Activity In Preclinical Tumor Models Research Articles

Abstract 822: Thienoindoles, a novel class of DNA minor groove alkylating agents highly suited for the generation of novel antibody drug conjugates (ADCs)

Abstract The rapidly growing field of Antibody-Drug Conjugates (ADCs) has recently spurred the study of novel drug payloads. In particular, much interest has been paid to the identification of novel cytotoxic agents, which differ in mechanism from anti-tubulin agents, currently the most commonly employed class of drug for ADC coupling. Novel drug payloads for ADC-based therapy should thus ideally possess highly potent cytotoxic activity with a diverse mechanism from tubulin agents, as well as physicochemical properties, which facilitate coupling to antibodies. Duocarmycins are a widely-described class of DNA minor groove binding alkylating agents, examples of which can exhibit picomolar activity against human tumor cell lines, as well as high activity in preclinical tumor models. The clinical development of duocarmycins as drugs in their own right has however been hampered by an extremely limited therapeutic window. Duocarmycins also typically exhibit poor physicochemical profiles, most importantly generally possessing limited aqueous solubility and a propensity to induce protein aggregation, which renders these molecules poorly tractable as optimal ADC payloads. Based on the premise that opportune modification of the duocarmycin scaffold could lead to the discovery of compounds better suited to antibody conjugation, we describe the identification and related proof of concept studies for a novel chemical series based on a proprietary thienoindole scaffold, characterized both by potent antitumor activity and by physicochemical properties that are highly compatible with deployment as antibody payloads. Extensive in vitro profiling within the thienoindole series led to selection of potent cytotoxic compounds suitable for test conjugation to humanized monoclonal antibodies, which were then submitted to in vitro/in vivo profiling. Typically, average drug/antibody ratios (DARs) of 4 could readily be achieved without induction of antibody aggregation/precipitation and without affecting natural antibody-ligand affinity. Efficacy and mechanism of action studies were carried out for thienoindole-ADCs prepared with trastuzumab and with rituximab showing target driven effects in Her2 positive vs. negative breast cancer cell lines and against CD20 positive vs. negative hematological cell lines. PK data indicated long, essentially unaffected plasma half life of our novel ADCs in the mouse. In vivo efficacy studies in the Her2-positive HCC1954 human breast cancer model showed complete tumor regression in mice treated with trastuzumab-ADC with no effects on body weight gain, while unarmed trastuzumab and armed control antibody had little and no effect, respectively. Citation Format: Barbara Valsasina, Fabio Gasparri, Italo Beria, Nicoletta Colombo, Paolo Orsini, Rita Perego, Simona Rizzi, Ulisse Cucchi, Clara Albanese, Aurelio Marsiglio, Ivan Fraietta, Marina Ciomei, Sabrina Cribioli, Carlo Visco, Eduard R. Felder, Antonella Isacchi, Enrico A. Pesenti, Arturo Galvani, Daniele Donati, Michele Caruso. Thienoindoles, a novel class of DNA minor groove alkylating agents highly suited for the generation of novel antibody drug conjugates (ADCs). [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 822. doi:10.1158/1538-7445.AM2014-822

A PLK4 inhibitor has single-agent activity in preclinical tumor models.

CFI-400945 is a potent, selective, orally bioavailable PLK4 inhibitor with antitumor activity in vivo.

Abstract 676: Dinitroazetidines are a novel class of anticancer agents and hypoxia-activated radiation sensitizers developed from highly energetic materials

Abstract Here we report the first biologic studies of a unique class of potentially clinically useful compounds, derived from highly energetic materials. In an effort to develop unique targeted cancer therapies which maximize cytotoxicity to cancer cells while minimizing unwanted side effects to normal tissue, we have studied a series of novel, non-explosive compounds, based on the highly energetic dinitroazetidine scaffold. ABDNAZ, 1-acetylbromo-3,3-dinitroazetidine is a lead compound for clinical development both as a stand-alone chemotherapeutic drug and as a radiation sensitizer. In vitro studies demonstrated that ABDNAZ generated reactive oxygen species (ROS) in a concentration- and time-dependent manner. Modulation of intracellular redox status by buthionine-(S,R)-sulfoxime or N-acetylcysteine significantly affected ABDNAZ-induced apoptosis of tumor cells. ABDNAZ administered as a single agent was as cytotoxic to tumor cells as cisplatin with IC50s of 2.6 ± 1.6uM and 4.4 ± 2.2uM for ABDNAZ and cisplatin, respectively. ABDNAZ was more cytotoxic to tumor cells than cisplatin or tirapazamine under hypoxic conditions. ABDNAZ and cisplatin had similar effects on well established SCC VII tumors in mice, but unlike cisplatin, ABDNAZ at well-tolerated sub-maximum tolerated doses (MTD) significantly inhibited tumor growth without systemic toxicity. When combined with local tumor radiation therapy, ABDNAZ significantly enhanced the efficacy of the radiation and improved the therapeutic index of radiation therapy. Toxicity studies demonstrated that ABDNAZ was not myelosuppressive and had no apparent dose limiting toxicity when administered at sub-MTD dose daily for 14 days. These data demonstrate that ABDNAZ is a promising novel anticancer drug with a very favorable toxicity profile at therapeutic doses with activity in preclinical tumor models as both a stand-alone chemotherapeutic agent and as a radiosensitizer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 676. doi:10.1158/1538-7445.AM2011-676

Abstract LB-406: Phase 1 study of the Smac mimetic TL32711 in adult subjects with advanced solid tumors and lymphoma to evaluate safety, pharmacokinetics, pharmacodynamics, and antitumor activity

Abstract Introduction: TL32711 is a small molecule Smac mimetic that potently and specifically antagonizes inhibitor of apoptosis proteins (IAPs), resulting in caspase-dependent apoptosis and inactivation of NF-kB signaling. In preclinical studies, single agent tumor regression was observed for multiple tumor types and potent anti-tumor activity was observed when TL32711 was combined with specific chemotherapies and death receptor ligands. This first-in-human study assesses the safety, pharmacokinetic (PK), and pharmacodynamic (PD) profile and anti-tumor activity of single agent TL32711. Methods: Using a standard 3+3 dose escalation design, subjects with refractory solid tumors and lymphoma received weekly intravenous TL32711 for 3 weeks on/ 1 week off, with disease assessment every 2 cycles. PK analysis was conducted using an intensive sampling approach. PD assays included measurement of cIAP1 and cIAP2 levels in lysates from serially collected PBMC and tumor tissue, serum levels of cleaved cytokeratin-18 (CK-18, requiring activated caspase-3) and activated caspase-3/7. Results: 27 patients were treated at doses 0.18–26 mg/m2 over 9 cohorts. TL32711 was well-tolerated, with no dose-limiting toxicities. Toxicities included reversible grade 2 lymphocytopenia and grade 1 rash at ≥11.5 mg/m2. TL32711 exhibited dose-proportional, uniform, non-accumulating PK, with a mean β-phase half-life of 35 hrs. Doses ≥2.88 mg/m2 achieved an AUC0-∞ exposure sufficient for single agent activity in preclinical tumor models. At doses ≥1.44 mg/m2, cIAP1 levels were suppressed >80% within 24 hrs and after 1 week >50% suppression was maintained. Based on PBMC drug levels, actual and predicted cIAP1 suppression was well-correlated. A dose-dependent increase in serum levels of cleaved CK-18 and activated caspase-3/7 was observed in patients treated at doses ≥2.88 mg/m2. At 11.5 mg/m2, > 90% cIAP1 suppression, increased activated caspase-8, and PARP cleavage in tumor lysates were observed in a tumor biopsy from a melanoma patient with progressive disease prior to TL32711, with stable disease after 2 cycles. One colon cancer subject with progressive disease after prior therapies, at 0.36 mg/m2 demonstrated tumor shrinkage in radiographic lesions, serum CEA decline, and elevated serum caspase-3/7. One colon cancer subject at 17.2 mg/m2 demonstrated serum CEA decline, elevated serum caspase-3/7, and a large photopenic lesion in a metastatic lesion within the first cycle. Conclusions: TL32711 is well-tolerated, has dose-proportional PK, and demonstrates potent and sustained target inhibition and apoptotic pathway activation in tumor and surrogate tissues. Preliminary evidence of antitumor activity was observed in colon cancer and melanoma. Updated results of final dose escalation cohorts will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-406. doi:10.1158/1538-7445.AM2011-LB-406

Implication of Heat Shock Protein 90 (HSP90) in Tumor Angiogenesis: A Molecular Target for Anti-Angiogenic Therapy?

The inhibition of oncogenic signaling pathways has gained great interest for cancer therapy. In this context, the molecular chaperone heat shock protein 90 (HSP90) has emerged as a promising molecular target, since it is critically involved in maintaining stability, integrity and functions of key oncogenic proteins. A variety of HSP90 inhibitors have been developed in the past decade and have shown convincing anti-neoplastic activity in pre-clinical tumor models. Importantly, HSP90 inhibitors are predominantly being recognized as "tumor cell targeting" agents since cancer cells a) overexpress HSP90 protein, b) highly rely on HSP90 function for maintaining oncogenic signaling, and c) HSP90 inhibitors bind with high affinity to HSP90 in tumor cells. Nevertheless, results from recent studies also suggest that HSP90 inhibitors elicit anti-angiogenic properties by affecting the PI-3K/Akt/eNOS signal transduction pathway in endothelial cells, as well as through down-regulation of VEGFR-2 expression, a crucial component of the angiogenic process. In addition, blocking HSP90 may also diminish the secretion and expression of tumor cell-derived pro-angiogenic growth factors and cytokines, thus leading to "indirect" anti-angiogenic effects. This review article focuses on the role of HSP90 in angiogenesis and on delineating the effects of HSP90 inhibitors on angiogenic signaling pathways involved in tumor vascularization.

376 Preliminary results from a Phase 1 study of D-3263 HCl, a TRPM8 calcium channel agonist, in patients with advanced cancer

D-3263 HCl is a calcium channel agonist that induces calcium influx and subsequent apoptosis in cells that express TRPM8. TRPM8 was originally discovered as a gene expressed in the prostate and upregulated in prostate cancer, and was subsequently identified as the cold-menthol receptor (Tsavaler 2001). D3263-HCl, an orally administered small molecule agonist, showed evidence of activity in preclinical tumor models. BACKGROUND

Vorinostat, a histone deacetylase inhibitor, combined with pelvic palliative radiotherapy for gastrointestinal carcinoma: the Pelvic Radiation and Vorinostat (PRAVO) phase 1 study

Histone deacetylase (HDAC) inhibitors have shown radiosensitising activity in preclinical tumour models. This phase 1 study assessed the use of vorinostat combined with pelvic palliative radiotherapy for gastrointestinal carcinoma. Between Feb 14, 2007, and May 18, 2009, eligible patients with histologically confirmed carcinoma, scheduled to receive pelvic palliative radiation to 30 Gy in 3 Gy daily fractions over 2 weeks, were enrolled into cohorts of escalating vorinostat dose. Vorinostat was administered orally once daily, 3 h before each radiotherapy fraction, at the following dose levels: 100 mg (n=1), 200 mg (n=4), 300 mg (n=6), and 400 mg (n=6). Endpoints included safety, tolerability, and biological activity (tumour histone acetylation). This study is registered with ClinicalTrials.gov, number NCT00455351. One patient withdrew consent after one treatment day, leaving 16 patients evaluable for tolerability. Most recorded adverse events were grade 1 and 2, among which fatigue (all patients) and gastrointestinal events (all patients) were most common. Grade 3 adverse events included fatigue (n=5), anorexia (n=3), diarrhoea (n=2), hyponatraemia (n=1), hypokalaemia (n=1), and acneiform rash (n=1). Of these, treatment-related grade 3 events (ie, dose-limiting toxicities) were observed in one of six patients at vorinostat 300 mg once daily (fatigue and anorexia), and in two of six patients at vorinostat 400 mg once daily (two events of diarrhoea and one each of fatigue, anorexia, hyponatraemia, and hypokalaemia). The maximum-tolerated dose of vorinostat in combination with palliative radiotherapy was thus determined to be 300 mg once daily. Histone hyperacetylation was detected, indicating biological activity of vorinostat. Vorinostat can be safely combined with short-term pelvic palliative radiotherapy. This study highlights the potential use of HDAC inhibitors with radiation, and suggests investigation of vorinostat in long-term curative pelvic radiotherapy--eg, as a component of preoperative chemoradiotherapy for rectal cancer. Merck & Co, Inc, Norwegian Cancer Society, Norwegian Health and Rehabilitation Foundation.

Abstract C106: Discovery and characterization of INCB024360, a potent and selective inhibitor of indoleamine 2,3-dioxygenase (IDO1) as a novel agent for cancer immunotherapy

Abstract Indoleamine 2,3-dioxygenase (IDO or IDO1) mediates the oxidation of tryptophan, an amino acid essential for cell proliferation and survival. Inhibition of IDO activity or expression has shown therapeutic potential in preclinical models of immunodeficiency-associated abnormalities including cancer. Here we report the identification of INCB024360, a novel, potent and selective small molecule inhibitor of IDO1, and the investigation of its effects on the proliferation and activation of various immune cells in vitro as well as its anti-tumor activity in preclinical tumor models. In multiple cell-based assays, INCB024360 potently inhibits human IDO1 with an IC50 of approximately 10 nM - the most potent IDO1 inhibitor published thus far. INCB024360 is IDO1 selective and has little activity against other related enzymes including IDO2, TDO or tryptophan transporters. In co-culture systems of human allogeneic lymphocytes with either dendritic cells or tumor cells, inhibition of IDO1 by INCB024360 increases the proliferation of T and NK cells, as well as IFN- production. INCB024360 addition also reduces the generation of regulatory T cells. Interestingly, IDO1 expression promotes dendritic cell apoptosis while addition of INCB024360 reverses this and increases the number of CD86high cells within the IDO+ DC population, potentially representing a novel mechanism by which IDO1 inhibition can promote T cell activation. In vivo, INCB024360 inhibits tryptophan catabolism in tumors and tumor draining lymph nodes and controls tumor growth either alone or in combination with chemotherapeutic agents in multiple tumor models. The ability to reduce tumor growth is dependent on a functional immune system, consistent with the proposed mechanism of action. Finally, an analysis of kynurenine/tryptophan levels in patient blood samples affirms that the IDO activity is elevated in multiple tumor types. Collectively, these data suggest that INCB024360, a potent IDO1 selective inhibitor, has the potential to be a novel and effective immunotherapeutic agent for cancer treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C106.

Membrane type matrix metalloproteinases (MMPs) show differential expression in non-small cell lung cancer (NSCLC) compared to normal lung: Correlation of MMP-14 mRNA expression and proteolytic activity

Improved understanding of the involvement of matrix metalloproteinases (MMPs), including membrane-type MMPs (MT-MMPs), in human tumours has potential diagnostic, prognostic and therapeutic implications. We assessed the relationship between MT-MMP expression and clinicopathological parameters in human non-small cell lung cancer (NSCLC) and histologically normal lung tissue by quantitative Real Time PCR (qRT-PCR). All MT-MMPs (MMPs 14–17, 24 and 25) were detected by qRT-PCR with significantly higher MMP-14, -15 and -17 expression observed in tumour relative to normal lung specimens. MMP-16 was undetectable in normal lung but expressed in 8% tumours. MMP-15 demonstrated significant overexpression in adenocarcinomas relative to squamous cell carcinomas and normal lung tissue. MMP-14 mRNA expression strongly correlated to MMP-14 proteolytic activity in preclinical tumour models, indicating that qRT-PCR may predict MMP-14 activity levels in NSCLC. These data suggest that MMP-14, -15 and -17 may be good markers of disease, or therapeutic targets for treatment of human NSCLC.

Phase I study of CYT997, a novel cytotoxic and vascular disrupting agent, given as a 24-hour intravenous infusion to patients with advanced solid tumours

14115 Background: CYT997 is a novel tubulin-binding small molecule which inhibits microtubule assembly and also demonstrates potent vascular-disrupting activity in preclinical tumour models. Methods: CYT997 was administered by continuous infusion over 24 hours every 3 weeks to patients with advanced cancer. Dose escalation proceeded by a standard phase I design (3 patients per dose level) for the first 18 patients; subsequently, an accelerated titration design (1 patient per dose level) was utilized. Intrapatient dose escalation was permitted. Pharmacokinetic (PK) analyses were performed in the first cycle. Tumour response was determined every second cycle using RECIST criteria. Pharmacodynamic effects on the tumour vasculature were assessed with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Results: 22 patients (M/F: 11/11; median age 57.5, range 28–75) were enrolled with tumour types including melanoma (4), renal cell (4), colorectal (2), non-small cell lung (2) and adenoid cystic (2) carcinomas, mesothelioma (2) and others (6). A total of 66 cycles of CYT997 were administered (median 2/patient, range 1–6) over 10 dose levels (7, 14, 23, 35, 49, 65, 86, 114, 152 and 202 mg/m2). No dose-limiting toxicity was observed. Because of grade-2 injection site reactions in 2 patients (one each at dose levels 3 and 4), all subsequent patients received CYT997 via a central venous access device. Other toxicities included grade-2 renal toxicity at dose- level 8 in one patient with abnormal baseline renal function and grade-1 QTc prolongation in one patient at dose-level 10. No myelosuppression, gastrointestinal toxicity or clinically-significant cardiac toxicity were observed. PK data revealed dose-related increases in Cmax and AUC values. Six patients had stable disease after 4 cycles of CYT997. Conclusions: CYT997 was well tolerated at the doses studied and accrual to the 269 mg/m2 dose level will now proceed. No significant financial relationships to disclose.

Discovery of a Fluoroindolo[2,3-a]carbazole Clinical Candidate with Broad Spectrum Antitumor Activity in Preclinical Tumor Models Superior to the Marketed Oncology Drug, CPT-11

A series of fluoroglycosylated fluoroindolocarbazoles was examined with respect to their topoisomerase I activity, cytotoxicity, and selectivity. The lead clinical candidate from this series, BMS-250749, displays broad spectrum antitumor activity superior to CPT-11 against some preclinical xenograft models, including curative antitumor activity against Lewis lung carcinoma.

TLR9-targeted CpG immunostimulatory treatment of metastatic melanoma: A phase II trial with CpG 7909 (ProMune)

7513 Background: Malignant melanoma is widely accepted as an immunologically responsive tumor, but large recently reported chemobiotherapy trials have been disappointing and prognosis is poor. The new synthetic oligodeoxynucleotide CpG 7909 activates plasmacytoid dendritic cells (pDC) and B cells through specific interaction with Toll-like receptor 9 (TLR9) and is a strong activator of both innate and specific immunity. CPG 7909 crossreacts with mouse TLR9 and has shown impressive antitumor activity in preclinical tumor models when used as monotherapy. Methods: Patients with confirmed metastatic melanoma (stage IV without CNS metastases) were enrolled into an open-label, multicenter, single arm study. Pts received 6 mg CPG 7909 weekly by SC injection for 24 weeks or until disease progression in an outpatient setting. Disease status was assessed at screening and weeks 8, 16, and 24 according to RECIST. Results: 20 pts (5 female, 15 male, aged between 37 and 74) were enrolled. Two pts achieved a confirmed p...

TLR9-targeted CpG immunostimulatory treatment of metastatic melanoma: A phase II trial with CpG 7909 (ProMune)

7513 Background: Malignant melanoma is widely accepted as an immunologically responsive tumor, but large recently reported chemobiotherapy trials have been disappointing and prognosis is poor. The new synthetic oligodeoxynucleotide CpG 7909 activates plasmacytoid dendritic cells (pDC) and B cells through specific interaction with Toll-like receptor 9 (TLR9) and is a strong activator of both innate and specific immunity. CPG 7909 crossreacts with mouse TLR9 and has shown impressive antitumor activity in preclinical tumor models when used as monotherapy. Methods: Patients with confirmed metastatic melanoma (stage IV without CNS metastases) were enrolled into an open-label, multicenter, single arm study. Pts received 6 mg CPG 7909 weekly by SC injection for 24 weeks or until disease progression in an outpatient setting. Disease status was assessed at screening and weeks 8, 16, and 24 according to RECIST. Results: 20 pts (5 female, 15 male, aged between 37 and 74) were enrolled. Two pts achieved a confirmed partial response (PR) and one has maintained this response with continuing therapy for over 13 months. Three pts achieved stable disease (SD). CPG 7909 was well tolerated. Adverse events included transient injection site reactions (erythema, swelling, induration), fever and arthralgias. Hematological and non-hematological toxicities were limited, transient and did not result in any withdrawals. Phenotyping of PBMC revealed activation of pDC under CPG 7909 therapy consistent with the mode of action and pts exhibiting PR or SD could be distinguished from non-R pts by differential dynamics in NK cell-cytotoxicity (1.6-fold increase vs. 1.7-fold decrease in lytic units, p<0.05) during the first 8 weeks of treatment. Conclusions: CPG 7909 exerts anti-tumor activity in pts with metastatic melanoma, can be administered safely and induces a phenotypic signature in PBMC associated with exposure and, possibly, response to therapy. A randomised phase II/III trial has been initiated to compare efficacy and safety of two dose levels of CPG 7909, CPG 7909 in combination with DTIC, and DTIC alone. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Coley Pharmaceutical Group Coley Pharmaceutical Group Coley Pharmaceutical Group Coley Pharmaceutical Group Coley Pharmaceutical Group

Emerging drug treatments for solid tumours.

A number of novel anticancer agents have emerged during the past few decades, which show high activity in preclinical tumour models and promising activity in early trials in patients with solid tumours. Most of the agents have novel and unique mechanisms of action, and show activity against a variety of malignancies, including tumours which are notoriously resistant to systemic treatment. Recently, our understanding of the molecular basis of cancer has increased considerably. This is reflected in the development of agents that are directed at well defined molecular targets, such as the mitotic tubulin/microtubuli system (taxoids), nuclear enzymes (topoisomerase I inhibitors) and cell signal transduction pathways (protein kinase C inhibitors). In addition, significant advances have been made in our understanding of mechanisms of toxicity, especially of cisplatin. This has resulted in the development of agents modulating cisplatin toxicity, among which amifostine (WR-2721) is one of the most promising. The outlined emerging drug therapies with novel anticancer agents and treatment modalities will, it is hoped, result in increased response rates of advanced tumours, longer disease-free and total survival and better palliative care.

Synthesis of water-soluble (aminoalkyl)camptothecin analogs: inhibition of topoisomerase I and antitumor activity

Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.