Activity In Multiple Cancer Models Research Articles

Abstract LB174: OKI-219 is an inhibitor of PI3Kα H1047R that has brain penetrance and anti-tumor activity in a preclinical intracranial model of metastatic breast cancer

Abstract Background: Dysregulation of PI3K signaling is a common driver of solid tumors, with the PI3KαH1047R activating mutation found in 4% of all cancers. Because many cancers, including breast and lung cancers, can metastasize to the CNS, brain penetration should be a key feature of new therapies. The FDA-approved PI3K inhibitor alpelisib lacks CNS activity, and brain-penetrant alternatives such as buparlisib and paxalasib were not approved due to toxicity associated with a lack of mutant selectivity. OKI-219 is a potent, highly selective PI3KαH1047R inhibitor that has shown preclinical anti-tumor activity in multiple cancer models, both as a monotherapy and in combination with HER2- and ER-mediated therapeutics. Here, we demonstrate its brain penetrance across multiple animal species and anti-tumor activity in an intracranial mouse model of PI3KαH1047R mutant breast cancer. Materials and methods: Brain penetrance in mouse, rat, dog, and monkey was quantified by the ratio of unbound drug concentrations in brain to plasma (Kp,uu). Plasma, brain, and cerebrospinal fluid (CSF) concentrations of OKI-219 were measured by LC/MS, and protein binding in plasma, CSF, and brain homogenate were measured by rapid equilibrium dialysis. Additionally, free drug levels in the brain were directly measured by microdialysis in rats. Mice were implanted subcutaneously and intracranially with PI3KαH1047R xxT47D-luc tumors and tumor growth was measured by calipers (subcutaneous tumors) and luminescence (intracranial tumors). Results: At the Cmax of a pharmacologically active 10 mg/kg PO dose, OKI-219 had a Kp,uu of 0.34, 0.61, and 0.58 in rat, dog, and monkey CSF, respectively. Additionally, OKI-219 had an average Kp,uu of 0.67 over 24h post-dose as measured by brain microdialysis in rats at 30 mg/kg, with a dose-proportionate increase in brain free drug levels at 300 mg/kg. After 21 days of treatment, OKI-219 induced a statistically significant dose-dependent anti-tumor effect on both flank and brain xxT47D-luc tumors. Conclusion: Here we show that OKI-219 had consistently good brain penetrance across multiple species by multiple methods of measuring free drug levels in the brain. We further demonstrated that OKI-219 had anti-tumor activity against xxT47D-luc tumors in an intracranial mouse model that is similar to activity in the same tumor when implanted subcutaneously, demonstrating that the brain levels achieved were pharmacologically active. These data indicate the potential for activity of OKI-219 in CNS disease settings, including metastatic brain cancer, which are not currently addressed with existing PI3K pathway inhibitors. OnKure has initiated the PIKture Phase I clinical study (OKI-219-101) to evaluate OKI-219 in patients with PI3KαH1047R-mutated solid tumors. Citation Format: Alexander A. Strait, Molly A. Taylor, Kevin S. Litwiler, Qian Zhao, David A. Mareska, Mark L. Boys, Yevgeniy Izrayelit, Richard Woessner, James D. Winkler. OKI-219 is an inhibitor of PI3Kα H1047R that has brain penetrance and anti-tumor activity in a preclinical intracranial model of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB174.

Targeting Hippo signaling in cancer: novel perspectives and therapeutic potential.

As highly conserved among diverse species, Hippo signaling pathway regulates various biological processes, including development, cell proliferation, stem cell function, tissue regeneration, homeostasis, and organ size. Studies in the last two decades have provided a good framework for how these fundamental functions of Hippo signaling are tightly regulated by a network with numerous intracellular and extracellular factors. The Hippo signaling pathway, when dysregulated, may lead to a wide variety of diseases, especially cancer. There is growing evidence demonstrating that dysregulated Hippo signaling is closely associated with tumorigenesis, cancer cell invasion, and migration, as well as drug resistance. Therefore, the Hippo pathway is considered an appealing therapeutic target for the treatment of cancer. Promising novel agents targeting the Hippo signaling pathway for cancers have recently emerged. These novel agents have shown antitumor activity in multiple cancer models and demonstrated therapeutic potential for cancer treatment. However, the detailed molecular basis of the Hippo signaling-driven tumor biology remains undefined. Our review summarizes current advances in understanding the mechanisms by which Hippo signaling drives tumorigenesis and confers drug resistance. We also propose strategies for future preclinical and clinical development to target this pathway.

A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8+ T cells

Strategies to increase intratumoral concentrations of an anticancer agent are desirable to optimize its therapeutic potential when said agent is efficacious primarily within a tumor but also have significant systemic side effects. Here, we generate a bifunctional protein by fusing interleukin-10 (IL-10) to a colony-stimulating factor-1 receptor (CSF-1R)-blocking antibody. The fusion protein demonstrates significant antitumor activity in multiple cancer models, especially head and neck cancer. Moreover, this bifunctional protein not only leads to the anticipated reduction in tumor-associated macrophages but also triggers proliferation, activation, and metabolic reprogramming of CD8+ Tcells. Furthermore, it extends the clonotype diversity of tumor-infiltrated Tcells and shifts the tumor microenvironment (TME) to an immune-active state. This study suggests an efficient strategy for designing immunotherapeutic agents by fusing a potent immunostimulatory molecule to an antibody targeting TME-enriched factors.

Abstract 95: In vitro, in vivo and molecular effects of triptolide and minnelide in renal cell carcinoma

Abstract Background: The worldwide incidence of Renal Cell Carcinoma (RCC) is increasing. Although new therapies have improved outcomes in advanced RCC, most patients eventually fail treatment and succumb to this devastating disease. Systemic therapy strategies have focused on TKI and immunotherapy, alone or in combination. Triptolide, a diterpenoid epoxide extracted from Tripterygium wilfordii Hook f (TWHf), has potent antitumor activity in multiple cancer models. Few studies have focused on the potential of triptolide as a novel therapeutic option in RCC. Objective: the objectives of this study are to characterize the in vitro and in vivo effects of Triptolide (T) and its water-soluble form, Minnelide (M), in models of renal cell carcinoma. Methods: The in vitro effects of triptolide (T) on human and murine RCC (786-0, A498, Caki-1, ACHN, and RENCA) cell proliferation was assessed using cell count and xCelligence assays at 24, 48, 72 and 96h. Molecular and mechanistic characterization of triptolide’s effects in 786-0 cells were analyzed by the (Reverse Phase Protein Array) and validated by western blot analysis. The in vivo effects (tumor progression and survival) of M were assessed in nude mice bearing 786-0 tumors (8x106 cells per mouse). M was administered to tumor-bearing mice at two different doses (0.21mg/kg and 0.42mg/kg daily), intraperitoneally, for 21 days. Correlative studies to explain the in vivo effect of M and the correlation with the molecular changes in vitro seen are being performed and will be shown in the poster. Results: T significantly inhibited, in a dose-response manner, cell proliferation in all human and murine renal cancer cell lines, with IC50 ranging between 12.5 and 25 nM). T was associated with significant negative modulation of proliferation, cell cycle, survival, increased apoptosis, and ER stress pathways in 786-0 cells, as demonstrated by RPPA analysis and validated by western Blot of selected pathway proteins, such as apoptosis (PARP cleavage and Caspase 3 activation), ER stress induction (pEIF2a, CHOP), and down-regulation of survival and proliferation pathways (pAKT). In vivo, M was associated with significant antitumor effects in 786-0 xenografts, with complete responses in the majority of mice while on treatment. These effects were associated with significant prolongation in overall survival in M treated vs. control mice. No significant toxicity or treatment-related deaths were observed. Conclusion: Our results have shown for the first time the potent in vitro and in vivo antitumor effects of T in RCC and the molecular changes associated with these effects. The profound antitumor effects in the aggressive 786-0 RCC xenograft model are highly encouraging and warrant further preclinical studies and potential clinical trials of M this devastating disease. Correlative tumor studies to understand the mechanisms of M in vivo antitumor effects are underway and will be presented at the meeting. Citation Format: Valery A. Chavez, Floritza Bustamante, Abner Murray, Ashok Saluja, Jaime Merchan. In vitro, in vivo and molecular effects of triptolide and minnelide in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 95.

Abstract B88: Mechanisms of resistance to the Hsp90 inhibitor ganetespib in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC), which accounts for 10-17% of all breast cancers, is challenging to treat due to its heterogeneity and the absence of identified targeted therapies. Heat shock protein 90 kDa (Hsp90) is a molecular chaperone protein essential for regulating the stability and activity of several client proteins required for oncogenesis, and selective Hsp90 inhibitors have shown anti-cancer activity in multiple cancer models including TNBC. Ganetespib, a second generation Hsp90 inhibitor, has shown promising clinical activity in TNBC patients but responders develop acquired resistance. Here, we generated an in vitro model of ganetespib-resistant TNBC cells and investigated the mechanisms of resistance to ganetespib in TNBC using a 96-well based small molecule screen. Methods: Ganetespib treatment sensitivity in a panel of seven TNBC cell lines (HS578T, HCC1143, HCC1937, MDA-MB-468, MDA-MB-231, MDA-MB-453 and BT20) was assessed using a resazurin-based cell proliferation assay. HS578T cells were incubated in a high concentration of ganetespib (>IC50) to generate ganetespib-resistant clones. The effects on Hsp90 oncogenic client proteins (AKT and EGFR) and Hsp70 were determined by western blotting. Cross resistance to other Hsp90 inhibitors was also assessed. 96-well based small molecule screen with a library of 326 bioactive small molecule inhibitors was performed on parental HS578T and ganetespib-resistant clones in the absence and presence of ganetespib. Resazurin-based cell proliferation assay was used as an end point. Positive hits were identified as log10(percentage of proliferation in parental/resistant) ≥ 2SD. Results: TNBC cell lines showed differential sensitivity to ganetespib (IC50 range: 4.75 to 20.16 nM), with HS578T being the most sensitive and HCC1143 the most resistant. Independent ganetespib-resistant HS578T clones (CR2 and CR3) were successfully generated by incubating parental HS578T cells in ganetespib (30 nM) for 42 days. IC50 values for the ganetespib-resistant clones (CR2: 15.57 ± 1.90 nM and CR3: 20.28 ± 2.75 nM) were 3 to 4-fold higher than the parental HS578T cells (IC50: 4.79 ± 0.32 nM). The client proteins were not further down-regulated after ganetespib treatment in the ganetespib-resistant clones compared to the parental HS578T cells. Resistant clones also showed cross resistance to 1st generation (17-AAG) and 2nd generation (NVP-AUY922) Hsp90 inhibitors. The small molecule screen was reproducible as the independent runs and duplicates strongly correlated (R2 >0.7). The ganetespib-resistant CR3 clones showed selective sensitivity to 11/326 compounds (Z score ≥ 2), only in the presence of ganetespib when compared to the parental HS578T cells, most notable being LY2784544, a JAK2 inhibitor. Conclusion: Ganetespib-resistant HS578T cells provide a promising model to study the resistance mechanisms to ganetespib in TNBC. Our current data suggest that TNBC cells are responsive to JAK2 inhibition in the presence of ganetespib despite prior acquisition of resistant to ganetespib. Activation of a JAK2/STAT3 survival pathway may be a potential resistance mechanism to Hsp90 inhibition and combined Hsp90 and JAK2 inhibition could potentially be a useful therapeutic strategy for TNBC. Citation Format: Nuramalina H. Mumin, Sivan Bokobza, Fiona Cahill, Neele Drobnitzky, Yanyan Jiang, Luiza Madia Lourenco, Aoife Vaughan, Anika Weber, Anthony Kong, Anderson Ryan. Mechanisms of resistance to the Hsp90 inhibitor ganetespib in triple negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B88.

ATPS-68COMBINATION OF TOPOISOMERASE II POISON BERUBICIN WITH STAT3 INHIBITOR WP1066BLOCKS POTENTLY TUMOR GROWTH IN GMB MODELS

BACKGROUND: Berubicin (WP744) is a DNA binding agent and potent topoisomerase II poison designed and synthesized in our laboratory. Preclinical studies demonstrated that WP744 is a potent anticancer agent with nanomolar potency in glioma cells. WP744 was shown to effectively cross blood-brain barrier (BBB) and displayed antitumor activity in orthotopic glioma models. WP744 gained IND approval and subsequent Phase I clinical trials shows clinical benefits (including complete response) in selected brain tumor patients. Signal Transducer and Activator of Transcription 3 (STAT3) is an oncogenic transcription factor that is activated in numerous tumors including GBM. WP1066, an analog of caffeic acid benzyl ester (CABE, a component of propolis), is a potent inhibitor of activated STAT3 (STAT3-Y705) designed in our laboratory. WP1066 is currently undergoing preclinical development and has demonstrated anticancer activity in multiple cancer models including GBM. WP1066 similarly to WP744 is able to cross BBB. The goal of this study is to assess therapeutic effects of WP744 in combination with WP1066 in the in vitro and in vivo GBM models. RESULTS: WP744 and WP1066 were tested in a panel of GBM cell lines including GSC cell lines. Both, WP744 and WP1066, effectively induce apoptosis and inhibit cells growth at low nanomolar and micromolar concentrations, respectively. Dosing schedule for a combination in vivo therapy was tested in CD-1 mice. The proof-of-principle efficacy study was performed in subcutaneous U87 GBM tumor model and demonstrated superior tumor growth inhibition in a group receiving combination of WP1066 and WP744. CONCLUSION: Proposed novel therapeutic strategy involving two compounds (WP744 and WP1066) with significant CNS uptake that are mechanistically complementary is highly promising and our results warrant further evaluation in different brain tumor models. Consistent activity of WP744 combination with WP1066 in a broader range of tumors would open possibility for its future clinical evaluation.

Resistance to the Translation Initiation Inhibitor Silvestrol is Mediated by ABCB1/P-Glycoprotein Overexpression in Acute Lymphoblastic Leukemia Cells

Protein synthesis is a powerful therapeutic target in leukemias and other cancers, but few pharmacologically viable agents are available that affect this process directly. The plant-derived agent silvestrol specifically inhibits translation initiation by interfering with eIF4A/mRNA assembly with eIF4F. Silvestrol has potent in vitro and in vivo activity in multiple cancer models including acute lymphoblastic leukemia (ALL) and is under pre-clinical development by the US National Cancer Institute, but no information is available about potential mechanisms of resistance. In a separate report, we showed that intraperitoneal silvestrol is approximately 100% bioavailable systemically, although oral doses were only 1% bioavailable despite an apparent lack of metabolism. To explore mechanisms of silvestrol resistance and the possible role of efflux transporters in silvestrol disposition, we characterized multi-drug resistance transporter expression and function in a silvestrol-resistant ALL cell line generated via culture of the 697 ALL cell line in gradually increasing silvestrol concentrations. This resistant cell line, 697-R, shows significant upregulation of ABCB1 mRNA and P-glycoprotein (Pgp) as well as cross-resistance to known Pgp substrates vincristine and romidepsin. Furthermore, 697-R cells readily efflux the fluorescent Pgp substrate rhodamine 123. This effect is prevented by Pgp inhibitors verapamil and cyclosporin A, as well as siRNA to ABCB1, with concomitant re-sensitization to silvestrol. Together, these data indicate that silvestrol is a substrate of Pgp, a potential obstacle that must be considered in the development of silvestrol for oral delivery or targeting to tumors protected by Pgp overexpression.

Abstract 1082: BI 811283, a potent inhibitor of the mitotic kinase Aurora B, shows dose- and schedule-dependent efficacy in human cancer xenograft models

Abstract Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. The essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for therapeutic intervention. BI 811283, a potent inhibitor of this key mitotic regulator, inhibits proliferation of a wide range of cultured human cancer cells at low nanomolar concentrations by inducing polyploidy, senescence and apoptosis. Methods. BomTac:NMRI-Foxn1nu mice were grafted subcutaneously with NCI-H460 non-small cell lung carcinoma (mutant KRAS, wild-type p53), HCT 116 colon carcinoma (mutant KRAS, wild-type p53) or BxPC-3 pancreas adenocarcinoma cells (wild-type KRAS, mutant p53). Treatment was initiated when the tumors had reached a volume of ∼50 mm3. BI 811283 was injected intravenously once or twice weekly as a single bolus or b.i.d. Alternatively, the compound was administered once-weekly by a continuous 24 h infusion via subcutaneously implanted osmotic mini-pumps. Multiple dose levels and dosing schedules were evaluated. Results. In models of human non-small cell lung cancer, colon carcinoma and pancreas carcinoma, multiple cycles of treatment with BI 811283 at total weekly doses of 20 to 75 mg/kg resulted in dose-dependent inhibition of tumor growth or tumor regression. Continuous s.c. infusion at 20 mg/kg over 24 h once-weekly was clearly superior to all bolus injection schedules delivering weekly doses up to 75 mg/kg. Furthermore, regression of large tumors (350 mm3) was induced in the HCT 116 colon carcinoma model. Biomarker analyses of HCT 116 tumors revealed that therapeutic doses of BI 811283 inhibited phosphorylation of histone H3, a direct substrate of Aurora B. Histological examination showed an accumulation of enlarged, multinucleated cells in accordance with the expected mechanism of action. Conclusions. BI 811283 has demonstrated potent antitumor activity in multiple cancer models at well-tolerated doses; treated tumors show hallmarks of Aurora B inhibition. Continuous infusion over 24 h provides a superior therapeutic index compared with bolus administration. The compound is currently under investigation in Phase I clinical trials. 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 1082.

BI 6727, A Polo-like Kinase Inhibitor with Improved Pharmacokinetic Profile and Broad Antitumor Activity

Antimitotic chemotherapy remains a cornerstone of multimodality treatment for locally advanced and metastatic cancers. To identify novel mitosis-specific agents with higher selectivity than approved tubulin-binding agents (taxanes, Vinca alkaloids), we have generated inhibitors of Polo-like kinase 1, a target that functions predominantly in mitosis. The first compound in this series, suitable for i.v. administration, has entered clinical development. To fully explore the potential of Polo-like kinase 1 inhibition in oncology, we have profiled additional compounds and now describe a novel clinical candidate. BI 6727 is a highly potent (enzyme IC(50) = 0.87 nmol/L, EC(50) = 11-37 nmol/L on a panel of cancer cell lines) and selective dihydropteridinone with distinct properties. First, BI 6727 has a pharmacokinetic profile favoring sustained exposure of tumor tissues with a high volume of distribution and a long terminal half-life in mice (V(ss) = 7.6 L/kg, t(1/2) = 46 h) and rats (V(ss) = 22 L/kg, t(1/2) = 54 h). Second, BI 6727 has physicochemical and pharmacokinetic properties that allow in vivo testing of i.v. as well as oral formulations, adding flexibility to dosing schedules. Finally, BI 6727 shows marked antitumor activity in multiple cancer models, including a model of taxane-resistant colorectal cancer. With oral and i.v. routes of administration, the total weekly dose of BI 6727 is most relevant for efficacy, supporting the use of a variety of well-tolerated dosing schedules. These findings warrant further investigation of BI 6727 as a tailored antimitotic agent; clinical studies have been initiated.

Bendamustine Is Effective in p53-Deficient B-Cell Neoplasms and Requires Oxidative Stress and Caspase-Independent Signaling

Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are two incurable B-cell lymphoid neoplasms characterized by distinct clinical presentation and evolution. Bendamustine hydrochloride is a multifunctional, alkylating agent with a purine-like ring system that exhibits activity in multiple cancer models, including CLL and MCL, but whose mechanism is only partially described. Our aim was to analyze the apoptotic pathways activated by bendamustine in CLL and MCL together with the relevance of p53 mutation in determining the response to this drug. Thirteen CLL/MCL cell lines and primary tumor cells from 8 MCL and 25 CLL patients were cultured for up to 24 h with bendamustine followed by cytotoxic assays, flow cytometry, immunofluorescence, and Western blot analysis of p53 response pathway and apoptosis-related factors. Bendamustine displayed cytotoxic activity on most CLL and MCL primary cells and cell lines irrespective of ZAP-70 expression and p53 status. Bendamustine was found to act synergistically with nucleoside analogues in both CLL and MCL, this combination being effective in p53 mutated cases resistant to standard chemotherapy. Bendamustine cytotoxicity was mediated by the generation of reactive oxygen species and triggering of the intrinsic apoptotic pathway involving up-regulation of PUMA and NOXA, conformational activation of BAX and BAK, and cytosolic release of caspase-related and caspase-unrelated mitochondrial apoptogenic proteins. Our findings support the use of bendamustine as a therapeutic agent, alone or in combination, for CLL and MCL with p53 alterations and describe the molecular basis of its activity in these entities.

BOOK REVIEW: Dietrich Frohne and Hans J�rgen Pf�nder. POISONOUS PLANTS: A HANDBOOK FOR DOCTORS, PHARMACISTS, TOXICOLOGISTS, BIOLOGISTS, AND VETERINARIANS

<h3>Background</h3> Neuropilin-1 (NRP1) is a co-receptor that complexes with diverse ligands and their cognate receptors. As such, it plays a role in multiple different biological processes, including axon guidance and angiogenesis. NRP1 contains two CUB domains (a1 and a2) involved in binding the ligand Semaphorin3A (SEMA3A), two Factor V/VIII domains (b1 and b2) involved in VEGF ligand binding and one MAM domain (c domain). While functional antibodies with anti-tumor activity have been generated against the SEMA3A and VEGF binding domains, little attention has been paid to the c domain of NRP1, which has been implicated in the dimerization of NRP1, a prerequisite for functionality. We therefore hypothesized that c domain-binding antibodies would offer an opportunity to generate functional inhibitors of both SEMA3A and VEGF signaling and therefore improved anti-tumor activity. <h3>Methods</h3> Recombinant human NRP1 comprising all subdomains was used to identify fully human anti-NRP1 antibodies. Specific antibodies were tested for their ability to block NRP1 interactions with recombinant SEMA3A and VEGF protein in vitro. Blocking antibodies were subsequently assessed for their functional effects, such as inhibition of SEMA3A-mediated growth cone collapse. Antibodies with diverse binding characteristics were then tested for in vivo anti-tumor activity in multiple cancer models of interest. <h3>Results</h3> Recombinant NRP1 containing the a1, a2, b1, b2 and c subdomains was used to successfully identify a series of specific monoclonal antibodies that cross-reacted with Cynomolgus monkey and mouse NRP1, but not human NRP2. Except for the a2 domain, epitope mapping showed an even distribution of mAbs for binding to each of the NRP1 subdomains, including the c domain that has been proposed to play a role in dimerization. Using biolayer interferometry, we identified antibody classes with direct SEMA3A and/or VEGF blocking properties. Further optimization of these antibodies yielded mAbs with subnanomolar affinities that showed significant tumor growth inhibition in multiple mouse models, including anti-PD1 non-responsive models. <h3>Conclusions</h3> Here we demonstrate the identification of fully human monoclonal antibodies that specifically bind to the c domain of human NRP1. A subset of these c domain binders do not block either SEMA3A or VEGF binding to NRP1 but do show in vivo efficacy, suggesting a role for the c domain of NRP1 in the formation of functional (dimeric) complexes. Thus, c domain binding antibodies show remarkable inhibition of tumor growth in mouse cancer models and offer a novel means of therapeutic intervention in patients who are refractory to immune checkpoint inhibition.

615. Anti-Tumor Activities of IL-12 and IL-23 Are Mediated by Similar, yet Divergent, Effector Mechanisms

Th1-polarizing cytokine IL-12 exhibits potent anti-tumor activity in multiple cancer models; however, therapeutic use of this cytokine is limited due to severe IFN-|[gamma]|-mediated toxicity. To reduce the amount of IL-12 needed to elicit a therapeutic response, and thereby decrease associated toxicity, it is necessary to characterize novel cytokines to use in conjunction with IL-12. Newly described IL-12 family member IL-23 shares the IL-12 p40 subunit and promotes Th1 immunity by specifically stimulating proliferation of memory CD4+ T-cells. We propose that adenoviral delivery of IL-23 into the tumor microenvironment will result in tumor rejection and generation of tumor-specific anti-tumor immunity utilizing mechanisms similar to IL-12. To establish the anti-tumor activity of IL-23, C57BL/6 mice bearing 7-day MCA205 fibrosarcoma tumors were treated with adenovirus expressing either IL-12 (Ad.IL-12) or 23 (Ad.IL- 23). Ad.IL-23 treatment resulted in tumor rejection in the majority of mice, elicited protective immunity and had no associated toxicity. To determine if IL-12 and IL-23 employ similar anti-tumor mechanisms, mice deficient in various cytokines and effector cells were inoculated with MCA205 tumors and treated with Ad.IL-12 or Ad.IL-23. IL-23 anti-tumor activity was found to be independent of IL-17, but completely dependent on perforin, Fas ligand, CD8+ T-cells, CD4+ T-cells, IFN-|[gamma]| and IL-12/23 p40. IL-12 anti-tumor activity was independent of Fas ligand, perforin and IL-17, but, like IL-23, completely dependent on IFN-|[gamma]| and CD8+ T-cells. Therefore, IL-12 activity appears to be mediated primarily by CD8+ T-cells and IFN-|[gamma]|, while IL-23 utilizes Fas ligand and perforin in addition to IFN-|[gamma]|, CD4+ and CD8+ T-cells to eradicate tumors. We currently are examining the possible synergy between Ad.IL-12 and Ad.IL-23 in conferring anti-tumor effects.