Significant Antitumor Efficacy Research Articles

ET-04 * MEBENDAZOLE IS EFFICACIOUS IN DIVERSE MEDULLOBLASTOMA TUMOR MODELS AND INHIBITS TUMOR ANGIOGENESIS

Medulloblastoma is the leading cause of cancer death in children. Surgery, radiotherapy and chemotherapy regimens are the current standard for treatment. While effective in most patients, those have long-term neurological sequelae in survivors, and a significant fraction of patients still succumb to the disease. In this study, we found that mebendazole (MBZ), an FDA-approved antiparasitic, demonstrated significant anti-tumor efficacy in etiologically distinct medulloblastoma mouse models. MBZ significantly improved the survival of mice with orthotopic xenograft tumors derived from the SHH group and group 3 medulloblastomas and was also highly efficacious against a PTCH1-mutant medulloblastoma with acquired resistance to the SMO inhibitor vismodgib. Analysis of the vasculature in rodent tumors revealed that MBZ selectively inhibited tumor angiogenesis but not the normal brain vasculature, and inhibited the kinase activity of VEGFR2 in vitro and in vivo. This study demonstrates that MBZ could be a highly promising therapeutic for medulloblastoma with anti- angiogenesis activity.

Elucidating immunologic mechanisms of PROSTVAC cancer immunotherapy.

BackgroundPROSTVAC®, an active immunotherapy currently studied for the treatment of metastatic castration-resistant prostate cancer (mCRPC), consists of a heterologous prime-boost regimen with two different poxvirus-based vectors to provoke productive immune responses against prostate specific antigen (PSA) as the target tumor antigen. A Phase 2 study of PROSTVAC immunotherapy showed significantly improved median overall survival by 8.5 months and is currently being validated in a global Phase 3 study (PROSPECT; NCT01322490). Here, preclinical models were explored to investigate the mechanism of action and immune signatures of anti-tumor efficacy with PROSTVAC immunotherapy with the goal to identify potential immune correlates of clinical benefit.MethodsPROSTVAC-induced immune responses and anti-tumor efficacy were studied in male BALB/c mice. Functionality of the induced T cell response was characterized by interferon-gamma (IFNγ) ELISPOT, cytotoxic degranulation, multi-cytokine intracellular staining, and in vivo T cell depletion. Tumor infiltrating lymphocytes (TILs) were evaluated phenotypically by flow cytometry.ResultsThe heterologous prime-boost regimen of the two PROSTVAC vectors significantly enhanced the magnitude and quality of activated PSA-specific CD4 and CD8 T cell responses compared to homologous, single vector regimens. PROSTVAC-activated CD4 and CD8 T cells were highly functional as evidenced by expression of activation markers, production of multiple cytokines, and amplified cytotoxic T cell activity. Importantly, PROSTVAC immunotherapy resulted in significant anti-tumor efficacy in a transplantable prostate cancer mouse model. Antigen-spreading occurred in PROSTVAC-treated animals that rejected PSA-expressing tumors, as shown by subsequent rejection of PSA-negative tumors. In vivo CD4 and CD8 depletion revealed that both T cell subsets contributed to anti-tumor efficacy. Characterization of TILs demonstrated that PROSTVAC immunotherapy greatly increased the intra-tumoral ratio of activated effector to regulatory T cells.ConclusionsPROSTVAC immunotherapy activates broad, highly functional T cell immunity to PSA and to endogenous tumor antigens via immune-mediated antigen spreading. These preclinical results further elucidate the mode of action of PROSTVAC immunotherapy and its potential causal relationship to extended overall survival as observed in the PROSTVAC Phase 2 study. The clinical validation is ongoing in the PROSPECT Phase 3 clinical study.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-014-0034-0) contains supplementary material, which is available to authorized users.

Abstract B72: Upfront treatment of high-risk neuroblastoma with 131I-MIBG therapy and Topotecan. A phase II pilot window study (AMRO-NB-HR-2000/01)

Abstract Purpose: Patients with high-risk (HR) neuroblastoma (NBL) have a poor prognosis and new treatment strategies are needed. 131I- Meta-Iodobenzylguanidine (MIBG) therapy alone has a significant antitumor efficacy against NBL. Topotecan (TPT) is a radio-enhancer and can potentiate radiation-induced cytotoxicity and up-regulate the uptake of MIBG in vitro. We designed a clinical study with upfront 131I-MIBG therapy and Topotecan for HR-NBL. We evaluated short term clinical response rate and outcome. Clinical response was scored according to International Neuroblastoma response Criteria (INRC) and Curie score (MIBG) after 2 courses of MIBG/TPT and after induction chemotherapy and HD ASCT. Haematological toxicity and the effect on harvesting/ feasibility performing autologous stem cell transplantation (ASCT) was evaluated. Topoisomerase-1 activity (Topo-1) levels were measured. Methods: Multi-centre, prospective (window phase II) study performed between 2000-2003 in HR NBL patients treated with 2 cycles of upfront 131I-MIBG therapy followed by the addition of TPT 0.7 mg/m2 for 5 days after each course. Thereafter, patients received 4 courses of VECI chemotherapy, followed by surgery and HD ASCT. Results: Sixteen patients (10 male), median age 2, 8 yrs. NMYC amplified 8/14 (57%) and LOH1P in 7/15 (47%). Surgery performed in 12/16 (75%) patients. Clinical response after 2x MIBG/TPT 15/16 (94%, VGPR/ PR/ MR), 1 PD; after 4x VECI 11/16 (69%, CR/ VGPR/ PR/MR), 3 PD, 1 death and 1 unknown. More than 50% reduction in MIBG Curie score after 2x MIBG/ TPT and subsequent 4x VECI courses in most cases. Overall outcome 2/16 currently alive. Long term follow up was limited, possibly related to low intensity induction chemotherapy as used in this study. Harvesting of stem cells in 13/16 patients (79%), mean yield 2, 2 CD34 pos/ kg, 9/16 (56%) underwent HD ASCT. Haematological toxicity was limited, median platelet recovery after HD ASCT was 73 days. Topo-1 activity was measured in 10/16 tumors, increased compared to reference level. Conclusion: Upfront treatment of HR-NBL with MIBG/TPT is feasible and highly effective after 2 courses. The toxicity is limited (haematological). Stem cell harvesting and HD ASCT are manageable. Citation Format: Max M. van Noesel, Kathelijne CJM Kraal, Jan de Kraker, Berthe LF van Eck-Smit, Godelieve AM Tytgat, Huib N. Caron. Upfront treatment of high-risk neuroblastoma with 131I-MIBG therapy and Topotecan. A phase II pilot window study (AMRO-NB-HR-2000/01). [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B72.

Abstract 4289: Noninvasive monitoring of blood-brain barrier permeability for antibodies by apoptosis imaging: optimization of treatment strategies for maximal brain delivery

Abstract Purposes: Large molecules like antibodies cannot penetrate the intact blood-brain barrier (BBB) in adequate amounts to deploy efficacy. Brain shuttle antibodies for enhanced BBB crossing are considered to overcome this obstacle. Thus, we established a non-invasive technology that accelerates quantification of brain tumor delivery and pharmacodynamics of antibodies. Experimental Procedures: The technology implies intracranial implantation of a split-luciferase apoptosis reporter cell line (Caspase-3/7 GloSensor) in SHO mice. An agonistic Death Receptor 5 antibody (anti-DR5) was used as model substance. To quantify BBB crossing of systemically applied anti-DR5, we compared apoptosis inductions after i.c. and i.v. application at different dosages. Results: Assuming a 100 % drug delivery after i.c. application, the amount of antibody delivery after i.v. application was calculated relative to its apoptosis induction. We found that less than 1 % of the unmodified anti-DR5 passed the BBB dose-dependently. These data were confirmed by 3-dimensional fluorescence microscopy of explanted brain tissue. Significant anti-tumor efficacy was detected when anti-DR5 is given in adequately high dosages. We further demonstrate in different brain tumor models that unspecific IgG antibody penetration into brain tumors is presumably enhanced by a slightly impaired BBB. Conclusions: Our technology facilitates the quantification of BBB penetration by measuring apoptosis non-invasively without using radiolabeling approaches. This orthotopic model accelerates the assessment of i.v. dosage optimization and brain shuttle systems for maximal anti-tumor efficacy in brain tumors. Citation Format: Thomas G. Weber, Julia Mathejczyk, Stefanie Galbán, Alnawaz Rehemtulla, Werner Scheuer. Noninvasive monitoring of blood-brain barrier permeability for antibodies by apoptosis imaging: optimization of treatment strategies for maximal brain delivery. [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 4289. doi:10.1158/1538-7445.AM2014-4289

Abstract 3685: BAY 1125976, is a selective allosteric AKT1/2 inhibitor with high efficacy in AKT1-mutated cancers

Abstract The PI3K-AKT-mTOR signaling cascade is activated in human cancers by elevated membrane receptor activity, mutation, amplification, and deletion of genes encoding components of the pathway. An oncogenic, single hotspot mutation in the AKT1 gene, G49A:E17K, in the pleckstrin homology domain of AKT1, was described in human cancers with highest relative incidence in breast cancer. The E17K mutation in AKT1 results in PI3K-independent membrane recruitment of AKT1. Recently we reported the development of a highly selective, potent allosteric AKT1/2 inhibitor BAY 1125976 with strong in vitro and in vivo activity in tumor models with activated AKT signaling and strong synergistic activity in combination. The efficacy of BAY 1125976 was evaluated in tumor models carrying activation of the PI3K-AKT pathway by either deletion of PTEN or activating mutation in PI3K. We investigated whether BAY 1125976 can also inhibit AKT signaling in cell lines carrying an activating mutation in AKT. To this extend, KU-19-19 (AKT1E17K; NRASQ61R) bladder cancer as well as LAPC4 (AKT1E17K) prostate cancer cell lines both bearing the AKT1E17K mutation were profiled. BAY 1125976 potently inhibited AKT activation as well as downstream signaling in KU-19-19 and LAPC4 cells. Furthermore, anti-tumor efficacy of BAY 1125976 was tested in the patient-derived anal cancer xenograft AXF 984 (AKT1E17K). BAY 1125976 shows dose-dependent potent pathway activity in AKT1mut patient derived tumor model with inhibition of pAKT473 as well as downstream targets p- PRAS40 and p-FOXO3a compared to selected PI3K, AKT and mTOR inhibitors. Continuous daily treatment of AXF 984 (AKT1E17K) mouse xenografts with 25 mg/kg and 50 mg/kg QD p.o. BAY 1125976 resulted in very potent statistically significant anti-tumor efficacy. All animals of these treatment groups exhibited tumor shrinkage or disease control with response rates of 88% and 83%, respectively. Furthermore, for animals treated with 50 mg/kg BAY 1125976 the delay of reaching a relative tumor volume of 600% was statically significant compared to the vehicle-treated control group. In summary, BAY 1125976 showed superior anti-tumor activity in AKT1E17K mutated models compared to PI3K and mTOR inhibitors. These results indicate that the clinical development of BAY 1125976 in patients with activating mutation in AKT could result in an innovative and more effective alternative to current treatments. Citation Format: Oliver Politz, Arne Scholz, Andrea Haegebarth, Ningshu Liu, Lars Baerfacker, Stuart Ince, Roland Neuhaus, Ulf Boemer, Martin Michels, Dominik Mumberg. BAY 1125976, is a selective allosteric AKT1/2 inhibitor with high efficacy in AKT1-mutated cancers. [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 3685. doi:10.1158/1538-7445.AM2014-3685

Antitumor effect of the tyrosine kinase inhibitor nilotinib on gastrointestinal stromal tumor (GIST) and imatinib-resistant GIST cells.

Despite the benefits of imatinib for treating gastrointestinal stromal tumors (GIST), the prognosis for high risk GIST and imatinib-resistant (IR) GIST remains poor. The mechanisms of imatinib resistance have not yet been fully clarified. The aim of the study was to establish imatinib-resistant cell lines and investigate nilotinib, a second generation tyrosine kinase inhibitor (TKI), in preclinical models of GIST and imatinib-resistant GIST. For a model of imatinib-resistant GIST, we generated resistant cells from GK1C and GK3C cell lines by exposing them to imatinib for 6 months. The parent cell lines GK1C and GK3C showed imatinib sensitivity with IC50 of 4.59±0.97 µM and 11.15±1.48 µM, respectively. The imatinib-resistant cell lines GK1C-IR and GK3C-IR showed imatinib resistance with IC50 values of 11.74±0.17 µM (P<0.001) and 41.37±1.07 µM (P<0.001), respectively. The phosphorylation status of key cell signaling pathways, receptor tyrosine kinase KIT (CD117), platelet-derived growth factor receptor alpha (PDGFRA) and downstream signaling kinases: serine-threonine kinase Akt (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) or the non-receptor tyrosine kinase: proto-oncogene tyrosine-protein kinase Src (SRC), was analyzed in established cell lines and ERK1/2 phosphorylation was found to be increased compared to the parental cells. Nilotinib demonstrated significant antitumor efficacy against GIST xenograft lines and imatinib-resistant GIST cell lines. Thus, nilotinib may have clinical potential for patients with GIST or imatinib-resistant GIST.

Antimetastasis and antitumor efficacy promoted by sequential release of vascular disrupting and chemotherapeutic agents from electrospun fibers

The vasculature in tumor microenvironment plays important roles in the tumor growth and metastasis, and the combination of vascular disrupting agents with chemotherapeutic drugs should be effective in inhibiting tumor progression. But the dosing schedules are essential to achieve a balance between vascular collapse and intratumoral uptake of chemotherapeutic agents. In the current study, emulsion and blend electrospinning were used to create compartmental fibers accommodating both combretastatin A-4 (CA4) and hydroxycamptothecin (HCPT). The release durations of CA4 and HCPT were modulated through the structure of fibers for dual drug loadings and the inoculation of 2-hydroxypropyl-β-cyclodextrin in fiber matrices. Under a noncontact cell coculture in Transwell, the sequential release of CA4 and HCPT indicated a sequential killing of endothelial and tumor cells. In an orthotopic breast tumor model, all the CA4/HCPT-loaded fibers showed superior antitumor efficacy and higher survival rate than fibers with loaded individual drug. Compared with fibrous mats with infiltrated free CA4 and fibers with extended release of CA4 for over 30 days, fibers with sustained release of CA4 for 3–7 days from CA4/HCPT-loaded fibers resulted in the most significant antitumor efficacy, tumor vasculature destruction, and the least tumor metastasis to lungs. A judicious selection of CA4 release durations in the combination therapy should be essential to enhance the tumor suppression efficacy and antimetastasis activity.

The influence of bile salt on the chemotherapeutic response of docetaxel-loaded thermosensitive nanomicelles.

The primary aim of this work was to investigate the potential of bile salt, sodium taurocholate (NaTC), in improving the bioavailability and anti-tumor efficacy of docetaxel (DCT) upon rectal administration. Poloxamer-based nanomicelles with thermosensitive and mucoadhesive properties were prepared using the cold method. The optimized nanomicellar formulation was evaluated in terms of physicochemical and viscoelastic parameters. Nanomicelles containing bile salt maintained sufficient gelation strength (234×102 mPa·s) and mucoadhesive force (17.3×102 dyne/cm2) to be retained in the upper part of the rectum. They significantly enhanced the DCT internalization across the rectal mucosa and showed a high plasma level during the first 4 hours of the study period, compared to nanomicelles with no bile salt. As a result, a slightly higher rectal bioavailability of ~33% was observed in nanomicelles containing bile salt, compared to ~28% from the latter system. The higher pharmacokinetic parameters for rectally administered DCT/P407/P188/Tween 80/NaTC (0.25%/11%/15%/10%/0.1% by weight, respectively) resulted in significant anti-tumor efficacy. However, the tumor regression rate for the NaTC group was not statistically different from that for nanomicelles without NaTC. Therefore, overall results suggest that thermosensitive nanomicelles could be a potential dosage form for improvement of the bioavailability and chemotherapeutic profile of DCT.

SOMCL-863, a novel, selective and orally bioavailable small-molecule c-Met inhibitor, exhibits antitumor activity both in vitro and in vivo

Deregulation of HGF/c-Met signaling and its driven neoplastic phenotype are associated with a variety of human malignancies. We herein reported SOMCL-863 as a novel selective c-Met inhibitor which effectively abrogated c-Met signaling pathways, thereby leading to substantial impairment of c-Met-dependent cell proliferation, migration, invasion, cell scattering and invasive growth. In EBC-1 and NCI-H1993 xenografts, SOMCL-863 exerted significant anti-tumor efficacy through anti-proliferative effects and antiangiogenic mechanisms, including reduction of tumor cell proliferation and reductions of microvessel density and secretion of proangiogenic factor IL-8. Together with the optimal pharmacokinetic properties, SOMCL-863 is a promising candidate worthy for further evaluation as a treatment of c-Met-driven human cancers.

Engineered, self-assembled near-infrared photothermal agents for combined tumor immunotherapy and chemo-photothermal therapy

New combination therapy strategy, which takes the advantages of co-delivery two or more therapeutic agents in one nanocarrier platform, has been widely used in the clinic and achieved immense popularity in cancer treatment. Here, we have rationally developed a multifunctional platform using a self-assembly strategy to incorporate materials with specific functions of chemotherapeutics, hyperthermia, and especially immunotherapy, which can collectively contribute to the effective cancer treatment. We design the immunomodulatory CpG ODNs based platform that is conjugated with NIR-responsive gold nanorods and doxorubicin for cancer therapy. The gold nanorods can be applied as the nanocarrier to simultaneously address the three kinds of treatments, which lead to a significant benefit relative to the use of each method alone. Both in vitro and in vivo assays reveal that this engineered vehicle exhibits significant antitumor efficacy. Our studies provide strong evidence that the AuNRs-CpG-Dox conjugates can be utilized as efficient antitumor agents.

Discovery of novel 2,4-diarylaminopyrimidine analogues (DAAPalogues) showing potent inhibitory activities against both wild-type and mutant ALK kinases.

We have developed a series of new 2,4-diarylaminopyrimidine analogues (DAAPalogues) bearing a flexible amino acid side chain, different from the majority of the literature reported ALK inhibitors that often possess a structurally constrained arylpiperazine fragment or its equivalents in the solvent-interaction region. Extensive structural elaboration led to compound 15 possessing IC50 values of 2.7 and 15.3 nM, respectively, in the ALK wild-type and gate-keeper mutant L1196M enzymatic assays. This compound not only showed high proliferative inhibition against ALK-addicted cells across different oncogenic forms but also effectively suppressed several ALK secondary mutant cells, including the gate-keeper L1196M and F1174L. Significant antitumor efficacy was achieved in the ALK-driven SUP-M2 xenograft model.

OX40 engagement depletes intratumoral Tregs via activating FcγRs, leading to antitumor efficacy.

Antibodies targeting checkpoint inhibitors or co-stimulatory receptors on T cells have shown significant antitumor efficacy in preclinical and clinical studies. In mouse tumor models, engagement of activating Fcγ receptor (FcγR)-expressing immune cells was recently shown to be required for the tumoricidal activity of antibodies recognizing the tumor necrosis factor superfamily receptor (TNFR) GITR (CD357) and CTLA-4 (CD152). In particular, activating FcγRs facilitated the selective elimination of intratumoral T-cell populations. However, it remains unclear whether FcγRs contribute to the antitumor efficacy of other immunomodulatory antibodies. Here, we explored the mechanism of antitumor activity mediated by an agonistic antibody (clone OX86) to the co-stimulatory TNFR OX40 (CD134). OX40 was highly expressed by intratumoral T cells, particularly those of the FoxP3(+) regulatory T-cell (Treg) lineage. OX86 administration resulted in the depletion of intratumoral regulatory T cells in an activating FcγR-dependent manner, which correlated with tumor regression. Together with previous data from our group and others, these findings support a mechanism whereby antibodies targeting antigens highly expressed by intratumoral T cells can mediate their elimination by FcγR-expressing immune cells, and facilitate subsequent antitumor immunity.

Optimized rod length of polyplex micelles for maximizing transfection efficiency and their performance in systemic gene therapy against stroma-rich pancreatic tumors

Poly(ethylene glycol) (PEG) modification onto a gene delivery carrier for systemic application results in a trade-off between prolonged blood circulation and promoted transfection because high PEG shielding is advantageous in prolonging blood retention, while it is disadvantageous with regard to obtaining efficient transfection owing to hampered cellular uptake. To tackle this challenging issue, the present investigation focused on the structure of polyplex micelles (PMs) obtained from PEG–poly(l-lysine) (PEG–PLys) block copolymers characterized as rod-shaped structures to seek the most appreciable formulation. Comprehensive investigations conducted with particular focus on stability, PEG crowdedness, and rod length, controlled by varying PLys segment length, clarified the effect of these structural features, with particular emphasis on rod length as a critical parameter in promoting cellular uptake. PMs with rod length regulated below the critical threshold length of 200 nm fully exploited the benefits of cross-linking and the cyclic RGD ligand, consequently, exhibiting remarkable transfection efficiency comparable with that of ExGen 500 and Lipofectamine® LTX with PLUS™ even though PMs were PEG shielded. The identified PMs exhibited significant antitumor efficacy in systemic treatment of pancreatic adenocarcinoma, whereas PMs with rod length above 200 nm exhibited negligible antitumor efficacy despite a superior blood circulation property, thereby highlighting the significance of controlling the rod length of PMs to promote gene transduction.

Cycles of transient high-dose cyclophosphamide administration and intratumoral oncolytic adenovirus vector injection for long-term tumor suppression in Syrian hamsters.

Immune responses against oncolytic adenovirus (Ad) vectors are thought to limit vector anti-tumor efficacy. In Syrian hamsters, which are immunocompetent and whose tumors and normal tissues are permissive for replication of Ad5-based oncolytic Ad vectors, treating with high-dose cyclophosphamide to suppress the immune system and exert chemotherapeutic effects enhances Ad vector anti-tumor efficacy. However, long term cyclophosphamide treatment and immunosuppression can lead to anemia and vector spread to normal tissues. Here we employed three cycles of transient high-dose cyclophosphamide administration plus intratumoral injection of the oncolytic Ad vector VRX-007 followed by withdrawal from cyclophosphamide. Each cycle lasted 4-6 weeks. This protocol allowed the hamsters to remain healthy so the study could be continued for ~100 days. The tumors were very well suppressed throughout the study. With immunocompetent hamsters, the vector retarded tumor growth initially, but after 3-4 weeks the tumors resumed rapid growth and further injections of vector were ineffective. Preimmunization of the hamsters with Ad5 prevented vector spillover from the tumor to the liver yet still allowed for effective long term anti-tumor efficacy. Our results suggest that a clinical protocol might be developed with cycles of transient chemotherapy plus intratumoral vector injection to achieve significant anti-tumor efficacy while minimizing the side effects of cytostatic treatment.

Apoptosis imaging for monitoring DR5 antibody accumulation and pharmacodynamics in brain tumors noninvasively.

High-grade gliomas often possess an impaired blood-brain barrier (BBB), which allows delivery of large molecules to brain tumors. However, achieving optimal drug concentrations in brain tumors remains a significant hurdle for treating patients successfully. Thus, detailed investigations of drug activities in gliomas are needed. To investigate BBB penetration, pharmacodynamics, and tumor retention kinetics of an agonistic DR5 antibody in a brain tumor xenograft model, we utilized a noninvasive imaging method for longitudinal monitoring of apoptosis induction. Brain tumors were induced by intracranial (i.c.) implantation of a luciferase-expressing tumor cell line as a reporter. To quantify accumulation of anti-DR5 in brain tumors, we generated a dosage-response curve for apoptosis induction after i.c. delivery of fluorescence-labeled anti-DR5 at different dosages. Assuming 100% drug delivery after i.c. application, the amount of accumulated antibody after i.v. application was calculated relative to its apoptosis induction. We found that up to 0.20% to 0.97% of antibody delivered i.v. reached the brain tumor, but that apoptosis induction declined quickly within 24 hours. These results were confirmed by three-dimensional fluorescence microscopy of antibody accumulation in explanted brains. Nonetheless, significant antitumor efficacy was documented after anti-DR5 delivery. We further demonstrated that antibody penetration was facilitated by an impaired BBB in brain tumors. These imaging methods enable the quantification of antibody accumulation and pharmacodynamics in brain tumors, offering a holistic approach for assessment of central nervous system-targeting drugs.

Elucidating immunologic mechanisms of PROSTVAC cancer immunotherapy

<h3>Background</h3> PROSTVAC®, an active immunotherapy currently studied for the treatment of metastatic castration-resistant prostate cancer (mCRPC), consists of a heterologous prime-boost regimen with two different poxvirus-based vectors to provoke productive immune responses against prostate specific antigen (PSA) as the target tumor antigen. A Phase 2 study of PROSTVAC immunotherapy showed significantly improved median overall survival by 8.5 months and is currently being validated in a global Phase 3 study (PROSPECT; NCT01322490). Here, preclinical models were explored to investigate the mechanism of action and immune signatures of anti-tumor efficacy with PROSTVAC immunotherapy with the goal to identify potential immune correlates of clinical benefit. <h3>Methods</h3> PROSTVAC-induced immune responses and anti-tumor efficacy were studied in male BALB/c mice. Functionality of the induced T cell response was characterized by interferon-gamma (IFNγ) ELISPOT, cytotoxic degranulation, multi-cytokine intracellular staining, and <i>in vivo</i> T cell depletion. Tumor infiltrating lymphocytes (TILs) were evaluated phenotypically by flow cytometry. <h3>Results</h3> The heterologous prime-boost regimen of the two PROSTVAC vectors significantly enhanced the magnitude and quality of activated PSA-specific CD4 and CD8 T cell responses compared to homologous, single vector regimens. PROSTVAC-activated CD4 and CD8 T cells were highly functional as evidenced by expression of activation markers, production of multiple cytokines, and amplified cytotoxic T cell activity. Importantly, PROSTVAC immunotherapy resulted in significant anti-tumor efficacy in a transplantable prostate cancer mouse model. Antigen-spreading occurred in PROSTVAC-treated animals that rejected PSA-expressing tumors, as shown by subsequent rejection of PSA-negative tumors. <i>In vivo</i> CD4 and CD8 depletion revealed that both T cell subsets contributed to anti-tumor efficacy. Characterization of TILs demonstrated that PROSTVAC immunotherapy greatly increased the intra-tumoral ratio of activated effector to regulatory T cells. <h3>Conclusions</h3> PROSTVAC immunotherapy activates broad, highly functional T cell immunity to PSA and to endogenous tumor antigens via immune-mediated antigen spreading. These preclinical results further elucidate the mode of action of PROSTVAC immunotherapy and its potential causal relationship to extended overall survival as observed in the PROSTVAC Phase 2 study. The clinical validation is ongoing in the PROSPECT Phase 3 clinical study.

Serum-resistant complex nanoparticles functionalized with imidazole-rich polypeptide for gene delivery to pulmonary metastatic melanoma

To enhance serum-resistance and overcome the lysosomal barrier are effective and feasible strategies to increase the transfection efficiency of non-viral gene delivery system. For the systemic delivery of therapeutic gene, we previously developed self-assemble carboxymethyl poly(l-histidine) (CM-PLH)/poly(β-amino ester) (PbAE)/pDNA ternary complex nanoparticles based on electrostatic coating as an effective pDNA carrier. Recharging cationic PbAE/pDNA polyplexes with CM-PLH was a promising method to reduce the cytotoxicity and enhance the stability in vivo of positive charged polyplexes. In the present study, the transfection activities of ternary complex nanoparticles were further evaluated in vitro and in vivo. The transfection efficiency of ternary complex nanoparticles showed significant serum-resistance (CM-PLH-containing (51.9±4.35)% in 50% FBS>CM-PLH-free (14.7±5.66)% in 50% FBS), cell line dependent (HEK293>MCF-7>COS7>B16F10>A549>Hela>SPC-A1>CHO>SKOV3) and incubation period dependent (24 h, 20 h, 16 h>12 h>8 h>4 h>2 h>1 h>0.5 h). After transfected with ternary complex nanoparticles loading pGV240-MDA-7/IL-24, the B16F10 cells exhibited significant apoptosis and proliferation inhibition due to the expression of IL-24. Moreover, in the pulmonary metastatic melanoma model, ternary complex nanoparticles loading pGV240-MDA-7/IL-24 showed significant antitumor therapeutic efficacy in vivo. These results suggested that CM-PLH/PbAE/pDNA ternary complex nanoparticles were promising and challenging gene vector for practical application.

Abstract A275: Co-targeting PIM and PI3K/mTOR pathways with a single molecule: Novel orally available combined PIM/PI3K and PIM/PI3K/mTOR kinase inhibitors.

Abstract The PI3K/AKT pathway is commonly activated in human cancer. Multiple small-molecule inhibitors have been developed to target PI3K/mTOR or AKT kinases, but the efficacy of these drugs is compromised by the stimulation of compensatory signaling pathways. The redundancy of oncogenic signaling pathways provides back-up mechanisms that allow cancer cells to escape to targeted therapies. One example of such compensatory pathways is that driven by PIM kinases, which produce parallel oncogenic signals to AKT and mTOR and share several downstream molecular targets. In fact, in mouse lymphoma models chemoresistance to doxorubicin caused by AKT is readily reversed by rapamycin, but PIM mediated resistance is refractory to mTORC1 inhibition. Moreover, PIM2 kinase can confer resistance of primary hematopoietic cells to rapamycin treatment. Furthermore, PIM 1 mediates resistance to AKT and PI3K/mTOR inhibition and suggests co-targeting to improve the efficacy of PI3K/AKT/mTOR inhibitors in anticancer therapy. The PI3K/AKT/mTOR and PIM pathways are activated and seem to be relevant for tumor progression in many lymphomas, leukemias and in some solid tumors. We have previously reported that the combination of PI3K inhibitor GDC-0941 with a PIM selective inhibitor, ETP-45299, was strongly synergistic in antiproliferation experiments in MV-4-11 AML cells. Therefore, combination of such activities in a single molecule might have the potential to achieve better clinical efficacy and prevent/suppress resistance in comparison with classical PI3K/AKT/mTOR inhibitors. During the course of our investigations in this field we have identified a new chemical series of compounds with dual (PIM/PI3K) and triple activity (PIM/PI3K/mTOR inhibitors). (WO2012/156756). In this work, we show the biochemical, cellular and in vivo characterization of dual PIM/PI3K ETP-539/(IBL-202) and triple PIM/PI3K/mTOR ETP-339/(IBL-301) inhibitors. These optimized lead compounds are low nanomolar pan PIM/PI3K and pan PIM/PI3K/mTOR inhibitors respectively. These dual and triple inhibitors show excellent kinase selectivity profile against a panel of 456 kinases. Both compounds have been profiled for their antiproliferative behavior. We have identified leukemia, lymphoma, colon, and NSCLC lines which exhibit a strong sensitivity to dual and triple inhibition with GI80 between 5-10 times more potent than PIM or PI3K selective reference inhibitors. Mechanistically, cells respond to dual and triple inhibitors with a clear cell cycle arrest and marked apoptosis in AML and NSCLC cell lines, and strong down regulation of biomarkers. Our dual and triple inhibitors are optimized with respect to their in vitro ADME properties and have excellent oral bioavailability. These inhibitors have been tested in vivo in xenograft (MV4:11 AML) and transgenic (KRAsV12NSCLC) cancer mouse models. Both compounds have demonstrated down regulation of biomarkers associated with their targeted profile and significant antitumor efficacy in both models after oral administration. These inhibitors have been well tolerated, with no signs of toxicity even 20 times above the efficacious dose. These results provide the rationale for further preclinical development of ETP-539/(IBL-202 and ETP-339/(IBL-301) and the basis for a potential clinical use in AML and NSCLC tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A275. Citation Format: Carmen Blanco Aparicio, Oliver Renner, Elena Gomez-Casero, Antonio Cebriá, Nuria Ajenjo, Enara Aguirre, David Cebrián, Ma Carmen Rodriguez de Miguel, Belén Pequeño, MaIsabel Albarrán, Rosario Riesco, Ana Belén García, Rosa Alvarez, Michael O'Neill, Sonia Martinez, Joaquin Pastor. Co-targeting PIM and PI3K/mTOR pathways with a single molecule: Novel orally available combined PIM/PI3K and PIM/PI3K/mTOR kinase inhibitors. [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 A275.

Abstract C270: In vitro and in vivo antitumor activity of DCBCI0901, a potent PI3K/mTORC1/mTORC2 inhibitor.

Abstract The mammalian target of rapamycin (mTOR) is a highly conserved Ser/Thr protein kinase and a central controller of cell growth, proliferation, and survival. Studies have linked the hyperactive PI3K-AKT-mTOR signaling pathway in a variety of human cancers. Drugs targeting to PI3K and mTOR are likely to be an effective treatment for human malignancies that rely on these targets for their growth. Institute of Pharmaceutics at Development Center for Biotechnology in Taiwan has developed a novel and potent small molecule DCBCI0901 as PI3K/mTORC1/mTORC2 inhibitor. DCBCI0901 attenuated cell survival and proliferation in both in vitro and in vivo models. DCBCI0901 possessed IC50 of 8.4 nM against mTOR kinase and IC50 of 78 nM against PI3Kα (p110α/p85α) while exerting high selectivity against a panel of 20 other serine/ threonine and tyrosine kinases. In addition, DCBCI0901 is highly active in cellular assays. It causes a significant reduction in phospho-p70S6K (mTORC1 substrate), phospho-AKT (S473) (mTORC2 substrate), and phospho-AKT (T308) (PI3K substrate) levels. The antitumor efficacy of DCBCI0901 was evaluated in diverse human tumor cell lines. DCBCI0901 effectively inhibits cell proliferation of five well-established cell lines (one NSCLC, A549; two prostate cancers, PC-3 and LNCap; two breast cancers, HCC1954 and BT474), with IC50 values ranging from 6 to 48 nM. Notably, DCBCI0901 is effective against a panel of nine lung cancer cell lines (one cell line is EGFR-TKI sensitive model and eight cell lines are EGFR-TKI resistant model), with IC50 in the ten of nanomolar range. DCBCI0901 showed significant antitumor efficacy in mouse xenograft models including lung cancer, prostate cancer, and leukemia with tumor growth inhibition greater than 65%. DCBCI0901 also demonstrated effective suppression of tumor growth in advanced-stage xenograft models (lung cancer (A549), mean tumor volume &amp;gt; 350mm3 at the start of treatment). Furthermore, in an EGFR-TKI resistant lung cancer H1975 xenograft model, DCBCI0901 effectively suppressed tumor growth. Based on the in vitro/in vivo pharmacology data, DCBCI0901 was selected as a drug candidate for further development as anticancer agent. This publication is dedicated to the memory of Dr. Paonien Chen. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C270. Citation Format: Ying-Shuan E. Lee, Mann-Yan Kuo, Yi-Ting Huang, Yu-Wen Tseng, Yu Jie Lin, Yen His Liu, Shian-Yi Chiou, Li Jung Chen, Y.-Y. Lu, Chih-Hsin Yang, Pan-Chyr Yang. In vitro and in vivo antitumor activity of DCBCI0901, a potent PI3K/mTORC1/mTORC2 inhibitor. [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 C270.

Abstract C275: CEP-40783: A potent and selective AXL/c-Met inhibitor for use in breast, non-small cell lung (NSCLC), and pancreatic cancers.

Abstract The oncogenic receptor tyrosine kinases AXL and c-Met are over-expressed and constitutively activated in a variety of human cancers. Activation of these receptors contributes to multiple steps in tumor progression by promoting cancer cell migration and invasion, enhancing tumor angiogenesis, facilitating cancer cell survival and tumor growth, and contributing to tumor resistance to several chemotherapy and molecular-targeted therapeutic agents. CEP-40783 is an orally active, potent and selective AXL and c-Met kinase inhibitor, with enzyme IC50 values of 7 nM and 12 nM, respectively. In AXL-transfected 293GT cells, CEP-40783 was 27-fold more active compared to recombinant enzyme with an IC50 value of 0.26 nM. Comparably high cellular potency was observed in NCI-H1299 human NSCL cells. CEP-40783 also demonstrated superior activity against c-Met in GTL-16 cells (IC50 = 6 nM). The increased inhibitory activity of CEP-40783 in cells could be attributed to its extended residence time on both AXL and c-Met, consistent with a Type II mechanism. The prolonged residence time of CEP-40783 at the target may provide for improved in vivo efficacy, selectivity and therapeutic index. Additionally, CEP-40783 showed high kinome selectivity against 298 kinases with an S90 of 0.04 (fraction of kinases showing &amp;gt;90% inhibition at 1 µM). In PK/PD studies, CEP-40783 showed dose- and time-dependent inhibition of AXL phosphorylation using NCI-H1299 NSCL xenografts with ∼80% target inhibition at 0.3 mg/kg 6 h post dose and complete target inhibition to &amp;gt;90% inhibition at 1 mg/kg between 6-24 h, while a 10 mg/kg po dose resulted in complete AXL inhibition up to 48 h post dosing. In AXL/NIH3T3 xenografts, 0.3 mg/kg po resulted in complete tumor regressions. CEP-40783 was also efficacious in reducing spontaneous lymph node and pulmonary metastatic tumor burden in the MDA-MB-231-luc and 4T1-luc orthotopic breast cancer models, respectively, at 10 and 30 mg/kg po. PK/PD evaluation of the c-Met activity of CEP-40783 (10, 30, 55 mg/kg po qdX5d) showed significant to complete inhibition of c-Met phosphorylation in GTL-16 gastric carcinoma xenografts. Efficacy studies in GTL-16 xenografts demonstrated significant anti-tumor efficacy (tumor stasis and regressions) at 10 and 30 mg/kg po. In EBC-1 NSCL xenografts, administration of CEP-40783 (3, 10 and 30 mg/kg, po qd) resulted in dose-related efficacy, with tumor stasis at 3 mg/kg, tumor regressions and &amp;gt;96% TGI at 10 mg/kg. In all studies CEP-40783 was well tolerated with no compound-related body weight loss. CEP-40783 demonstrated potent AXL and c-Met pharmacodynamic and anti-tumor efficacy in established tumor xenograft models, having potential therapeutic utility in multiple human tumor types in which c-Met and AXL activity play a critical role in tumor formation, local invasion and metastasis. Studies in primary human tumorgrafts are in progress. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C275. Citation Format: Sheila Miknyoczki, Mangeng Cheng, Robert Hudkins, Thelma Angeles, Lisa Aimone, Jean Husten, Jie Qian, Seetha Murthy, Thomas Conners, Robert Bendesky, Amy Landis, Jennifer Grobelny, Hong Chang, Bruce Dorsey, Mark Ator, Bruce Ruggeri. CEP-40783: A potent and selective AXL/c-Met inhibitor for use in breast, non-small cell lung (NSCLC), and pancreatic cancers. [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 C275.