Efficacy In Multiple Preclinical Models Research Articles

Glutamine Deprivation Synergizes the Anticancer Effects of Cold Atmospheric Plasma on Esophageal Cancer Cells.

Esophageal cancer is a highly aggressive malignancy with a low response to standard anti-cancer therapies. There is an unmet need to develop new therapeutic strategies to improve the clinical outcomes of current treatments. Cold atmospheric plasma (CAP) is a promising approach for cancer treatment, and has displayed anticancer efficacy in multiple preclinical models. Recent studies have shown that the efficacy of CAP is positively correlated with intracellular reactive oxygen species (ROS) levels. This suggests that aggressively increasing intracellular ROS levels has the potential to further improve CAP-mediated anticancer efficacy. Glutamine plays an important role in cellular ROS scavenging after being converted to glutathione (GSH, a well-described antioxidant) under physiological conditions, so reducing intracellular glutamine levels seems to be a promising strategy. To test this hypothesis, we treated esophageal cancer cells with CAP while controlling the supply of glutamine. The results showed that glutamine did affect the anticancer effect of CAP, and the combination of CAP stimulation and glutamine deprivation significantly inhibited the proliferation of esophageal cancer cells compared to the control group (p < 0.05). Furthermore, flow cytometric analysis documented a significant increase in more than 10% in apoptosis and necrosis of esophageal cancer cells after this synergistic treatment compared to the control group (p < 0.05). Thus, these results provide the first direct evidence that the biological function of CAP can be modulated by glutamine levels and that combined CAP stimulation and glutamine deprivation represent a promising strategy for the future treatment of esophageal cancer.

597 Development of LY3454738, an agonistic antibody to human CD200R

CD200R is an immune receptor of the IgG family that is primarily expressed on cells of the myeloid lineage and was recently identified as a marker for Th2 biology (Blom et al 2017). In vivo studies with knockout mice of either the receptor or its ligand, CD200, have demonstrated that it is an inhibitory receptor capable of negatively regulating immune responses. Previous work using agonistic antibodies to mouse CD200R showed inhibition of mast cell activation in vitro and in vivo (Cherwinski et al 2005) as well as efficacy in multiple preclinical models of autoimmune diseases.

Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc-engineering

Agonistic CD27 monoclonal antibodies (mAb) have demonstrated impressive anti-tumour efficacy in multiple preclinical models but modest clinical responses. This might reflect current reagents delivering suboptimal CD27 agonism. Here, using a novel panel of CD27 mAb including a clinical candidate, we investigate the determinants of CD27 mAb agonism. Epitope mapping and in silico docking analysis show that mAb binding to membrane-distal and external-facing residues are stronger agonists. However, poor epitope-dependent agonism could partially be overcome by Fc-engineering, using mAb isotypes that promote receptor clustering, such as human immunoglobulin G1 (hIgG1, h1) with enhanced affinity to Fc gamma receptor (FcγR) IIb, or hIgG2 (h2). This study provides the critical knowledge required for the development of agonistic CD27 mAb that are potentially more clinically efficacious.

Abstract CT160: A Phase I, open-label, safety, pharmacokinetic, and preliminary efficacy study of STRO-002, an anti-folate receptor alpha (FolRα) antibody drug conjugate (ADC), in patients with advanced epithelial ovarian cancer (including fallopian tube or primary peritoneal cancers) and endometrial cancers

Abstract Background: Folate receptor alpha (FolRα) is overexpressed in 80% of epithelial ovarian cancer (EOC) and endometrial adenocarcinoma (EC), with minimal expression on normal tissues and is thus a relevant target for cancer therapy using antibody drug conjugates (ADCs). STRO-002 is a novel, site-specific FolRα-targeting ADC that was generated using Sutro’s XpressCF+™ cell free protein synthesis platform. STRO-002 has highly specific cytotoxic activity on cells expressing FolRα. STRO-002 demonstrated reproducible and significant therapeutic efficacy in multiple preclinical models in vivo. STRO-002 activity was evaluated in EC patient-derived xenograft (PDX) models with a range of FolRα expression. Statistically significant efficacy was observed in 60% of the FolRα-positive PDX models tested, with response appearing to positively correlate with FolRα expression levels. Toxicology studies in cynomolgus monkeys demonstrate dose-dependent and reversible neutropenia when STRO-002 is administered at up to 9 mg/kg, with no evidence of ocular toxicity (Solis et al. AACR 2019). Methods: This study is a first-in-human Phase 1, open-label, multicenter, dose escalation (Part 1) study with dose expansion (Part 2) to identify the maximum tolerated dose (MTD), recommended phase 2 doses (RP2D) and to evaluate the safety, tolerability, and preliminary anti-tumor activity of STRO-002 in adults with ovarian cancer and endometrial cancer who are refractory to, or intolerant of, therapies known to provide clinical benefit. STRO-002 is given to all patients on study via intravenous infusion on Day 1of each 21-day cycle until disease progression. Dose limiting toxicities will be assessed in the first cycle (Days 1-21) of dose escalation. Part 1 will enroll about 30 ovarian cancer patients to determine the MTD and RP2D for expansion while Part 2 will enroll 2 cohorts based on disease subtypes (ovarian or endometrial). Efficacy will be evaluated per RECIST 1.1 criteria. Key inclusion criteria include relapsed and or progressive disease, adequate bone marrow and renal function, and ability to comply with treatment, testing and pharmacokinetic (PK) schedules. Key exclusion criteria include clear cell, mucinous or sarcomatous ovarian carcinoma, prior treatment with a FolRα-targeting ADC, need for ongoing immunotherapy including systemic corticosteroids and a history of CNS involvement. Samples will be collected to assess FolRα expression levels, PK and immunogenicity. No formal statistical hypothesis testing will be conducted in this study. This study is currently open for enrollment in the US with the ClinicalTrials.gov Identifier: NCT03748186.: Citation Format: Robert Wendel Naumann, Denise S. Uyar, Russell J. Schilder, Michael A. Palumbo, Venita DeAlmeida, Shannon L. Matheny, Arturo Molina. A Phase I, open-label, safety, pharmacokinetic, and preliminary efficacy study of STRO-002, an anti-folate receptor alpha (FolRα) antibody drug conjugate (ADC), in patients with advanced epithelial ovarian cancer (including fallopian tube or primary peritoneal cancers) and endometrial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT160.

Abstract 4059: KPC34: a co-drug that combines a DNA damaging agent with a targeted therapy for the treatment of AML

Abstract AML occurs in the elderly with ~18,000 new US cases in 2014. Mortality is an estimated 10,050 US deaths in 2014. Elderly patient prognosis is a dismal 6.6%, 5 year survival rate. AML treatment involves induction chemotherapy with Ara-C followed by consolidation therapy or stem cell transplant. The major obstacle to long-term disease-free survival is relapse after treatment due to chemoresistance. Chemoresistance occurs by several mechanisms including reduced activation of Ara-C via down-regulated deoxycytidine kinase (dCK) levels or decreased uptake via lowered Equilibrative Nucleoside Transporter 1 (ENT-1) levels. The low expression of both dCK and ENT-1 correlates with a poor prognosis in patients with acute leukemias. Recurrent disease is more difficult to treat successfully. Elderly patients often have trouble tolerating repeated chemotherapy treatments. Activation of NF-κβ by protein kinase C (PKC) promotes proliferation and inhibits apoptosis in AML. Increased expression of PKC isoforms has been detected in AML. The activity of these isoforms promotes leukemic proliferation and survival by ERK activation and BCL-2. KPC34 is a novel synthetic phospholipid/deoxycytidine analog conjugate. KPC34's novel properties result in improved pharmacokinetics. It overcomes chemoresistance to Ara-C by bypassing ENT-1 uptake and dCK activation. KPC34 is administered orally. It crosses the blood-brain barrier, targeting CNS-infiltrating leukemias. Finally, KPC34’s lipid moiety inhibits PKC activity. The study’s hypothesis is that KPC34 is active against AML in vivo. KPC34 is highly cytotoxic to a variety of leukemic cell lines in vitro, with IC50s in the nM range. The mean IC50 was 45.92 nM (range from 7.39-175.6 nM). In order to assess the activity of KPC34 in vivo, we injected NSG mice with OCI-AML-3 cells expressing luciferase. Following engraftment, mice were treated with KPC34, Ara-C, or water on days 1, 4 and 7. A significant prolongation of survival was observed in the KCP34-treated mice (median survival 44.5 days vs. 25 days for Ara-C-treated mice, p=0.0033, by log-rank test). Retreatment of the mice with additional KPC34 resulted in a median survival time of 78.5 days (p=0.0028, single KPC34 course vs. 2 additional treatment courses). KPC34 + doxorubicin is superior to the treatment of mice with Ara-C + doxorubicin. In a PDX murine model, KPC 34 showed a reduction in leukemic burden in the liver and spleen. KPC34 is well tolerated in vivo. KPC34 is effective against multiple models of naïve and chemoresistant leukemia. It has two distinct anti-neoplastic mechanisms. Oral administration improves efficacy over i.v. dosing. These data demonstrate KPC34 efficacy in multiple preclinical models of leukemia, and support its continued translation to the clinic. This co-drug has the potential to replace Ara-C-based therapies as a single agent/combo in naïve AML, a single agent in relapsed leukemia, and it reduces the CNS leukemic burden. Citation Format: Gregory L. Kucera, Peter Alexander, Kristin Pladna, Timothy S. Pardee. KPC34: a co-drug that combines a DNA damaging agent with a targeted therapy for the treatment of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4059. doi:10.1158/1538-7445.AM2017-4059

Role of YAP1 as a Marker of Sensitivity to Dual AKT and P70S6K Inhibition in Ovarian and Uterine Malignancies

The PI3K/AKT/P70S6K pathway is an attractive therapeutic target in ovarian and uterine malignancies because of its high rate of deregulation and key roles in tumor growth. Here, we examined the biological effects of MSC2363318A, which is a novel inhibitor of AKT1, AKT3, and P70S6K. Orthotopic murine models of ovarian and uterine cancer were utilized to study the effect of MSC2363318A on survival and regression. For each cell line, 10 mice were treated in each of the experimental arms tested. Moreover, in vitro experiments in 21 cell lines (MTT, immunoblot analysis, plasmid transfection, reverse phase protein array [RPPA]) were carried out to characterize underlying mechanisms and potential biomarkers of response. All statistical tests were two-sided. MSC2363318A decreased tumor growth and metastases in multiple murine orthotopic models of ovarian (SKOV3ip1, HeyA8, and Igrov1) and uterine (Hec1a) cancer by reducing proliferation and angiogenesis and increasing cell death. Statistically significant prolonged overall survival was achieved with combination MSC2363318A and paclitaxel in the SKUT2 (endometrioid) uterine cancer mouse model ( P < .001). Mice treated with combination MSC2363318A and paclitaxel had the longest overall survival (mean = 104.2 days, 95% confidence interval [CI] = 97.0 to 111.4) compared with those treated with vehicle (mean = 61.9 days, 95% CI = 46.3 to 77.5), MSC2363318A alone (mean = 89.7 days, 95% CI = 83.0 to 96.4), and paclitaxel alone (mean = 73.6 days, 95% CI = 53.4 to 93.8). Regression and stabilization of established tumors in the Ishikawa (endometrioid) uterine cancer model was observed in mice treated with combination MSC2363318A and paclitaxel. Synergy between MSC2363318A and paclitaxel was observed in vitro in cell lines that had an IC50 of 5 µM or greater. RPPA results identified YAP1 as a candidate marker to predict cell lines that were most sensitive to MSC2363318A (R = 0.54, P = .02). After establishment of a murine ovarian cancer model of adaptive anti-angiogenic resistance (SKOV3ip1-luciferase), we demonstrate that resensitization to bevacizumab occurs with the addition of MSC2363318A, resulting in improved overall survival ( P = .01) using the Kaplan-Meier method. Mice treated with bevacizumab induction followed by MSC2363318A had the longest overall survival (mean = 66.0 days, 95% CI = 53.9 to 78.1) compared with mice treated with control (mean = 42.0 days, 95% CI = 31.4 to 52.6) and bevacizumab-sensitive mice (mean = 47.2 days; 95% CI = 37.5 to 56.9). MSC2363318A has therapeutic efficacy in multiple preclinical models of ovarian and uterine cancer. These findings support clinical development of a dual AKT/P70S6K inhibitor.

PD-1 or PD-L1 Blockade Restores Antitumor Efficacy Following SSX2 Epitope-Modified DNA Vaccine Immunization.

DNA vaccines have demonstrated antitumor efficacy in multiple preclinical models, but low immunogenicity has been observed in several human clinical trials. This has led to many approaches seeking to improve the immunogenicity of DNA vaccines. We previously reported that a DNA vaccine encoding the cancer-testis antigen SSX2, modified to encode altered epitopes with increased MHC class I affinity, elicited a greater frequency of cytolytic, multifunctional CD8(+) T cells in non-tumor-bearing mice. We sought to test whether this optimized vaccine resulted in increased antitumor activity in mice bearing an HLA-A2-expressing tumor engineered to express SSX2. We found that immunization of tumor-bearing mice with the optimized vaccine elicited a surprisingly inferior antitumor effect relative to the native vaccine. Both native and optimized vaccines led to increased expression of PD-L1 on tumor cells, but antigen-specific CD8(+) T cells from mice immunized with the optimized construct expressed higher PD-1. Splenocytes from immunized animals induced PD-L1 expression on tumor cells in vitro. Antitumor activity of the optimized vaccine could be increased when combined with antibodies blocking PD-1 or PD-L1, or by targeting a tumor line not expressing PD-L1. These findings suggest that vaccines aimed at eliciting effector CD8(+) T cells, and DNA vaccines in particular, might best be combined with PD-1 pathway inhibitors in clinical trials. This strategy may be particularly advantageous for vaccines targeting prostate cancer, a disease for which antitumor vaccines have demonstrated clinical benefit and yet PD-1 pathway inhibitors alone have shown little efficacy to date.

Discovery of VU0409551/JNJ-46778212: An mGlu5 Positive Allosteric Modulator Clinical Candidate Targeting Schizophrenia.

Herein, we report the structure-activity relationship of a novel series of (2(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridine-5(4H)-yl(aryl)methanones as potent, selective, and orally bioavailable metabotropic glutamate receptor subtype 5 (mGlu5) positive allosteric modulators (PAMs). On the basis of its robust in vitro potency and in vivo efficacy in multiple preclinical models of multiple domains of schizophrenia, coupled with a good DMPK profile and an acceptable therapeutic window, 17a (VU0409551/JNJ-46778212) was selected as a candidate for further development.

Therapeutic Efficacy of Suppressing the JAK/STAT Pathway in Multiple Models of Experimental Autoimmune Encephalomyelitis

Pathogenic Th cells and myeloid cells are involved in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The JAK/STAT pathway is used by numerous cytokines for signaling and is critical for development, regulation, and termination of immune responses. Dysregulation of the JAK/STAT pathway has pathological implications in autoimmune and neuroinflammatory diseases. Many of the cytokines involved in MS/EAE, including IL-6, IL-12, IL-23, IFN-γ, and GM-CSF, use the JAK/STAT pathway to induce biological responses. Thus, targeting JAKs has implications for treating autoimmune inflammation of the brain. We have used AZD1480, a JAK1/2 inhibitor, to investigate the therapeutic potential of inhibiting the JAK/STAT pathway in models of EAE. AZD1480 treatment inhibits disease severity in myelin oligodendrocyte glycoprotein-induced classical and atypical EAE models by preventing entry of immune cells into the brain, suppressing differentiation of Th1 and Th17 cells, deactivating myeloid cells, inhibiting STAT activation in the brain, and reducing expression of proinflammatory cytokines and chemokines. Treatment of SJL/J mice with AZD1480 delays disease onset of PLP-induced relapsing-remitting disease, reduces relapses and diminishes clinical severity. AZD1480 treatment was also effective in reducing ongoing paralysis induced by adoptive transfer of either pathogenic Th1 or Th17 cells. In vivo AZD1480 treatment impairs both the priming and expansion of T cells and attenuates Ag presentation functions of myeloid cells. Inhibition of the JAK/STAT pathway has clinical efficacy in multiple preclinical models of MS, suggesting the feasibility of the JAK/STAT pathway as a target for neuroinflammatory diseases.

Glutamate Carboxypeptidase II in Diagnosis and Treatment of Neurologic Disorders and Prostate Cancer

Glutamate carboxypeptidase II (GCPII) is a membrane-bound binuclear zinc metallopeptidase with the highest expression levels found in the nervous and prostatic tissue. Throughout the nervous system, glia-bound GCPII is intimately involved in the neuron-neuron and neuron-glia signaling via the hydrolysis of N-acetylaspartylglutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. The inhibition of the GCPII-controlled NAAG catabolism has been shown to attenuate neurotoxicity associated with enhanced glutamate transmission and GCPII-specific inhibitors demonstrate efficacy in multiple preclinical models including traumatic brain injury, stroke, neuropathic and inflammatory pain, amyotrophic lateral sclerosis, and schizophrenia. The second major area of pharmacological interventions targeting GCPII focuses on prostate carcinoma; GCPII expression levels are highly increased in androgen-independent and metastatic disease. Consequently, the enzyme serves as a potential target for imaging and therapy. This review offers a summary of GCPII structure, physiological functions in healthy tissues, and its association with various pathologies. The review also outlines the development of GCPII-specific small-molecule compounds and their use in preclinical and clinical settings.

Abstract 5047: Synergistic anti-cancer activity of the CDK4/6 inhibitor PD-0332991 in combination with 5-fluorouracil-based chemotherapy in human colon cancer cells

Abstract Background: The cell cycle activator cyclin-dependent kinase 4 (CDK4) is overexpressed in many cancers and is a promising target for novel anti-cancer therapies. PD-0332991 (Pfizer, Inc) is a highly specific CDK4/6 inhibitor that has proven anti-cancer efficacy in multiple pre-clinical models, and is currently undergoing Phase II clinical testing in humans. PD-0332991 inhibits tumor cell growth by inducing cell cycle arrest. Sequential therapy with PD-0332991 followed by DNA damaging cytotoxic chemotherapy could produce additive or synergistic anti-cancer effects. Our goal was to assess the anti-tumor efficacy of a combination of PD-0332991 and cytotoxic chemotherapy in vitro, and in vivo. We also sought to determine the mechanisms of anti-cancer activity seen with combination therapy. Methods: First, using human colorectal cancer (CRC) cell lines, we performed in vitro cell growth assays to determine the effects of a combination of PD-0332991 and 5-fluorouracil (5-FU) or oxaliplatin. Next, we tested the activity of PD-0332991, oral capecitabine (an oral 5-FU prodrug), or both in mice bearing human CRC xenografts. Finally, we assessed the mechanisms of cell death in vitro and in resected xenograft tumor samples. Results: The combination of PD-0332991 and 5-FU or oxaliplatin exhibited additive/synergistic anti-cancer activity, in vitro. Furthermore, synergistic anti-cancer activity was observed in mice bearing xenografts of HCT 116 human CRC cells upon treatment with a combination of PD-0332991 and capecitabine. Virtually no anti-tumor activity was observed in mice treated with PD-0332991 or capecitabine alone. In assessing the mechanism of anti-tumor activity, in vitro and in resected xenograft samples, no increase in apoptosis was observed, as determined by annexin V and TUNEL staining. Even when cells were synchronized in G1 with PD-0332991 and then released and treated with 5-FU or oxaliplatin, there was no increase in apoptosis. Thus, assessment of alternative mechanisms of cell death, including cell senescence and autophagy are ongoing. Conclusions: Our results demonstrate an additive/synergistic anti-tumor activity with the combination of PD-0332991 and traditional cytotoxic chemotherapy. The mechanisms of anti-tumor activity have yet to be determined, but are not dependent upon apoptosis. Given the synergistic anti-tumor activity, these results suggest that a clinical trial of PD-0332991 and 5-FU-based chemotherapy is warranted. 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 5047.

Targeting leukocyte integrins in human diseases

As our understanding of integrins as multifunctional adhesion and signaling molecules has grown, so has their recognition as potential therapeutic targets in human diseases. Leukocyte integrins are of particular interest in this regard, as they are key molecules in immune-mediated and inflammatory processes and are thus critically involved in diverse clinical disorders, ranging from asthma to atherosclerosis. Antagonists that interfere with integrin-dependent leukocyte trafficking and/or post-trafficking events have shown efficacy in multiple preclinical models, but these have not always predicted success in subsequent clinical trials (e.g., ischemia-reperfusion disorders and transplantation). However, recent successes of integrin antagonists in psoriasis, inflammatory bowel disease, and multiple sclerosis demonstrate the tremendous potential of antiadhesion therapy directed at leukocyte integrins. This article will review the role of the leukocyte integrins in the inflammatory process, approaches to targeting leukocyte integrins and their ligands, and the results of completed clinical trials.

Inhibition of lipid peroxidation in central nervous system trauma and ischemia

A novel group of compounds, the 21-aminosteroids (“lazaroids”), have been designed that are potent inhibitors of oxygen free radical-induced, iron-catalyzed lipid peroxidation (LP) in microvascular and nervous tissue. One of these, tirilazad mesylate (U-74006F), has been selected for clinical evaluation as a cerebroprotective agent. In vitro studies suggest that tirilazad exerts its antioxidant activity by multiple mechanisms including: increasing membrane stability, scavenging of lipid peroxyl radicals, reducing LP-induced arachidonic acid release, decreased formation or scavenging of hydroxyl radicals, and maintenance of the levels of endogenous vitamin E. The major site of action appears to be the blood—brain barrier based upon its known localization in cerebrovascular endothelium and numerous studies showing an attenuation of subarachnoid hemorrhage (SAH), injury, and ischemia-induced blood—brain barrier permeability. Tirilazad has demonstrated neuroprotective efficacy in multiple preclinical models of spinal cord and head injury, SAH, and focal cerebral ischemia, as measured by a decrease in cerebral vasospasm, blood—brain barrier compromise, post-traumatic ischemia, edema, ischemic neuronal necrosis and infarction, and improved neurological recovery. This efficacy is correlated with a reduction in markers of oxygen radical-induced LP. Phase III clinical trials are currently ongoing in spinal cord and head injury, SAH, and ischemic stroke. Initial results from a European/Australian/New Zealand trial in SAH have shown a significant decrease in mortality and an increase in the incidence of good recovery.