Tumoricidal Activity Research Articles

Targeted Delivery of TNF‐Alpha and IFN‐Gamma by Tumor Targeting Peptide Inhibited Orthotopic Colorectal Cancer Growth

ObjectiveTo determine the in vivo anti‐tumor activities of TNF‐alpha and IFN‐ gamma given alone or in combination and compare with the effects of their conjugates with tumor targeting peptide in a mouse colorectal tumor model.MethodsA mouse orthotopic colorectal tumor model was established. Treatment was given by a single i.v. injection. Mice were sacrificed and examined for their tumor sizes and other immuno and biological parameters in tumors 7 days after treatment. Apoptosis was determined by the TUNEL assay. Immunotherapeutic response was determined by immunofluorecence using antibodies against T lymphocytes, macrophages and NK cells.ResultsTNF‐alpha and IFN‐gamma showed marginal inhibitory effects on tumor growth, while their conjugates with tumor targeting peptide further decreased tumor sizes. Compared with the treatment with non‐targeted TNF‐alpha plus IFN‐gamma, targeted delivery by the peptide markedly alleviated the systemic toxicity as shown by a significant reduction in mortality after treatment. Moreover, targeted delivery of TNF‐alpha plus IFN‐gamma synergistically and significantly inhibited tumor growth.ConclusionsTargeted delivery of TNF‐alpha plus IFN‐gamma exhibited promising tumoricidal activity and at the same time alleviated the systematic toxicity induced by the immuno‐modulators in mice.Acknowledgements: The study was supported by the Innovation and Technology Support Programme and the General Research Fund from the Innovation and Technology Commission and the Research Grant Council from Hong Kong.

ADP-ribose/TRPM2-mediated Ca2+ signaling is essential for cytolytic degranulation and antitumor activity of natural killer cells.

Natural killer (NK) cells are essential for immunosurveillance against transformed cells. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel gated by ADP-ribose (ADPR). However, the role of TRPM2-mediated Ca2+ signaling in the antitumor response of NK cells has not been explored. Here, we show that ADPR-mediated Ca2+ signaling is important for cytolytic granule polarization and degranulation but not involved in target cell recognition by NK cells. The key steps of this pathway are: 1) the activation of intracellular CD38 by protein kinase A following the interaction of the NK cell with a tumor cell results in the production of ADPR, 2) ADPR targets TRPM2 channels on cytolytic granules, and 3) TRPM2-mediated Ca2+ signaling induces cytolytic granule polarization and degranulation, resulting in antitumor activity. NK cells treated with 8-Br-ADPR, an ADPR antagonist, as well as NK cells from Cd38−/− mice showed reduced tumor-induced granule polarization, degranulation, granzyme B secretion, and cytotoxicity of NK cells. Furthermore, TRPM2-deficient NK cells showed an intrinsic defect in tumoricidal activity. These results highlight CD38, ADPR, and TRPM2 as key players in the specialized Ca2+ signaling system involved in the antitumor activity of NK cells.

Splenectomy restores tumoricidal activity to promote elimination of intraocular tumors

We recently demonstrated that splenectomy restores an interaction between CD8+ T cells and macrophages necessary for intraocular tumor elimination. Taking into consideration other studies indicating that intraocular tumor growth does not induce tumor-specific CD8+ T-cell tolerance, our data suggest that splenectomy may influence the phenotype of tumor-associated macrophages.

C-type lectin-like molecule-1 (CLL1)-targeted TRAIL augments the tumoricidal activity of granulocytes and potentiates therapeutic antibody-dependent cell-mediated cytotoxicity

The therapeutic effect of anti-cancer monoclonal antibodies stems from their capacity to opsonize targeted cancer cells with subsequent phagocytic removal, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) or induction of complement-mediated cytotoxicity (CDC). The major immune effector cells involved in these processes are natural killer (NK) cells and granulocytes. The latter and most prevalent blood cell population contributes to phagocytosis, but is not effective in inducing ADCC. Here, we report that targeted delivery of the tumoricidal protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to granulocyte marker C-type lectin-like molecule-1 (CLL1), using fusion protein CLL1:TRAIL, equips granulocytes with high levels of TRAIL. Upon CLL1-selective binding of this fusion protein, granulocytes acquire additional TRAIL-mediated cytotoxic activity that, importantly, potentiates antibody-mediated cytotoxicity of clinically used therapeutic antibodies (e.g., rituximab, cetuximab). Thus, CLL1:TRAIL could be used as an adjuvant to optimize the clinical potential of anticancer antibody therapy by augmenting tumoricidal activity of granulocytes.

SapC-DOPS nanovesicles: a novel targeted agent for the imaging and treatment of glioblastoma.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor. Classified by the World Health Organization (WHO) as grade IV astrocytoma, GBMs are extremely aggressive, almost always recur, and despite our best efforts, remain incurable. This review describes the traditional treatment approaches that led to moderate successes in GBM patients, discusses standard imaging modalities, and presents data supporting the use of SapC-DOPS, a novel proteoliposomal formulation with tumoricidal activity, as a promising diagnostic imaging tool and an innovative anti-cancer agent against GBM.

Interferon regulatory factor (IRF)-1 is a master regulator of the cross talk between macrophages and L929 fibrosarcoma cells for nitric oxide dependent tumoricidal activity.

Macrophage tumoricidal activity relies, mainly, on the release of Tumor Necrosis Factor alpha (TNFα) and/or on reactive oxygen or nitrogen intermediates. In the present work, we investigated the cytotoxic activity of resident peritoneal macrophages against L929 fibrosarcoma cell line in vitro and in vivo. Resident macrophages lysed L929 cells in a mechanism independent of TNFα and cell-to-cell contact. The cytotoxic activity was largely dependent on nitric oxide (NO) release since treatment with L-NAME (NOS inhibitor) inhibited L929 cells killing. Macrophages from mice with targeted deletion of inducible NO synthase (iNOS) together with L929 cells produced less NO and displayed lower, but still significant, tumoricidal activity. Notably, NO production and tumor lysis were abolished in co-cultures with macrophages deficient in Interferon Regulatory Factor, IRF-1. Importantly, the in vitro findings were reproduced in vivo as IRF-1 deficient animals inoculated i.p with L929 cells were extremely susceptible to tumor growth and their macrophages did not produce NO, while WT mice killed L929 tumor cells and their macrophages produced high levels of NO. Our results indicate that IRF-1 is a master regulator of bi-directional interaction between macrophages and tumor cells. Overall, IRF-1 was essential for NO production by co-cultures and macrophage tumoricidal activity in vitro as well as for the control of tumor growth in vivo.

Pomalidomide – An Appraisal of Its Clinical Development and Role in the Treatment of Relapsed/Refractory Multiple Myeloma

Pomalidomide is a distinct immunomodulatory agent with significant activity in relapsed/refractory multiple myeloma (RRMM). The optimal treatment schedule in patients with RRMM who have received multiple lines of treatment, including bortezomib and lenalidomide, is 4 mg/day on days 1–21 of a 28-day cycle in combination with weekly low-dose dexamethasone. Improved responses and outcomes relative to traditional therapies continue to be confirmed in recently completed and ongoing trials. Pomalidomide exhibits direct tumoricidal, immunomodulatory, anti-angiogenic and anti-inflammatory activities, which facilitate combination therapy with agents with complementary mechanisms of action, resulting in greater anti-myeloma effects than single-agent therapy or previous combination therapies. For example, in combination with proteasome inhibitors and traditional chemotherapeutic agents in doublet or triplet regimens, pomalidomide provides high rates of durable response, and represents an important new treatment option for patients with RRMM requiring effective new therapies. Additionally, pomalidomide maintains its efficacy and tolerability profile in difficult-to-treat patients, including the elderly, patients with poor cytogenetics and those with renal impairment. This review summarises the clinical development of pomalidomide and discusses this effective agent for the treatment of patients with RRMM in the context of current myeloma treatment options, as well as potential future directions to further improve patient outcomes.

The cytotoxicity of BAMLET complexes is due to oleic acid and independent of the α-lactalbumin component

Lipid–protein complexes comprised of oleic acid (OA) non-covalently coupled to human/bovine α-lactalbumin, named HAMLET/BAMLET, display cytotoxic properties against cancer cells. However, there is still a substantial debate about the role of the protein in these complexes. To shed light into this, we obtained three different BAMLET complexes using varying synthesis conditions. Our data suggest that to form active BAMLET particles, OA has to reach critical micelle concentration with an approximate diameter of 250nm. Proteolysis experiments on BAMLET show that OA protects the protein and is probably located on the surface, consistent with a micelle-like structure. Native or unfolded α-lactalbumin without OA lacked any tumoricidal activity. In contrast, OA alone killed cancer cells with the same efficiency at equimolar concentrations as its formulation as BAMLET. Our data show unequivocally that the cytotoxicity of the BAMLET complex is exclusively due to OA and that OA alone, when formulated as a micelle, is as toxic as the BAMLET complex. The contradictory literature results on the cytotoxicity of BAMLET might be explained by our finding that it was imperative to sonicate the samples to obtain toxic OA.

Immunomodulatory effect of β-glucan on peritoneal macrophages of Babl/c mice

We assessed the effect of β-Glucan on macrophages by Griess reagent and viability by MTT assay and cytotoxicity. Assay of macrophages culture supernatants were carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay were done. NO release was increased at the dose of 10 µg/ml (P = 0.001) of β-Glucan while the viability of macrophages in all concentrations was the same. In TNF-α bioassay, the supernatant of macrophages stimulated with β-Glucan had a significant cytotoxic effect on WEHI-164 cells (P = 0.023). β-Glucan had a positive effect on increasing tumoricidal activity of macrophages which may help in anti-cancer immune responses.

TNFerade, an innovative cancer immunotherapeutic.

Tumor necrosis factor-α (TNF-α) is a chemokine with effective tumoricidal properties. However, severe systemic toxicity limits its use of as anticancer agent. TNFerade is a novel replication deficient adenovector based gene therapy, which enables the radiation inducible translation of human TNF-α gene specifically in cancer cells. When injected intratumorally, it has least systemic distribution. Consequently, it lacks TNF-α related systemic toxicity. Evidence suggests that it has superior tumoricidal activity and tolerability with minimum adverse effects. It has demonstrated its beneficial role in the treatment of a variety of cancers in terms of improving the disease free and overall survival, delaying tumor progression, and inducing tumor regression when used with concurrent radiotherapy or chemotherapy.

Chimeric antigen receptor–engineered T cells as oncolytic virus carriers

The use of engineered T cells in adoptive transfer therapies has shown significant promise in treating hematological cancers. However, successes treating solid tumors are much less prevalent. Oncolytic viruses (OVs) have the capacity to induce specific lysis of tumor cells and indirectly impact tumor growth via vascular shutdown. These viruses bear natural abilities to associate with lymphocytes upon systemic administration, but therapeutic doses must be very high in order to evade antibodies and other components of the immune system. As T cells readily circulate through the body, using these cells to deliver OVs directly to tumors may provide an ideal combination. Our studies demonstrate that loading chimeric antigen receptor–engineered T cells with low doses of virus does not impact receptor expression or function in either murine or human T cells. Engineered T cells can deposit virus onto a variety of tumor targets, which can enhance the tumoricidal activity of the combination treatment. This concept appears to be broadly applicable, as we observed similar results using murine or human T cells, loaded with either RNA or DNA viruses. Overall, loading of engineered T cells with OVs represents a novel combination therapy that may increase the efficacy of both treatments.

The natural tumorcide Manumycin-A targets protein phosphatase 1α and reduces hydrogen peroxide to induce lymphoma apoptosis

Numerous compounds for treating human disease have been discovered in nature. Manumycin-A (Man-A) is a natural, well-tolerated microbial metabolite and a potent experimental tumoricide. We recently showed that Man-A stimulated reactive oxygen species (ROS) which were upstream of serine/threonine (Ser/Thr) dephosphorylation and caspase-dependent cleavage of MEK and Akt in lymphoma apoptosis. Conversely, activation-specific, Ser/Thr phosphorylation of MEK and Akt proteins was stable in Man-A-resistant tumors suggesting that stimulation of Ser/Thr PPase activity might be required for Man-A tumoricidal activity. Pre-treatment with Calyculin-A, an equipotent inhibitor of PP1 and PP2A, blocked all downstream effects of Man-A whereas, the PP2A-selective inhibitor, Okadaic acid did not, suggesting that PP1 and not PP2A played a role in Man-A action. Phosphorylation of PP1α on Thr320 inhibits its activity. Hence, we posited that if PP1α was important for Man-A action, then Man-A treatment should promote dephosphorylation of PP1α on Thr320. Indeed, T320 was only dephosphorylated in the tumors that underwent apoptosis. Lastly, stable over-expression of a constitutively active PP1α mimetic (PP1αT320A mutant), elevated basal ROS levels and enhanced Man-A-stimulated apoptosis. Taken together, we conclude that PP1α is an important proximal effector of Man-A mediated lymphoma apoptosis and that the mechanisms of Man-A action warrant further investigation.

Vitamin D-Dependent Induction of Cathelicidin in Human Macrophages Results in Cytotoxicity Against High Grade B-Cell Lymphoma

Infiltration by macrophages represents a characteristic morphological hallmark in high grade lymphatic malignancies such as Burkitt’s lymphoma (BL). Although macrophages can in principle target neoplastic cells and mediate antibody-dependent cytotoxicity (ADCC), tumor associated macrophages (TAMs) regularly fail to exert direct cytotoxic functions. The underlying mechanisms responsible for this observation remain unclear. Here we demonstrate that inflammatory M1 macrophages kill proliferating high grade B-cell lymphoma cells by releasing the antimicrobial peptide cathelicidin in a vitamin D-dependent fashion. Moreover, we show that cathelicidin directly induces cell death by targeting mitochondria of BL-cells. In contrast, anti-inflammatory M2 macrophages and M2-like TAMs in BL exhibit an altered vitamin D metabolism resulting in a reduced production of cathelicidin and consequently in inability to lyse BL cells. However, treatment of M2 macrophages with the bioactive form of vitamin D, 1,25D, or a vitamin D receptor agonist effectively induces cathelicidin production and triggers tumoricidal activity against BL cells. Furthermore, rituximab-mediated cytotoxicity of M2 macrophages is cathelicidin dependent and vitamin D treatment of 25D deficient volunteers improves rituximab-mediated ADCC against BL cells. These data indicate that activation of the vitamin D signaling pathway may activate antitumor activity of TAMs and improve the efficacy of ADCC. DisclosuresNo relevant conflicts of interest to declare.

Concomitant Administration of Gene-Modified T Cells Expressing a Chimeric CD16-CD3ζ Receptor with Mogamulizmab Synergistically Suppresses Adult T Cell Leukemia Cells in Vivo

[Background and purpose] Mogamulizumab, a newly developed monoclonal antibody (mAb) targeting the receptor for C-C chemokine 4 (CCR4) has initially demonstrated a promising clinical outcome for the treatment of therapy-resistant Adult T cell leukemia (ATL) of which tumor cells broadly express CCR4. However, in practice, we sometimes encounter unsuccessful cases of ATL treated with mogmulizumab, especially in the setting of combination therapy with multiple anticancer agents. In those cases, ATL tumor cells were still positive for CCR4, but the chemotherapy-induced lymphocytopenia was reproducibly noticeable. Considering the pharmacologic action of mogamulizumab, the defucosylation-enhanced antibody-dependent cellular cytotoxicity (ADCC) exerted by FcγRIIIa (CD16) expressing effector cells in vivo, as observed for Natural Killer (NK) cells, we hypothesized that adoptive transfer of ADCC effector cells might be able to regain the anti-ATL efficacy of mogamulizumab. Accordingly, we examined in vivo the feasibility of T cells gene-modified to express a newly generated chimeric CD16-CD3ζ receptor as a transferable alternative effector cell for mogamulizumab.[Methods] A novel affinity-matured chimeric CD16 with a 158V/V-CD3ζ (cCD16ζ ) gene construct was lentivirally introduced into CD3+ T cells from both healthy individuals (n=4) and patients with adult T cell leukemia (ATL) (n=3) (cCD16ζ-T cells). Using ATL or HTLV-1 infected cell lines variably expressing CCR4 on the cell surface (n=7), and primary ATL tumor cells (n=3), in the context of ADCC activity, functional properties of cCD16ζ-T cells were extensively examined both in vitro and in vivo, compared with those of NK cells from healthy donors (n=3). Next, we examined the in vivo therapeutic efficacy of concomitantly infused cCD16ζ-T cells with mogamulizumab via tail vein using a xenografted mouse model. Finally, we examined the feasibility of double gene-modified T cells to express human telomerase reverse transcriptase (hTERT)-specific TCR and cCD16ζ receptor, because we recently demonstrated that HLA-A*24:02-restricted and hTERT461-469 (VYGFVRACL)-specific TCR gene-modified CD8+ T cells displayed the cytocidal activity against ATL tumor cells (Blood, 2014).[Results] cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human (rh)IL-2, and they successfully displayed ADCC-mediated tumoricidal activity against CCR4+ MT-4 and ATN-1 (ATL cell lines) cells, but not CCR4- K562 cells with mogamulizumab, in a effector cell number and an antibody dose dependent manner. Pharmacological dose of 0.1μg/ml mogamulizumab could bind to ATL cell line cells expressing variable extent of CCR4 ranging from MFI 1.01 for HUT102 cell line to 12.3 for MT-2 (0.57 for K562 as a negative control), and could mediate the similar degree of ADCC activity exerted by cCD16ζ-T cells. The magnitude of ADCC activity mediated by cCD16ζ-T cells against opsonized ATN-1 with mogamulizumab was almost similar to that by activated NK cells using rhIL-2. This cytotoxicity was inhibited by anti-CD16 mAb. During ADCC, ligation of opsonized cancer cells with mogamulizumab to cCD16ζ receptor stimulated cCD16ζ-T cells to release toxic granules shown by CD107a expression. cCD16ζ-T cells generated from patients with ATL (n=3) successfully displayed ADCC activity against autologous tumor cells in vitro. Human cCD16ζ-T cells infused concomitantly with mogamulizumab synergistically inhibited the growth of disseminated luciferase gene-modified ATN-1 cells in immunodeficient mice, demonstrated using an in vivo bioluminescence assay. Additionally, this tumor suppressive effect contributed to the longer survival of treated mice. Finally, the double-gene modified CD3+ T cells could successfully recognize ATN-1 through both the mogamulizumab-opsonized CCR4 and hTERT epitope/HLA-A24*02 complex on the cell surface. DisclosuresOkamoto:CDM Center, Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment. Shiku:Takara Bio Inc.: Research Funding.

Splenectomy promotes indirect elimination of intraocular tumors by CD8+ T cells that is associated with IFNγ- and Fas/FasL-dependent activation of intratumoral macrophages.

Ocular immune privilege (IP) limits the immune surveillance of intraocular tumors as certain immunogenic tumor cell lines (P815, E.G7-OVA) that are rejected when transplanted in the skin grow progressively when placed in the anterior chamber of the eye. As splenectomy (SPLNX) is known to terminate ocular IP, we characterized the immune mechanisms responsible for rejection of intraocular tumors in SPLNX mice as a first step toward identifying how to restore tumoricidal activity within the eye. CD8(+) T cells, IFNγ, and FasL, but not perforin, or TNFα were required for the elimination of intraocular E.G7-OVA tumors that culminated in destruction of the eye (ocular phthisis). IFNγ and FasL did not target tumor cells directly as the majority of SPLNX IFNγR1(-/-) mice and Fas-defective lpr mice failed to eliminate intraocular E.G7-OVA tumors that expressed Fas and IFNγR1. Bone marrow chimeras revealed that IFNγR1 and Fas expression on immune cells was most critical for rejection, and SPLNX increased the frequency of activated macrophages (Mϕ) within intraocular tumors in an IFNγ- and Fas/FasL-dependent manner, suggesting an immune cell target of IFNγ and Fas. As depletion of Mϕs limited CD8 T cell-mediated rejection of intraocular tumors in SPLNX mice, our data support a model in which IFNγ- and Fas/FasL-dependent activation of intratumoral Mϕs by CD8(+) T cells promotes severe intraocular inflammation that indirectly eliminates intraocular tumors by inducing phthisis, and suggests that immunosuppressive mechanisms that maintain ocular IP interfere with the interaction between CD8(+) T cells and Mϕs to limit the immunosurveillance of intraocular tumors.

Preparation of esterified derivatives of α-lactalbumin and investigation of their antibacterial activity

α-lactalbumin (α-La) is one of the major milk proteins that in addition of nutritional value it can get many other useful novel functions such as antiviral and tumoricidal activities via structural modifications by enzyme or chemical methods. Several researches show antiviral activity of esterified milk proteins because of their new functional properties followed by structural modifications. In this research, the hypothesis based on antibacterial activity of esterified α-La was examined. The esterification reaction as an important tool for modifying food proteins was employed in this study by using different alcohols under strong acidic condition at 4 °C for 3 days. α-La was esterified to the extent of 53.8%, 34.95%, 32.2% , 6.07% and 46.8% with methanol, ethanol, 1-butanol, 2-pentanol and 1-hexanol, respectively, showed the influence of molar ratio, type of used alcohol and time of reaction on the reaction yield. Then the effects of esterified derivatives of α-La were tested on the growth of some positive and negative gram bacteria(including Bacillus cereus, methicylin resistant Staphylococcus aureus (MRSA), Escherichia coli, and Klebsiella) by disc assay. After 24-48 h incubation, no inhibition zone surrounding the discs was observed which represents the fail of any antibacterial activity of α-La and its esterified derivatives.

Abstract 1069: M1 macrophages suppress tumorigenesis via accumulated p53 and upregulated STAT1 in lung cancer

Abstract Purpose Macrophages involved in innate immunity and possess cytotoxicity via NO, ROS and pro-inflammatory cytokines, such as TNF-α and IFN-γ. But long-term effects of M1 macrophages on cancer cells, especially their tumoricidal activity, remain unclear. In our previous study, conditioned medium (CM) from M1 macrophages can induce A549 apoptosis and senescence, but detail mechanism is unexplored. Materials & Methods Lung cancer cell lines, A549 and H460, are cells with wild-type p53, but H1299 is p53-null. Cytokine and cDNA microarray were used to evaluate the content of M1 macrophages and their effects on A549 cell. Flow cytometry was assessed cell apoptosis; real-time PCR and Western blot were assessed the expression of identified genes and proteins. Clinical statistical analysis for predicting survival of lung cancer patients was applied by a published clinical microarray dataset. Results According to our previous results of cDNA microarray, several significantly differential genes were the down-stream of p53, the important initiator for apoptosis. After M1 CM treatment, A549 and H460 cells went apoptosis and p53 was abundantly accumulated. But cell death was not observed in H1299 cells, unless exogenous wt-p53 was expressed and M1 CM treated. Another transcription factor STAT1 was also up-expressed at translational and transcriptional levels after M1 CM stimulation. STAT1 may interact and assist with p53 to cause cell death. IFN-γ which is a well known cytokine for STAT1 activation highly expresses in M1 macrophages, but antibody neutralization can not reverse cell apoptosis. M1 macrophages from human PBMCs also had tumoricidal activity for A549. Moreover, the M1/M2 gene signature from A549 cDNA microarray can predict the patients' survival. Conclusion Accumulated data show that p53 and STAT1 are the main tumoricidal regulators induced by M1 macrophages in long-term culture and that is IFNs independent signaling pathway. Those data imply that M1 macrophage plays an important role in the immune surveillance for cancer progression. That may be a potential immunol target therapy. Citation Format: Yi-Jing Hsiao, Kang-Yi Su, Jian-Wei Chen, Sung-Liang Yu. M1 macrophages suppress tumorigenesis via accumulated p53 and upregulated STAT1 in lung cancer. [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 1069. doi:10.1158/1538-7445.AM2014-1069

Abstract 2773: AD-O56.9: A fusion of TRAIL/Apo2L with a membrane disrupting peptide as a novel anticancer therapeutic

Abstract Background The tumor necrosis factor related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF superfamily that initiates apoptosis of tumor cells through the activation of their death receptors. The ability of TRAIL to selectively induce apoptosis of tumor cells but not normal cells makes it an attractive agent for cancer therapy. However, many cancer types have developed resistance mechanisms, such as dysfunctions of proapoptotic proteins. Hereby, we report a novel molecule - AD-O56.9, which is composed of the soluble fragment of TRAIL (acting as both carrier and also effector) fused with a cationic, alpha-helical (KLAKLAK)2 antimicrobial peptide (acting as effector). The (KLAKLAK)2 peptide fused to protein transduction domain can induce cancer cell death by triggering mitochondrial membrane permeabilization and swelling, resulting in the release of cytochrome c and induction of apoptosis. It creates also the capacity to cause aggregation of mitochondria that is also a mechanism of cytotoxic action. (KLAKLAK)2 peptide is equipped with protein transduction domain domain, to increase its internalization. To allow separation of TRAIL fragment from the effector peptide domain specifically in the tumor environment, we linked these two domains with a sequence motif recognized by MMP and uPa proteases, present in tumor cells membranes or their proximity. Methods AD-O56.9 protein was produced in E. coli and purified by IEC. The molecule was characterized biochemically and biophysically. MTT assay was used to estimate killing of carcinoma cells. Flow cytometric analysis was used to evaluate influence of the AD-O56.9 on plasma and mitochondrial membrane integrity, caspase 3 activation, PARP cleavage, as well as on the cell cycle of cancer cells. The tumoricidal activity of AD-O56.9 was evaluated in NOD/SCID mice bearing different types of tumor xenografts. Results AD-O56.9 exhibited cytotoxic effect on various cancer cell lines, both TRAIL-sensitive and TRAIL-resistant, but showed no toxic effect on normal cells. This protein was also highly cytotoxic against primary cancer cells. The component that overcomes resistance to TRAIL is RRRRRRRR(KLAKLAK)2 peptide, but only as a component of AD-O56.9 fusion protein. Analyzing cell cycle and plasma membrane integrity in relatively sensitive cell line (NCI-H460) and TRAIL-resistant cell line (A549) we showed that AD-O56.9 induced apoptosis in those cells. This protein led to activation of caspase 3, cleavage of PARP as well as caused strong depolarization of mitochondrial membrane. AD-O56.9 administration caused significant regression of TRAIL-sensitive MIA PaCa-2, OE19, Colo205 and TRAIL-resistant HepG2. Conclusions AD-O56.9 is able to induce cell death in many cancer cell lines, even TRAIL resistant and causes tumor regression in mice bearing human tumors. Obtained results make this molecule worth of further preclinical development. Citation Format: Bartlomiej Maciej Zerek, Michal Szymanik, Piotr Kamil Rozga, Anna Pieczykolan, Marlena Galazka, Katarzyna Bukato, Albert Jaworski, Katarzyna Poleszak, Sebastian Dominik Pawlak, Malgorzata Teska-Kaminska, Wojciech Strozek, Jerzy Szczepan Pieczykolan. AD-O56.9: A fusion of TRAIL/Apo2L with a membrane disrupting peptide as a novel anticancer therapeutic. [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 2773. doi:10.1158/1538-7445.AM2014-2773

Abstract 1663: CD40 agonists drive the anti-tumor functions of macrophages and induce systemic immune activation survival the Pan02 orthotopic pancreatic model

Abstract Pancreatic cancer represents an area of high unmet medical need. Antibodies agonising CD40 (αCD40) inhibit tumor growth in preclinical models, and are showing activity in early clinical trials in pancreatic cancer. αCD40 antibodies can directly drive apoptosis and promote antibody-dependent cell cytotoxicity of CD40 positive tumor cells, and may also drive antitumor effects through endothelial cells and B cells. However, data from preclinical models indicate that a major mechanism of action for αCD40 antibodies is through targeting of macrophages and dendritic cells, in some cases promoting long term CD8 T cell-dependent anti-tumor immunity. Pancreatic tumors often become highly infiltrated with immunosuppressive tumor macrophages. Our aim was therefore to test the hypothesis that αCD40 inhibits pancreatic tumor growth and spread by re-educating tumor macrophages to have anti-tumor functions, promoting a Th1 anti-tumor immune response, and gaining direct tumoricidal activity. We found that CD40 activation licenses human macrophage anti-tumor functions in vitro, and that this was dependent on the presence of T cell derived cytokines. In a syngeneic orthotopic model of pancreatic cancer (Pan02), we found that αCD40 significantly improved survival. This correlated with a significant enhancement in macrophage activation and in systemic immune activation (reduction in myeloid derived suppressor cells and increase in CD8 T cells) as determined by flow cytometry analysis. These data add support to the hypothesis that αCD40 inhibits pancreatic tumor growth through promoting anti-tumor functions of tumor macrophages. Citation Format: Nadia Luheshi, James Harper, Jane Coates-Ulrichsen, Richard Sainson, Robert W. Wilkinson, Gareth Davies, James William Legg. CD40 agonists drive the anti-tumor functions of macrophages and induce systemic immune activation survival the Pan02 orthotopic pancreatic model. [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 1663. doi:10.1158/1538-7445.AM2014-1663

Abstract 3804: CPA-7, an inhibitor of STAT3, is a potent tumoricidal agent in peripheral tumor models and is impermeable to the CNS

Abstract Analogues of the prototypical cancer drug cisplatin number by the hundreds with variable effects on DNA and signal transduction. Of these compounds, CPA-7 has been shown to elicit potent tumoricidal activity by preferentially targeting STAT3 in transformed cells. In this study, we characterized the response of rodent and human glioma cells to STAT3 inhibition by CPA-7. In addition, we determined if CPA-7 administration is sufficient for the induction of tumor regression in intracranial and peripheral mouse tumor models. Cultured glioma cells treated with CPA-7 ceased to proliferate and underwent apoptosis. Induction of apoptosis was associated with reduction of phosphorylated STAT3 and downstream transcriptional products such as cyclin D, survivin, and Bcl-xl. Although CPA-7 inhibits STAT3 preferentially, we observed an inhibition of STAT1 at doses higher than 30uM. Furthermore, mice bearing GL26 flank tumors underwent complete tumor regression in response to CPA-7 administration. This potent therapeutic effect of CPA-7 could not be duplicated in mice bearing intracranial GL26 brain tumors as that group became moribund at the same time as vehicle treated mice. Western blot and immunohistochemistry analysis of tumors isolated from CPA-7 treated mice demonstrated a reduction of phosphorylated STAT3 in peripheral but not intracranial tumors. This data suggests that CPA-7 is impermeable to the brain as a consequence of its molecular structure. Permeability measurements of CPA-7 using the PAMPA assay support this notion. These observations were corroborated in the B16-f0 and B16-f10 mouse melanoma models. Inhibition of STAT3 has been shown to be an effective strategy at inducing tumor regression in multiple cancer types. Although the discovery of effective and specific inhibitors has been limited, we believe that CPA-7 is a compound with potential therapeutic activity for peripheral solid tumors and warrants further evaluation and pharmacological characterization. Supported by NIH/NINDS Citation Format: Hikmat Assi, Chris Paran, Jon Savakus, James Hoeschle, Leda Raptis, Pedro R. Lowenstein, Maria G. Castro. CPA-7, an inhibitor of STAT3, is a potent tumoricidal agent in peripheral tumor models and is impermeable to the CNS. [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 3804. doi:10.1158/1538-7445.AM2014-3804