either at the N-terminal end (linear HER-GLP-1) or in the middle between the CD4+ and CD8+ T cell epitopes (branched HER-GLP-2). We have investigated the uptake, processing and cross-presentation pathways of the two HER-GLP vaccine constructs, and assessed whether the position of linkage of the lipid moiety would affect the B- and T-cell immunogenicity and protective efficacy. Immunization of mice revealed that the linear HER-GLP-1 induced a stronger and longer lasting HER420–429-specific IFN-γ producing CD8+ T cell response, while the branched HER-GLP-2 induced a stronger tumor-specific IgG response. The linear HER-GLP-1 was taken up easily by dendritic cells (DCs), induced stronger DCs maturation and produced a potent TLR- 2-dependent T-cell activation. The linear and branched HER-GLP molecules appeared to follow two different cross-presentation pathways. While regression of established tumors was induced by both linear HER-GLP-1 and branched HER-GLP-2, the inhibition of tumor growth was significantly higher in HER-GLP-1 immunized mice (p<0.005).SignificanceThese findings have important implications for the development of effective GLP based immunotherapeutic strategies against cancers.

Abstract LB-387: MYCN dependence of tumor propagating cells in an orthotopic model of medulloblastoma

Abstract Medulloblastoma (MB) is the most common paediatric brain tumor and is a major cause of cancer death in children. MYCN expression is critical to cerebellar development, regulates the fate of neural progenitor cells (NPCs) and drives the expansion of tumor propagating cells (TPCs) thought to represent the cell-of-origin for this disease. To examine the role of MYCN in MB tumorigenesis, we established neurosphere cell lines enriched for putative TPCs from the cerebella of GTML mice (transgenic for doxycycline-regulable overexpression of MYCN and luciferase). These mice are predisposed to anaplastic/large-cell MB (AACR abstract LB-79). Neurospheres derived from GTML cerebella with positive luciferase imaging signals but no visible tumor tissue exhibited several characteristics consistent with enrichment for TPCs and/or NPCs. Spheroids proliferated efficiently in the absence of exogenous growth factor, exhibited self-renewal in clonal dilution assays, expressed high levels of Mycn protein and the NPC markers nestin and SOX2, and in response to serum, differentiated along neuronal and glial lineages (increased GFAP and III tubulin expression by immunofluorescence). Finally, orthotopic reimplantation at low titer (&amp;lt;250 cells) recapitulated anaplastic/large-cell MB tumor formation with short latency (&amp;lt;14 days) and complete penetrance. Suppression of MYCN with doxycycline (confirmed by loss of luciferase imaging) caused growth arrest via a G1 blockade of neurospheres in vitro. Pretreatment of implanted spheres significantly reduced tumor penetrance (&amp;lt;60%) and increased latency (2-3 months). Efficient regression of established tumors was observed with concomitant loss of nestin expression and increases in differentiated glial and neuronal markers. Taken together, these data suggest that MYCN expression drives MB tumor formation by maintenance or expansion of a population of relatively undifferentiatedTPCs present in normal cerebella. This model represents a significant tool for further investigation of the role of MYCN in MB initiation and progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-387.

Immunization with lentivector expressing HBV Ag markedly improves the tumor microenvironment and result in regression of established tumors (131.44)

Abstract One of the main reasons for the limited antitumor efficacy of current tumor vaccines is the failure of immune effector cells inside the tumor environment due to the infiltration of immune suppressor cells and immune dysfunction of effector cells. In this study, we investigated if the immune suppressive tumor environment can be improved by lentivector immunization. We found that lentivector immunization could not only stimulate potent T cell responses against HBV surface Ag and cause tumor influx of effector T cells, but also markedly changed the tumor microenvironment. Immunization with lentivector induces strong proliferation of CD4 and CD8 T effector cells but does not increase the proliferation of Treg in the vaccine draining LN. More importantly, lentivector immunization profoundly increase proliferation of the CD4 Teff cells in the tumor lesions, resulting in more CD4 Teff cells accumulation in the tumor lesions and decrease of Treg ratio in the tumor. Analysis of the tumor environment showed that lentivector immunization can skew immune suppressive tumor environment to Th1 and Tc1 like environment, with marked increase of T-bet, IFNγ, Perforin, and granzyme in the tumor lesions. Lentivector HBV-S-lv immunized mice were completely protected from HBV positive B16 tumor cells (B16-S) challenge. Furthermore, established B16-S tumor can be eradicated by lentivector immunization.

Naïve tumor-specific CD4+ T cells differentiated in vivo eradicate established melanoma (131.12)

Abstract In vitro differentiated CD8+ T cells have been the primary focus of immunotherapy of cancer with little focus on CD4+ T cells. Immunotherapy involving in vitro differentiated T cells given after lymphodepleting regimens significantly augments antitumor immunity in animals and human patients with cancer. However, the mechanisms by which lymphopenia augments adoptive cell therapy and the means of properly differentiating T cells in vitro are still emerging. We demonstrate that naïve tumor/self-specific CD4+ T cells naturally differentiated into T helper type 1 (Th1) cytotoxic T cells in vivo and caused the regression of established tumors and depigmentation in lymphopenic hosts. Therapy was independent of vaccination, exogenous cytokine support, CD8+-, B-, NK and NK-T cells. Proper activation of CD4+ T cells in vivo was important for tumor clearance as naïve tumor-specific CD4+ T cells could not completely treat tumor in lymphopenic common gamma chain deficient (γc-/-) hosts. γc signaling in the tumor-bearing host was important for survival and proper differentiation of adoptively transferred tumor-specific CD4+ T cells. Thus, these data provide a platform for designing immunotherapies that incorporate tumor/self-reactive CD4+ T cells.

Naive tumor-specific CD4+ T cells differentiated in vivo eradicate established melanoma

In vitro differentiated CD8+ T cells have been the primary focus of immunotherapy of cancer with little focus on CD4+ T cells. Immunotherapy involving in vitro differentiated T cells given after lymphodepleting regimens significantly augments antitumor immunity in animals and human patients with cancer. However, the mechanisms by which lymphopenia augments adoptive cell therapy and the means of properly differentiating T cells in vitro are still emerging. We demonstrate that naive tumor/self-specific CD4+ T cells naturally differentiated into T helper type 1 cytotoxic T cells in vivo and caused the regression of established tumors and depigmentation in lymphopenic hosts. Therapy was independent of vaccination, exogenous cytokine support, CD8+, B, natural killer (NK), and NKT cells. Proper activation of CD4+ T cells in vivo was important for tumor clearance, as naive tumor-specific CD4+ T cells could not completely treat tumor in lymphopenic common gamma chain (γc)–deficient hosts. γc signaling in the tumor-bearing host was important for survival and proper differentiation of adoptively transferred tumor-specific CD4+ T cells. Thus, these data provide a platform for designing immunotherapies that incorporate tumor/self-reactive CD4+ T cells.

Pten Inactivating Mutations Promote Loss of MYC “Oncogene Addiction” in a Conditional Zebrafish Model of T-ALL.

Abstract 3977Poster Board III-913The MYC oncogene is overexpressed in most human cases of T-cell acute lymphoblastic leukemia (T-ALL), and our laboratory previously developed a zebrafish model of MYC-induced T-ALL that closely recapitulates the most common subtype of human T-ALL, which demonstrates overexpression of TAL1 and LMO2. We have now developed a conditional zebrafish model of MYC-induced T-ALL, based on the rag2-driven expression of a MYC-estrogen receptor (MYC-ER) fusion transgene, in which T-ALL is induced by 4-hydroxytamoxifen treatment. Most cases of T-ALL in this model exhibit 'oncogene addiction' to MYC, as evidenced by the regression of established tumors when 4HT treatment is discontinued. However, the genetic determinants of 'addiction' to the MYC oncogene are unknown. Deletion of the PTEN tumor suppressor has been found to be a recurrent abnormality in human T-ALL, and we have now identified abnormalities in PTEN, PI3K or AKT in 48% of human T-ALL. Due to a partial genome duplication that occurred during evolution, zebrafish have two pten genes, pten-A and pten-B, which are both ubiquitously expressed and are largely redundant. In order to examine the consequences of pten inactivation in MYC-induced T-ALL, we mated zebrafish loss-of-function mutations in pten-A and pten-B into our rag2-MYC-ER line that also carries the rag2-GFP transgene, allowing the in vivo monitoring of tumor onset and regression. Zebrafish were raised in 4-hydroxytamoxifen beginning at 5 days post-fertilization, and tumor onset was monitored via fluorescence microscopy. At the time of tumor onset, zebrafish were removed from 4-hydroxytamoxifen, and tumor regression was monitored weekly. Although pten loss-of-function mutations did not accelerate the onset of MYC-induced T-ALL, these did affect tumor regression. We found that T-ALL underwent regression in 68% (21/31) of zebrafish whose germline pten status was wild-type, while regression occurred in only 21% (7/32) of zebrafish harboring loss of any of their germline pten alleles (p=0.0003). The effect of pten inactivation on tumor regression was phenocopied by the expression of a constitutively active murine Akt2 transgene in the MYC-ER line, indicating that the effect of pten inactivation is mediated through the activation of AKT. Our studies provide new evidence that the PTEN-PI3K-AKT pathway is an important modulator of MYC ‘oncogene addiction' in zebrafish T-ALL. Disclosures:No relevant conflicts of interest to declare.