Picomolar Potency Research Articles

Abstract 695: The generation of a mono-selective CDK7 inhibitor.

Abstract Cyclin-depending kinase (CDK) family members that trigger passage through the cell cycle have been considered as attractive cancer targets for at least two decdes Yet, the first generation of pan-specific CDK inhibitors failed to show clinical efficacy due to their small therapeutic window. CDK family members that control processes such as transcription have caught less attention, although experimental evidence for an involvement in different pathological processes is emerging. CDK7 (or CAK) has essential roles in both the cell-division cycle and transcription, and serves as a direct link between the regulation of basal transcription and the cell cycle. In addition to its role as a transcription activator, CDK7 has been reported to act on specific transcription events by phosphorylating a series of specific transcription factors, such as the estrogen receptor and p53. These findings indicate that CDK7 is a potential target for cancer therapy, and perhaps inflammation. The detailed molecular characterization of the biological role of CDK7, as well as its therapeutic validation, is dependent on the development of highly specific and potent CDK7 inhibitors. We have developed a series of CDK7 inhibitors with picomolar potency on CDK7, more than 1000-fold selectivity over any other CDK, and single digit nM inhibition of sensitive cancer cell lines. Interestingly, a broad screen of >200 different tumor cell lines lead to the identification of responder and non-responder cell lines. Non-responder tumor lines, similar to primary cells are at least by a factor of 1000 less sensitive to the inhibition with the mono-selective CDK7 inhibitor. Our findings further indicate that CDK7 is neither essential for general transcription, nor for the progression through the cell cycle. It rather coordinates cell line specific biological processes. The effects of selective CDK7 inhibitors on tumor cell lines are clearly distinct from existing, nonselective pan-CDK inhibitors. The responder status for a subset of cancer cell lines can be predicted with a distinct biomarker. Chemical optimization of the selective inhibitor lead to a compound which shows dose-dependent pharmacological efficacy in mouse xenograft models after oral administration, with no signs of toxicity at the efficacious doses. Citation Format: Jan E. Eickhoff, Gunther Zischinsky, Axel Choidas, Peter Habenberger, Alexander Wolf, Carsten Degenhardt, Matthias Baumann, Anke Unger, Ahmad Ghallab, Doreen Werchau, Jan Hengstler, Peter Nussbaumer, Bert M. Klebl. The generation of a mono-selective CDK7 inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 695. doi:10.1158/1538-7445.AM2013-695

Pharmacological Characterization of GSK573719 (Umeclidinium): A Novel, Long-Acting, Inhaled Antagonist of the Muscarinic Cholinergic Receptors for Treatment of Pulmonary Diseases

Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.2]octane; umeclidinium). The affinity (Ki) of GSK573719 for the cloned human M1-M5 mAChRs ranged from 0.05 to 0.16 nM. Dissociation of [(3)H]GSK573719 from the M3 mAChR was slower than that for the M2 mAChR [half-life (t1/2) values: 82 and 9 minutes, respectively]. In Chinese hamster ovary cells transfected with recombinant human M3 mAChRs, GSK573719 demonstrated picomolar potency (-log pA2 = 23.9 pM) in an acetylcholine (Ach)-mediated Ca(2+) mobilization assay. Concentration-response curves indicate competitive antagonism with partial reversibility after drug washout. Using isolated human bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA2 = 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED50 value was 0.02 μg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 μg elicited 50% bronchoprotection for >24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.

Picomolar inhibition of cholera toxin by a pentavalent ganglioside GM1os-calix[5]arene

Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells. Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding domain (CTB). This pentavalent inhibitor contains five GM1os moieties linked to a calix[5]arene scaffold. When evaluated by an inhibition assay, it achieved a picomolar inhibition potency (IC50 = 450 pM) for CTB. This represents a significant multivalency effect, with a relative inhibitory potency of 100,000 compared to a monovalent GM1os derivative, making GM1os-calix[5]arene one of the most potent known CTB inhibitors.

β‐D‐Glucosyl Conjugates of Highly Potent Inhibitors of Blood Coagulation Factor Xa Bearing 2‐Chorothiophene as a P1 Motif

We synthesized a novel O-glucoside of the recently reported potent factor Xa (fXa) inhibitor 1, which bears a 5-chlorothien-2-yl moiety and 1-isopropylpiperidine as fragments that bind the S1 and S4 enzyme pockets, respectively. A β-D-glucosyl unit was conjugated through an ether-linked C3-alkyl spacer to the central phenyl ring of 1. The synthesized β-D-glucose-based compound 16 achieved picomolar inhibitory potency against human fXa (K(i)=60 pM) and high selectivity over thrombin and other serine proteases. In addition to the chlorothienyl S1 binder, a large gain in ΔG resulted from the addition of protonated 1-isopropylpiperidine (ΔΔG=29.7-30.5 kJ mol(-1)), which should bind to the aromatic S4 pocket through efficient cation-π and C-H···π interactions. Instead, the C3-alkyl-linked glucose fragment, which is likely directed toward the solvent outside the enzyme binding site, improves ΔG by an average of 2.9-3.8 kJ mol(-1) . Compound 16 showed sub-micromolar in vitro anticoagulant activity, as assessed by prothrombin time (PT) and activated thromboplastin time (aPTT) clotting assays in pooled human plasma (PT(2) and aPTT(2) equal to 0.135 and 0.389 μM, respectively). Although compound 16 was 1.4-fold less active than parent compound 1 in the ex vivo anticoagulant assay in mice, it showed a significant (1.6-fold) prolongation of PT relative to controls (P<0.05) 60 min after oral dosing (75 mg kg(-1)).

ChemInform Abstract: Discovery of Oxazole‐Based PDE4 Inhibitors with Picomolar Potency.

AbstractA series of quinolyloxazoles with 4‐benzylcarboxamide and 5‐α‐aminoethyl groups, which show picomolar potency against PDE4, is discovered.

Protection Against Clostridium difficile Infection With Broadly Neutralizing Antitoxin Monoclonal Antibodies

The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50 and antitoxin B mAb PA-41 have picomolar potencies in vitro and bind to novel regions of the respective toxins. In a hamster model for CDI, 95% of animals treated with a combination of humanized PA-50 and PA-41 showed long-term survival relative to 0% survival of animals treated with standard antibiotics or comparator mAbs. These humanized mAbs provide insight into C. difficile intoxication and hold promise as potential nonantibiotic agents for improving clinical management of CDI.

Bi-specific TCR anti-CD3 fusions for cancer immunotherapy (53.19)

Abstract Although cancer patients frequently mount antigen specific T cell responses, the low affinity of T cell Receptors (TCRs) to self-antigens coupled with low levels of HLA-peptide on cancer cells contribute to the failure of the immune system to clear tumours. Peptide epitopes presented by Class I HLA represent the largest class of tumor associated antigens and are ideal targets for cancer immunotherapy, providing that the issues of low TCR affinity and immune-suppression in the tumor microenvironment can be overcome. The most attractive targets for minimising toxicity are expressed on a restricted number of normal tissue cells, for example cancer testis antigens. We have engineered ImmTAC reagents comprising soluble, high affinity TCRs fused to an anti-CD3 scFv domain that redirects a potent, polyclonal T cell response to kill tumour cells expressing as few as 10-20 epitopes. IMCgp100 targets the HLA-A2 presented epitope gp100 280-288 and kills malignant melanoma cells with pico-Molar potency, leaving other HLA-A2 positive cells untouched. This agent is undergoing clinical testing in a Phase I dose-escalation trial and is well tolerated. A second ImmTAC, IMCmage1, targets MAGE-A3 168-176 in the context of HLA-A1. It specifically kills the myeloma population within CD138+ cells extracted from the marrow of a stage III multiple myeloma patient and can re-direct T cells extracted from the tumor microenvironment. IMCmage1 will enter the clinic in 2012.

Inhibition of hepatitis C virus NS5A by fluoro-olefin based γ-turn mimetics

The HCV non-structural protein NS5A has been established as a viable target for the development of direct acting antiviral therapy. From computational modeling studies strong intra-molecular hydrogen bonds were found to be a common structural moiety within known NS5A inhibitors that have low pico-molar replicon potency. Efforts to reproduce these γ-turn-like substructures provided a novel NS5A inhibitor based on a fluoro-olefin isostere. This fluoro-olefin containing inhibitor exhibited picomolar activity (EC50=79pM) against HCV genotype 1b replicon without measurable cytotoxicity. This level of activity is comparable to the natural peptide-based inhibitors currently under clinic evaluation, and demonstrates that a peptidomimetic approach can serve as a useful strategy to produce potent and structurally unique inhibitors of HCV NS5A.

Discovery of oxazole-based PDE4 inhibitors with picomolar potency

Optimization of oxazole-based PDE4 inhibitors has led to the discovery of a series of quinolyl oxazoles, with 4-benzylcarboxamide and 5-α-aminoethyl groups which exhibit picomolar potency against PDE4. Selectivity profiles and in vivo biological activity are also reported.

Pyridyl aminothiazoles as potent inhibitors of Chk1 with slow dissociation rates

Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region.

Anti-Obesity Effect of Shk-186, a K+ Channel Blocker

Obesity is a global health problem and there is a pressing need for safe therapeutics. Here, we describe the anti-obesity effects of ShK-186, a Kv1.3-selective peptide inhibitor with picomolar potency and an excellent safety profile in rodents and monkeys. C57BL/J mice fed a diet containing high fat (21%) diet and fructose solution (60%) were administered ShK-186 (20, 100 or 500 μg/kg) or vehicle on alternate days by subcutaneous injection. ShK-186-treatment caused a dose-dependent reduction in weight gain over the 45-day trial, the maximal weight difference being 30%. ShK-186-treatment reduced plasma levels of triglyceride and free fatty acids without affecting cholesterol, HDL, LDL or glucose. CT scan revealed significant reduction in volume of white adipose tissue (WAT) in ShK-186-treated mice compared to vehicle-controls. However, histology revealed no difference in cell size and fat content of white fat cells. In metabolic cage experiments, ShK-186-treatment increased cumulative intake of food and fluid compared to vehicle-treated mice, especially during the light-cycle. Furthermore, their respiratory exchange ratio and energy expenditure were also higher. In PET experiments, ShK-186 treatment increased uptake of 18-fluoro-deoxyglucose (18FDG) by brown adipose tissue (BAT) and skeletal muscle compared to vehicle-controls; pre-administration of the beta-adrenoceptor blocker propranolol (5mg/kg) inhibited 18FDG uptake by BAT but not skeletal muscle. ShK-186's anti-obesity effect appears to be mediated by activation of BAT and/or skeletal muscle, two important thermogenic tissues in the body.

6-Position optimization of tricyclic 4-quinolone-based inhibitors of glycogen synthase kinase-3β: Discovery of nitrile derivatives with picomolar potency

We previously disclosed tricylic, 6-carboxylic acid-bearing 4-quinolones as GSK-3β inhibitors. Herein we discuss the optimization of this series to yield a series of more potent 6-nitrile analogs with insignificant anti-microbial activity. Finally, kinase profiling indicated that members of this class were highly specific GSK-3 inhibitors.

Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS-790052 in Humans: In Vitro and In Vivo Correlations

The NS5A replication complex inhibitor, BMS-790052, inhibits hepatitis C virus (HCV) replication with picomolar potency in preclinical assays. This potency translated in vivo to a substantial antiviral effect in a single-ascending dose study and a 14-day multiple-ascending dose (MAD) monotherapy study. However, HCV RNA remained detectable in genotype 1a-infected patients at the end of the MAD study. In contrast, viral breakthrough was observed less often in patients infected with genotype 1b, and, in several patients, HCV RNA declined and remained below the level of quantitation (<25 IU/mL) through the duration of treatment. Here, we report on the results of the genotypic and phenotypic analyses of resistant variants in 24 genotype 1-infected patients who received BMS-790052 (1, 10, 30, 60, and 100 mg, once-daily or 30 mg twice-daily) in the 14-day MAD study. Sequence analysis was performed on viral complementary DNA isolated from serum specimens collected at baseline and days 1 (4, 8, and 12 hours), 2, 4, 7, and 14 postdosing. Analyses of the sequence variants (1) established a correlation between resistant variants emerging in vivo with BMS-790052 treatment and those observed in the in vitro replicon system (major substitutions at residues 28, 30, 31, and 93 for genotype 1a and residues 31 and 93 for genotype 1b); (2) determined the prevalence of variants at baseline and the emergence of resistance at different times during dosing; and (3) revealed the resistance profile and replicative ability (i.e., fitness) of the variants. Although resistance emerged during monotherapy with BMS-790052, the substantial anti-HCV effect of this compound makes it an excellent candidate for effective combination therapy.

A Critical Role for PIM2 Kinase in Multiple Myeloma Through NF-κB Activation

Abstract 1839The PIM kinases are a family of 3 growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. Although PIM-1, -2 have been noted as highly expressed in multiple myeloma (MM) (Claudio JO et al., 2002), there are few data to support potential therapeutic utility of PIM inhibition in this indication. Here we show that the myeloma cell lines express all PIM protein isoforms to varying extents, and we describe the properties of a novel pan-PIM inhibitor GNE-652 with picomolar biochemical potency, an excellent selectivity profile, and favorable ADME properties. Myeloma cell lines and patient samples exhibit a striking prevalence of response to GNE-652 (23 of 25 lines with IC50 < 1 micromolar, median < 0.1 micromolar) and synergy in combination with the PI3K inhibitor GDC-0941 (mean combination index values ∼0.2 (n=25)). MM cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. Conversely, a PIM-1, -3 selective inhibitor, GNE-568, failed to suppress MM cell growth and also failed to provide synergy in combination with PI3K inhibition, suggesting PIM-2 is a critical driver of MM cell growth & survival. Additional results suggest that PIM signaling converges on both TORC1 and AKT to generate differential synergies with PI3K/AKT/mTOR pathway inhibitors. PIM has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and resulted in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in all the myeloma cell lines tested. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5 × IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. It has been hypothesized that a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, including a number of oncogenes such as cyclin D1. We noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1, and D3 that were further decreased by combination treatment of PIM and PI3K inhibition. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergy in multiple myeloma cell lines.As PIM isoforms are highly expressed in MM cells, we hypothesized that this could be due to proteosomal-mediated stability, and interestingly, MG132 and velcade each stabilized all PIM isoforms. It is commonly known that the JAK/STAT pathway regulates PIM transcription, but we show JAK inhibitors failed to abolish the expression of PIM in myeloma cells, suggesting a role for additional regulators. Recent genome sequencing studies from human myeloma samples (Chapman MA et al., 2011) confirmed the prevalence of NF-kB pathway activation, consistent with prior observations made in MM cell lines (Demchenko YN et al., 2010). The relationship of PIM and NF-kB is controversial in the literature (Hammerman PS et al., 2004 & Zhu N et al., 2002), with some groups placing PIM upstream of NF-kB and others the converse. Using an IκBα inhibitor, BMS-345541, we have examined the role for NF-kB in the regulation of PIM kinases. Here, we show that the BMS-345541 could preferentially suppress PIM2 expression in a dose dependent manner while PIM 1, 3 levels are modestly affected, suggesting that the high levels of PIM2 expression observed are partly driven by deregulation of the NF-kB pathway in MM. In conclusion, we provide pharmacological and biochemical evidence to suggest that PIM2 differentially regulate growth and survival of myeloma cells. Our results provide the rationale for further preclinical development of PIM inhibitors and the basis for a possible clinical development plan in multiple myeloma. Disclosures:Munugalavadla:Genentech: Employment. Berry:Genentech: Employment. Chang:Genentech: Employment. Rosario:Genentech: Employment. Drummond:Genentech: Employment. Du:Genentech: Employment. Fitzgerald:Genentech: Employment. Friedman:Genentech: Employment. Gould:Genentech: Employment. Maecker:Genentech: Employment. Moffat:Genentech: Employment. Slaga:Genentech: Employment. Xiaojing:Genentech: Employment. West:Genentech: Employment. Yu:Genentech: Employment. Ebens:Genentech: Employment.

A Novel Kinase Inhibitor, INCB28060, Blocks c-MET–Dependent Signaling, Neoplastic Activities, and Cross-Talk with EGFR and HER-3

The c-MET receptor tyrosine kinase plays important roles in the formation, progression, and dissemination of human cancer and presents an attractive therapeutic target. This study describes the preclinical characterization of INCB28060, a novel inhibitor of c-MET kinase. Studies were conducted using a series of in vitro and in vivo biochemical and biological experiments. INCB28060 exhibits picomolar enzymatic potency and is highly specific for c-MET with more than 10,000-fold selectivity over a large panel of human kinases. This inhibitor potently blocks c-MET phosphorylation and activation of its key downstream effectors in c-MET-dependent tumor cell lines. As a result, INCB28060 potently inhibits c-MET-dependent tumor cell proliferation and migration and effectively induces apoptosis in vitro. Oral dosing of INCB28060 results in time- and dose-dependent inhibition of c-MET phosphorylation and tumor growth in c-MET-driven mouse tumor models, and the inhibitor is well tolerated at doses that achieve complete tumor inhibition. In a further exploration of potential interactions between c-MET and other signaling pathways, we found that activated c-MET positively regulates the activity of epidermal growth factor receptors (EGFR) and HER-3, as well as expression of their ligands. These effects are reversed with INCB28060 treatment. Finally, we confirmed that circulating hepatocyte growth factor levels are significantly elevated in patients with various cancers. Activated c-MET has pleiotropic effects on multiple cancer-promoting signaling pathways and may play a critical role in driving tumor cell growth and survival. INCB28060 is a potent and selective c-MET kinase inhibitor that may have therapeutic potential in cancer treatment.

Superpotent [Dmt1]Dermorphin Tetrapeptides Containing the 4-Aminotetrahydro-2-benzazepin-3-one Scaffold with Mixed μ/δ Opioid Receptor Agonistic Properties

Novel dermorphin tetrapeptides are described in which Tyr(1) is replaced by Dmt(1), where d-Ala(2) and Gly(4) are N-methylated, and where Phe(3)-Gly(4) residue is substituted by the constrained Aba(3)-Gly(4) peptidomimetic. Most of these peptidic ligands displayed binding affinities in the nanomolar range for both μ- and δ-opioid receptors but no detectable affinity for the κ-opioid receptor. Measurements of cAMP accumulation, phosphorylation of extracellular signal-regulated kinase (ERK1/2) in HEK293 cells stably expressing each of these receptors individually, and functional screening in primary neuronal cultures confirmed the potent agonistic properties of these peptides. The most potent ligand H-Dmt-NMe-d-Ala-Aba-Gly-NH(2) (BVD03) displayed mixed μ/δ opioid agonist properties with picomolar functional potencies. Functional electrophysiological in vitro assays using primary cortical and spinal cord networks showed that this analogue possessed electrophysiological similarity toward gabapentin and sufentanil, which makes it an interesting candidate for further study as an analgesic for neuropathic pain.

Structure-based design of new DHFR-based antibacterial agents: 7-aryl-2,4-diaminoquinazolines

Dihydrofolate reductase (DHFR) inhibitors such as trimethoprim (TMP) have long played a significant role in the treatment of bacterial infections. Not surprisingly, after decades of use there is now bacterial resistance to TMP and therefore a need to develop novel antibacterial agents with expanded spectrum including these resistant strains. In this study, we investigated the optimization of 2,4-diamnoquinazolines for antibacterial potency and selectivity. Using structure-based drug design, several 7-aryl-2,4-diaminoquinazolines were discovered that have excellent sub-100 picomolar potency against bacterial DHFR. These compounds have good antibacterial activity especially on gram-positive pathogens including TMP-resistant strains.

Abstract 1787: ImmTACs: Bi-functional reagents for redirected tumour cell killing

Abstract The human immune system can theoretically identify malignant cells by inspecting cell surface Class I HLA -peptide complexes for the presence of disease-associated epitopes. Indeed, many cancer patients generate CD8 cyto-toxic T cell responses to tumour-associated antigens; the majority of patients, however, fail to clear tumours since T cell avidity for self-antigens tends to be weak, and cancer cells employ escape mechanisms for avoiding destruction by T cells. To overcome these issues, we have engineered novel, bi-functional protein therapeutics termed ImmTACs (Immune Mobilising mTCR Against Cancer) which re-direct the immune system to target and destroy tumour cells with a high degree of potency and specificity. An ImmTAC comprises a high affinity ‘monoclonal’ T cell Receptor (mTCR) targeting a cancer-associated HLA-peptide complex, fused to an anti-CD3 scFv domain which activates an anti-tumour T cell response. We demonstrate that ImmTACs against a number of different cancer-associated antigens can target and kill tumour cells expressing as few as 10-20 epitopes per cell with pico-Molar potency. ImmTACs preferentially activate effector memory CD8 T cells, resulting in secretion of multiple cytokines and tumour cell killing; a single activated T cell can kill multiple antigen positive tumour cells. Furthermore, we demonstrate that the reagents are able to inhibit tumour growth in mouse xenograft models. In vitro ImmTAC potency translates to a dose of less than 1mg in humans, representing a significant advance over existing targeted anti-cancer therapies. Currently we are conducting a phase I dose-escalation trial and Phase 0 exploratory trial using ImmTAC-gp100 in late stage melanoma patients. In summary, ImmTACs offers a novel therapeutic approach for the treatment of various cancers with the potential to provide major benefits over current treatments including reduction in dose to sub-mg quantities and an improved safety profile. 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 1787. doi:10.1158/1538-7445.AM2011-1787

Development of an online SPE-LC-MS-based assay using endogenous substrate for investigation of soluble epoxide hydrolase (sEH) inhibitors.

Soluble epoxide hydrolase (sEH) is a promising therapeutic target for the treatment of hypertension, pain, and inflammation-related diseases. In order to enable the development of sEH inhibitors (sEHIs), assays are needed for determination of their potency. Therefore, we developed a new method utilizing an epoxide of arachidonic acid (14(15)-EpETrE) as substrate. Incubation samples were directly injected without purification into an online solid phase extraction (SPE) liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) setup allowing a total run time of only 108 s for a full gradient separation. Analytes were extracted from the matrix within 30 s by turbulent flow chromatography. Subsequently, a full gradient separation was carried out on a 50X2.1 mm RP-18 column filled with 1.7 μm core–shell particles. The analytes were detected with high sensitivity by ESI–MS–MS in SRM mode. The substrate 14(15)-EpETrE eluted at a stable retention time of 96 ± 1 s and its sEH hydrolysis product 14,15-DiHETrE at 63 ± 1 s with narrow peak width (full width at half maximum height: 1.5 ± 0.1 s). The analytical performance of the method was excellent, with a limit of detection of 2 fmol on column, a linear range of over three orders of magnitude, and a negligible carry-over of 0.1% for 14,15-DiHETrE. The enzyme assay was carried out in a 96-well plate format, and near perfect sigmoidal dose–response curves were obtained for 12 concentrations of each inhibitor in only 22 min, enabling precise determination of IC50 values. In contrast with other approaches, this method enables quantitative evaluation of potent sEHIs with picomolar potencies because only 33 pmol L−1 sEH were used in the reaction vessel. This was demonstrated by ranking ten compounds by their activity; in the fluorescence method all yielded IC50 ≤ 1 nmol L−1. Comparison of 13 inhibitors with IC50 values >1 nmol L−1 showed a good correlation with the fluorescence method (linear correlation coefficient 0.9, slope 0.95, Spearman’s rho 0.9). For individual compounds, however, up to eightfold differences in potencies between this and the fluorescence method were obtained. Therefore, enzyme assays using natural substrate, as described here, are indispensable for reliable determination of structure–activity relationships for sEH inhibition.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-4861-2) contains supplementary material, which is available to authorized users.

Bi-specific TCR anti-CD3 fusion for the immunotherapy of malignant melanoma (156.16)

Abstract The human immune system can theoretically identify malignant cells by ‘scanning’ peptide antigens presented on the cell surface by Class I HLA molecules, and indeed many cancer patients generate CD8 cyto-toxic T cell responses to tumour-specific antigens. However, the majority of patients fail to clear tumours, since T cell avidity for self-antigens tends to be weak, and cancer cells employ escape mechanisms for avoiding destruction by T cells. We have engineered novel, bi-functional protein therapeutics termed ImmTACs which re-direct the immune system to target and destroy tumour cells with a high degree of potency and specificity. IMCgp100 comprises a soluble, high affinity T cell Receptor specific for the HLA-A2 presented melanoma-associated epitope gp100 280-288, fused to an anti-CD3 scFv domain. The anti-CD3 moiety activates a polyclonal T cell response when targeted to melanoma cells presenting as few as twenty gp100 epitopes, predominantly through activation of effector memory CD8 lymphocytes, leading to killing of the tumour cells. Extensive in vitro testing of IMCgp100 with a panel of tumour cells and normal human tissue cells demonstrates pico-Molar potency against cancer cells and specificity for HLA-A2 gp100 positive cells. The agent is able to inhibit tumour growth in mouse xenograft models. A phase I dose-escalation trial in late stage melanoma patients was initiated in October 2010 and interim results will be presented at this meeting.