Showing papers in "Molecular Cancer Therapeutics in 2012"

Identification and Characterization of NVP-BKM120, an Orally Available Pan-Class I PI3-Kinase Inhibitor

Abstract: Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional phosphoinositide 3-kinase (PI3K) inhibitors from different chemical classes with a different selectivity profile. The key to achieve these objectives was to couple a structure-based design approach with intensive pharmacologic evaluation of selected compounds during the medicinal chemistry optimization process. Here, we report on the biologic characterization of the 2-morpholino pyrimidine derivative pan-PI3K inhibitor NVP-BKM120. This compound inhibits all four class I PI3K isoforms in biochemical assays with at least 50-fold selectivity against other protein kinases. The compound is also active against the most common somatic PI3Kα mutations but does not significantly inhibit the related class III (Vps34) and class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic models and relevant tumor cell lines, as well as downstream effectors in a concentration-dependent and pathway-specific manner. Tested in a panel of 353 cell lines, NVP-BKM120 exhibited preferential inhibition of tumor cells bearing PIK3CA mutations, in contrast to either KRAS or PTEN mutant models. NVP-BKM120 shows dose-dependent in vivo pharmacodynamic activity as measured by significant inhibition of p-Akt and tumor growth inhibition in mechanistic xenograft models. NVP-BKM120 behaves synergistically when combined with either targeted agents such as MEK or HER2 inhibitors or with cytotoxic agents such as docetaxel or temozolomide. The pharmacological, biologic, and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is undergoing phase II clinical trials in patients with cancer.

A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates Glycolysis, Induces Cell-Cycle Arrest, and Inhibits Cancer Cell Growth In Vitro and In Vivo

Abstract: The functional and therapeutic importance of the Warburg effect is increasingly recognized, and glycolysis has become a target of anticancer strategies. We recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation-like mechanism. We hypothesized that the compounds target Glut1 and are efficacious in vivo as anticancer agents. Here, we report that a novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 at 10 mg/kg resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP-sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule. Senescence induction and the essential role of ATP were reported for the first time in Glut1 inhibitor-treated cancer cells. Thus, WZB117 is a prototype for further development of anticancer therapeutics targeting Glut1-mediated glucose transport and glucose metabolism.

Combinations of BRAF, MEK, and PI3K/mTOR Inhibitors Overcome Acquired Resistance to the BRAF Inhibitor GSK2118436 Dabrafenib, Mediated by NRAS or MEK Mutations

Abstract: Recent results from clinical trials with the BRAF inhibitors GSK2118436 (dabrafenib) and PLX4032 (vemurafenib) have shown encouraging response rates; however, the duration of response has been limited. To identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated GSK2118436 drug-resistant clones from the A375 BRAF(V600E) and the YUSIT1 BRAF(V600K) melanoma cell lines. These clones also showed reduced sensitivity to the allosteric mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor GSK1120212 (trametinib). Genetic characterization of these clones identified an in-frame deletion in MEK1 (MEK1(K59del)) or NRAS mutation (NRAS(Q61K) and/or NRAS(A146T)) with and without MEK1(P387S) in the BRAF(V600E) background and NRAS(Q61K) in the BRAF(V600K) background. Stable knockdown of NRAS with short hairpin RNA partially restored GSK2118436 sensitivity in mutant NRAS clones, whereas expression of NRAS(Q61K) or NRAS(A146T) in the A375 parental cells decreased sensitivity to GSK2118436. Similarly, expression of MEK1(K59del), but not MEK1(P387S), decreased sensitivity of A375 cells to GSK2118436. The combination of GSK2118436 and GSK1120212 effectively inhibited cell growth, decreased ERK phosphorylation, decreased cyclin D1 protein, and increased p27(kip1) protein in the resistant clones. Moreover, the combination of GSK2118436 or GSK1120212 with the phosphoinositide 3-kinase/mTOR inhibitor GSK2126458 enhanced cell growth inhibition and decreased S6 ribosomal protein phosphorylation in these clones. Our results show that NRAS and/or MEK mutations contribute to BRAF inhibitor resistance in vitro, and the combination of GSK2118436 and GSK1120212 overcomes this resistance. In addition, these resistant clones respond to the combination of GSK2126458 with GSK2118436 or GSK1120212. Clinical trials are ongoing or planned to test these combinations.

Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background

Abstract: AKT is a key node in the most frequently deregulated signaling network in human cancer. AZD5363, a novel pyrrolopyrimidine-derived compound, inhibited all AKT isoforms with a potency of 10 nmol/L or less and inhibited phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 μmol/L. AZD5363 monotherapy inhibited the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of 3 μmol/L or less. Cell lines derived from breast cancers showed the highest frequency of sensitivity. There was a significant relationship between the presence of PIK3CA and/or PTEN mutations and sensitivity to AZD5363 and between RAS mutations and resistance. Oral dosing of AZD5363 to nude mice caused dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC(50) ~ 0.1 μmol/L total plasma exposure), reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[18F]fluoro-2-deoxy-D-glucose ((18)F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of AZD5363 caused dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2(+) breast cancer models that are resistant to trastuzumab. AZD5363 also significantly enhanced the antitumor activity of docetaxel, lapatinib, and trastuzumab in breast cancer xenografts. It is concluded that AZD5363 is a potent inhibitor of AKT with pharmacodynamic activity in vivo, has potential to treat a range of solid and hematologic tumors as monotherapy or a combinatorial agent, and has potential for personalized medicine based on the genetic status of PIK3CA, PTEN, and RAS. AZD5363 is currently in phase I clinical trials.

Ponatinib (AP24534), a Multitargeted Pan-FGFR Inhibitor with Activity in Multiple FGFR-Amplified or Mutated Cancer Models

Abstract: Members of the fibroblast growth factor receptor family of kinases (FGFR1-4) are dysregulated in multiple cancers. Ponatinib (AP24534) is an oral multitargeted tyrosine kinase inhibitor being explored in a pivotal phase II trial in patients with chronic myelogenous leukemia due to its potent activity against BCR-ABL. Ponatinib has also been shown to inhibit the in vitro kinase activity of all four FGFRs, prompting us to examine its potential as an FGFR inhibitor. In Ba/F3 cells engineered to express activated FGFR1-4, ponatinib potently inhibited FGFR-mediated signaling and viability with IC(50) values <40 nmol/L, with substantial selectivity over parental Ba/F3 cells. In a panel of 14 cell lines representing multiple tumor types (endometrial, bladder, gastric, breast, lung, and colon) and containing FGFRs dysregulated by a variety of mechanisms, ponatinib inhibited FGFR-mediated signaling with IC(50) values <40 nmol/L and inhibited cell growth with GI(50) (concentration needed to reduce the growth of treated cells to half that of untreated cells) values of 7 to 181 nmol/L. Daily oral dosing of ponatinib (10-30 mg/kg) to mice reduced tumor growth and inhibited signaling in all three tumor models examined. Importantly, the potency of ponatinib in these models is similar to that previously observed in BCR-ABL-driven models and plasma levels of ponatinib that exceed the IC(50) values for FGFR1-4 inhibition can be sustained in patients. These results show that ponatinib is a potent pan-FGFR inhibitor and provide strong rationale for its evaluation in patients with FGFR-driven cancers.

Antitumor Activity of a Novel Bispecific Antibody That Targets the ErbB2/ErbB3 Oncogenic Unit and Inhibits Heregulin-Induced Activation of ErbB3

Abstract: The prevalence of ErbB2 amplification in breast cancer has resulted in the heavy pursuit of ErbB2 as a therapeutic target. Although both the ErbB2 monoclonal antibody trastuzumab and ErbB1/ErbB2 dual kinase inhibitor lapatinib have met with success in the clinic, many patients fail to benefit. In addition, the majority of patients who initially respond will unfortunately ultimately progress on these therapies. Activation of ErbB3, the preferred dimerization partner of ErbB2, plays a key role in driving ErbB2-amplified tumor growth, but we have found that current ErbB2-directed therapies are poor inhibitors of ligand-induced activation. By simulating ErbB3 inhibition in a computational model of ErbB2/ErbB3 receptor signaling, we predicted that a bispecific antibody that docks onto ErbB2 and subsequently binds to ErbB3 and blocks ligand-induced receptor activation would be highly effective in ErbB2-amplified tumors, with superior activity to a monospecific ErbB3 inhibitor. We have developed a bispecific antibody suitable for both large scale production and systemic therapy by generating a single polypeptide fusion protein of two human scFv antibodies linked to modified human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling and showing antitumor activity in preclinical models that is dependent on ErbB2 overexpression. MM-111 can be rationally combined with trastuzumab or lapatinib for increased antitumor activity and may in the future complement existing ErbB2-directed therapies to treat resistant tumors or deter relapse.

Ganetespib, a Unique Triazolone-Containing Hsp90 Inhibitor, Exhibits Potent Antitumor Activity and a Superior Safety Profile for Cancer Therapy

Abstract: Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 μm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors.

Bispecific and Trispecific Killer Cell Engagers Directly Activate Human NK Cells through CD16 Signaling and Induce Cytotoxicity and Cytokine Production

Abstract: This study evaluates the mechanism by which bispecific and trispecific killer cell engagers (BiKEs and TriKEs) act to trigger human NK cell effector function and investigates their ability to induce NK cell cytokine and chemokine production against human B-cell leukemia. We examined the ability of BiKEs and TriKEs to trigger NK cell activation through direct CD16 signaling, measuring intracellular Ca2+ mobilization, secretion of lytic granules, induction of target cell apoptosis and production of cytokine and chemokines in response to the Raji cell line and primary leukemia targets. Resting NK cells triggered by the recombinant reagents led to intracellular Ca2+ mobilization through direct CD16 signaling. Co-culture of reagent-treated resting NK cells with Raji target cells resulted in significant increases in NK cell degranulation and target cell death. BiKEs and TriKEs effectively mediated NK cytotoxicity of Raji target cells at high and low effector-to-target (E:T) ratios and maintained functional stability after 24 and 48 hours of culture in human serum. NK cell production of IFN-γ, TNF-α, GM-CSF, IL-8, MIP-1α and RANTES was differentially induced in the presence of recombinant reagents and Raji targets. Moreover, significant increases in NK cell degranulation and enhancement of IFN-γ production against primary ALL and CLL targets were induced with reagent treatment of resting NK cells. In conclusion, BiKEs and TriKEs directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and cytokine production against tumor targets, demonstrating their therapeutic potential for enhancing NK cell immunotherapies for leukemias and lymphomas.

ERK Inhibition Overcomes Acquired Resistance to MEK Inhibitors

Abstract: The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor-resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients.

Rationale behind targeting fibroblast activation protein-expressing carcinoma-associated fibroblasts as a novel chemotherapeutic strategy.

Abstract: The tumor microenvironment has emerged as a novel chemotherapeutic strategy in the treatment of cancer. This is most clearly exemplified by the antiangiogenesis class of compounds. Therapeutic strategies that target fibroblasts within the tumor stroma offer another treatment option. However, despite promising data obtained in preclinical models, such strategies have not been widely used in the clinical setting, largely due to a lack of effective treatments that specifically target this population of cells. The identification of fibroblast activation protein α (FAP) as a target selectively expressed on fibroblasts within the tumor stroma or on carcinoma-associated fibroblasts led to intensive efforts to exploit this novel cellular target for clinical benefit. FAP is a membrane-bound serine protease of the prolyl oligopeptidase family with unique post-prolyl endopeptidase activity. Until recently, the majority of FAP-based therapeutic approaches focused on the development of small-molecule inhibitors of enzymatic activity. Evidence suggests, however, that FAP's pathophysiological role in carcinogenesis may be highly contextual, depending on both the exact nature of the tumor microenvironment present and the cancer type in question to determine its tumor-promoting or tumor-suppressing phenotype. As an alternative strategy, we are taking advantage of FAP's restricted expression and unique substrate preferences to develop a FAP-activated prodrug to target the activation of a cytotoxic compound within the tumor stroma. Of note, this strategy would be effective independently of FAP's role in tumor progression because its therapeutic benefit would rely on FAP's localization and activity within the tumor microenvironment rather than strictly on inhibition of its function.

The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo.

Abstract: Recent studies suggest that metformin, which is commonly used as an oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis, but the mechanisms by which metformin affects various cancers, including gastric cancer, remains unknown. The goal of the present study was to evaluate the effects of metformin on human gastric cancer cell proliferation in vitro and in vivo and to study microRNAs (miRNA) associated with antitumor effect of metformin. We used MKN1, MKN45, and MKN74 human gastric cancer cell lines to study the effects of metformin on human gastric cancer cells. Athymic nude mice bearing xenograft tumors were treated with or without metformin. Tumor growth was recorded after 4 weeks, and the expression of cell-cycle-related proteins was determined. In addition, we used miRNA array tips to explore the differences among miRNAs in MKN74 cells bearing xenograft tumors treated with or without metformin in vitro and in vivo. Metformin inhibited the proliferation of MKN1, MKN45, and MKN74 in vitro. Metformin blocked the cell cycle in G(0)-G(1)in vitro and in vivo. This blockade was accompanied by a strong decrease of G(1) cyclins, especially in cyclin D1, cyclin-dependent kinase (Cdk) 4, Cdk6 and by a decrease in retinoblastoma protein (Rb) phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor-1 receptor in vitro and in vivo. The miRNA expression was markedly altered with the treatment of metformin in vitro and in vivo. Various miRNAs altered by metformin also may contribute to tumor growth in vitro and in vivo.

Coexistence of PIK3CA and Other Oncogene Mutations in Lung Adenocarcinoma–Rationale for Comprehensive Mutation Profiling

Abstract: Phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA) encodes the p110α subunit of the mitogenic signaling protein phosphoinositide 3-kinase (PI3K). PIK3CA mutations in the helical binding domain and the catalytic subunit of the protein have been associated with tumorigenesis and treatment resistance in various malignancies. Characteristics of patients with PIK3CA-mutant lung adenocarcinomas have not been reported. We examined epidermal growth factor receptor (EGFR), Kirsten rate sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), human epidermal growth factor receptor 2 (HER2), PIK3CA, v-akt murine thymoma vial oncogene homolog 1 (AKT1), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen-activated protein kinase kinase 1 (MEK1), and anaplastic lymphoma kinase (ALK) in patients with adenocarcinoma of the lung to identify driver mutations. Clinical data were obtained from the medical records of individuals with mutations in PIK3CA. Twenty-three of 1,125 (2%, 95% CI: 1-3) patients had a mutation in PIK3CA, 12 in exon 9 (10 E545K and 2 E542K), and 11 in exon 20 (3 H1047L and 8 H1047R). The patients (57% women) had a median age of 66 at diagnosis (range: 34-78). Eight patients (35%) were never smokers. Sixteen of 23 (70%, 95% CI: 49-86) had coexisting mutations in other oncogenes-10 KRAS, 1 MEK1, 1 BRAF, 1 ALK rearrangement, and 3 EGFR exon 19 deletions. We conclude that PIK3CA mutations occur in lung adenocarcinomas, usually concurrently with EGFR, KRAS, and ALK. The impact of PIK3CA mutations on the efficacy of targeted therapies such as erlotinib and crizotinib is unknown. Given the high frequency of overlapping mutations, comprehensive genotyping should be carried out on tumor specimens from patients enrolling in clinical trials of PI3K and other targeted therapies.

Epithelial–Mesenchymal Transition and Stem Cell Markers in Patients with HER2-Positive Metastatic Breast Cancer

Abstract: Currently, there is extensive information about circulating tumor cells (CTC) and their prognostic value; however, little is known about other characteristics of these cells. In this prospective study, we assessed the gene transcripts of epithelial-to-mesenchymal transition-inducing transcription factors (EMT-TF) and cancer stem cell (CSC) features in patients with HER2(+) metastatic breast cancer (MBC). Epithelial cells were enriched from peripheral blood mononuclear cells (PBMC) using antibody-coated anti-CD326 antibody (CD326(+)) magnetic beads, and the residual CD326(-) PBMCs were further depleted of leukocytes using anti-CD45 antibody-coated magnetic beads (CD326(-)CD45(-)). RNA was extracted from all cell fractions, reverse transcribed to cDNA, and subjected to quantitative reverse transcription PCR to detect EMT-TFs (TWIST1, SNAIL1, ZEB1, and TG2) as a measure of CTCs undergoing EMT (EMT-CTCs). In addition, PBMCs were analyzed using multiparameter flow cytometry for ALDH activity and CSCs that express CD24, CD44, and CD133. Twenty-eight patients were included in this study. At least one EMT-TF mRNA was elevated in the CTCs of 88.2% of patients and in the CD326(-)CD45(-) cell fraction of 60.7% of patients. The CD326(-)CD45(-) fraction of patients with elevated SNAIL1 and ZEB1 transcripts also had a higher percentage of ALDH(+)/CD133(+) cells in their blood than did patients with normal SNAIL1 and ZEB1 expression (P = 0.038). Our data indicate that patients with HER2(+) MBCs have EMT-CTCs. Moreover, an enrichment of CSCs was found in CD326(-)CD45(-) cells. Additional studies are needed to determine whether EMT-CTCs and CSCs have prognostic value in patients with HER2(+) MBCs treated with trastuzumab-based therapy.

miRNA-29b Suppresses Prostate Cancer Metastasis by Regulating Epithelial–Mesenchymal Transition Signaling

Abstract: Prostate cancer remains the second leading cause of cancer deaths among American men. Early diagnosis increases survival rate in patients; however, treatments for advanced disease are limited to hormone ablation techniques and palliative care. Thus, new methods of treatment are necessary for inhibiting prostate cancer disease progression. Here, we have shown that miRNA-29b (miR-29b) expression was lower in prostate cancer cells (PC3 and LNCaP) as compared with immortalized prostate epithelial cells. Between these two prostate cancer cell lines, metastatic prostate cancer PC3 cells displayed lower expression of miR-29b. We also observed a significant downregulation of miR-29b expression in human prostate cancer tissues as compared with patient-matched nontumor tissues. PC3 cells ectopically expressing miR-29b inhibited wound healing, invasiveness, and failed to colonize in the lungs and liver of severe combined immunodeficient mice after intravenous injection, while PC3 cells expressing a control miRNA displayed metastasis. Epithelial cell marker E-cadherin expression was enhanced miR-29b transfected in prostate cancer cells as compared with cells expressing control miRNA. On the other hand, N-cadherin, Twist, and Snail expression was downregulated in PC3 cells expressing miR-29b. Together these results suggested that miR-29b acts as an antimetastatic miRNA for prostate cancer cells at multiple steps in a metastatic cascade. Therefore, miR-29b could be a potentially new attractive target for therapeutic intervention in prostate cancer.

Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302

Abstract: TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of γH2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell–based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect. Mol Cancer Ther; 11(3); 740–51. ©2011 AACR .

Activation of IL-6R/JAK1/STAT3 Signaling Induces de novo Resistance to Irreversible EGFR Inhibitors in Non-Small Cell Lung Cancer with T790M Resistance Mutation

Abstract: The secondary T790M mutation in epidermal growth factor receptor (EGFR) is the major mechanism of acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in non-small cell lung cancer (NSCLC). Although irreversible EGFR TKIs, such as afatinib or dacomitinib, have been introduced to overcome the acquired resistance, they showed a limited efficacy in NSCLC with T790M. Herein, we identified the novel de novo resistance mechanism to irreversible EGFR TKIs in H1975 and PC9-GR cells, which are NSCLC cells with EGFR T790M. Afatinib activated interleukin-6 receptor (IL-6R)/JAK1/STAT3 signaling via autocrine IL-6 secretion in both cells. Inhibition of IL-6R/JAK1/STAT3 signaling pathway increased the sensitivity to afatinib. Cancer cells showed stronger STAT3 activation and enhanced resistance to afatinib in the presence of MRC5 lung fibroblasts. Blockade of IL-6R/JAK1 significantly increased the sensitivity to afatinib through inhibition of afatinib-induced STAT3 activation augmented by the interaction with fibroblasts, suggesting a critical role of paracrine IL-6R/JAK1/STAT3 loop between fibroblasts and cancer cells in the development of drug resistance. The enhancement of afatinib sensitivity by inhibition of IL-6R/JAK1/STAT3 signaling was confirmed in in vivo PC9-GR xenograft model. Similar to afatinib, de novo resistance to dacomitinib in H1975 and PC9-GR cells was also mediated by dacomitinib-induced JAK1/STAT3 activation. Taken together, these findings suggest that IL-6R/JAK1/STAT3 signaling can be a potential therapeutic target to enhance the efficacy of irreversible EGFR TKIs in patients with EGFR T790M.

Sorafenib-Induced Hepatocellular Carcinoma Cell Death Depends on Reactive Oxygen Species Production In Vitro and In Vivo

Abstract: Sorafenib is presently the only effective therapy in advanced hepatocellular carcinoma (HCC). Because most anticancer drugs act, at least in part, through the generation of reactive oxygen species, we investigated whether sorafenib can induce an oxidative stress. The effects of sorafenib on intracellular ROS production and cell death were assessed in vitro in human (HepG2) and murine (Hepa 1.6) HCC cell lines and human endothelial cells (HUVEC) as controls. In addition, 26 sera from HCC patients treated by sorafenib were analyzed for serum levels of advanced oxidation protein products (AOPP). Sorafenib significantly and dose-dependently enhanced in vitro ROS production by HCC cells. The SOD mimic MnTBAP decreased sorafenib-induced lysis of HepG2 cells by 20% and of Hepa 1.6 cells by 75% compared with HCC cells treated with 5 mg/L sorafenib alone. MnTBAP significantly enhanced by 25% tumor growth in mice treated by sorafenib. On the other hand, serum levels of AOPP were higher in HCC patients treated by sorafenib than in sera collected before treatment (P < 0.001). An increase in serum AOPP concentration ≥0.2 μmol/L chloramine T equivalent after 15 days of treatment is a predictive factor for sorafenib response with higher progression free survival (P < 0.05) and overall survival rates (P < 0.05). As a conclusion, sorafenib dose-dependently induces the generation of ROS in tumor cells in vitro and in vivo. The sera of Sorafenib-treated HCC patients contain increased AOPP levels that are correlated with the clinical effectiveness of sorafenib and can be used as a marker of effectiveness of the drug. .

The effect of different linkers on target cell catabolism and pharmacokinetics/pharmacodynamics of trastuzumab maytansinoid conjugates

Abstract: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the anti-HER2 antibody trastuzumab linked via a nonreducible thioether linker to the maytansinoid antitubulin agent DM1. T-DM1 has shown favorable safety and efficacy in patients with HER2-positive metastatic breast cancer. In previous animal studies, T-DM1 exhibited better pharmacokinetics (PK) and slightly more efficacy than several disulfide-linked versions. The efficacy findings are unique, as other disulfide-linked antibody-drug conjugates (ADC) have shown greater efficacy than thioether-linked designs. To explore this further, the in vitro and in vivo activity, PK, and target cell activation of T-DM1 and the disulfide-linked T-SPP-DM1 were examined. Both ADCs showed high in vitro potency, with T-DM1 displaying greater potency in two of four breast cancer cell lines. In vitro target cell processing of T-DM1 and T-SPP-DM1 produced lysine-N(e)-MCC-DM1, and lysine-N(e)-SPP-DM1 and DM1, respectively; in vivo studies confirmed these results. The in vitro processing rates for the two conjugate to their respective catabolites were similar. In vivo, the potencies of the conjugates were similar, and T-SPP-DM1 had a faster plasma clearance than T-DM1. Slower T-DM1 clearance translated to higher overall tumor concentrations (conjugate plus catabolites), but unexpectedly, similar levels of tumor catabolite. These results indicate that, although the ADC linker can have clear impact on the PK and the chemical nature of the catabolites formed, both linkers seem to offer the same payload delivery to the tumor.

Immunotherapy of Cancer with 4-1BB

Abstract: 4-1BB (CD137), a member of the TNF receptor superfamily, is an activation-induced T-cell costimulatory molecule. Signaling via 4-1BB upregulates survival genes, enhances cell division, induces cytokine production, and prevents activation-induced cell death in T cells. The importance of the 4-1BB pathway has been underscored in a number of diseases, including cancer. Growing evidence indicates that anti-4-1BB monoclonal antibodies possess strong antitumor properties, which in turn are the result of their powerful CD8+ T-cell activating, IFN-γ producing, and cytolytic marker-inducing capabilities. In addition, combination therapy of anti-4-1BB with other anticancer agents, such as radiation, has robust tumor-regressing abilities against nonimmunogenic or poorly immunogenic tumors. Furthermore, the adoptive transfer of ex vivo anti-4-1BB-activated CD8+ T cells from previously tumor-treated animals efficiently inhibits progression of tumors in recipient mice that have been inoculated with fresh tumors. In addition, targeting of tumors with variants of 4-1BBL directed against 4-1BB also have potent antitumor effects. Currently, a humanized anti-4-1BB is in clinical trials in patients with solid tumors, including melanoma, renal carcinoma, and ovarian cancer, and so far seems to have a favorable toxicity profile. In this review, we discuss the basis of the therapeutic potential of targeting the 4-1BB-4-1BBL pathway in cancer treatment.

STAT3 Inhibition Overcomes Temozolomide Resistance in Glioblastoma by Downregulating MGMT Expression

Abstract: Glioblastoma multiforme (GBM) is one of the most aggressive human tumors with a poor prognosis. Current standard treatment includes chemotherapy with the DNA-alkylating agent temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide-induced DNA damage due to elevated expression of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). Here, we show that upregulation of both MGMT and STAT3 was accompanied with acquisition of temozolomide resistance in the GBM cell line U87. Inactivation of STAT3 by inhibitor or short hairpin RNA (shRNA) downregulated MGMT expression in GBM cell lines. MGMT upregulation was not observed by the treatment of interleukin (IL)-6 which is a strong activator of STAT3. Contrarily, forced expressed MGMT could be downregulated by STAT3 inhibitor which was partially rescued by the proteasome inhibitor, MG132, suggesting the STAT3-mediated posttranscriptional regulation of the protein levels of MGMT. Immunohistochemical analysis of 44 malignant glioma specimens showed significant positive correlation between expression levels of MGMT and phosphorylated STAT3 (p-STAT3; P < 0.001, r = 0.58). Importantly, the levels of both MGMT and p-STAT3 were increased in the recurrence compared with the primary lesion in paired identical tumors of 12 cases. Finally, we showed that STAT3 inhibitor or STAT3 knockdown potentiated temozolomide efficacy in temozolomide-resistant GBM cell lines. Therefore, STAT3 inhibitor might be one of the candidate reagents for combination therapy with temozolomide for patients with temozolomide-resistant GBM.

The EGFR T790M Mutation in Acquired Resistance to an Irreversible Second-Generation EGFR Inhibitor

Abstract: Molecular target therapies using the first-generation reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib or erlotinib, have been shown to be effective for non-small cell lung cancer (NSCLC) patients who harbor activating mutations in EGFR. However, these patients eventually develop resistance to the reversible TKIs, and this has led to the development of second-generation irreversible EGFR inhibitors. Currently, the mechanism of acquired resistance to irreversible EGFR inhibitors is not clear. Using an in vitro cell culture system, we modeled the acquired resistance to first-line treatment with second-generation EGFR-TKIs, using an EGFR mutant NSCLC cell line. Here we report a mechanism of resistance involving T790M secondary mutation as well as a corresponding clinical case. The results of these findings suggest that inhibition of EGFR by currently available second-generation EGFR-TKIs may not be sufficient to physiologically prevent the emergence of cells that are still dependent on EGFR signaling. This finding bears important implications on the limitations of currently available second-generation EGFR TKIs.

MET Activation Mediates Resistance to Lapatinib Inhibition of HER2-Amplified Gastric Cancer Cells

Abstract: HER2 amplification is found in >15% of gastric cancers (GC), and is associated with poor clinical outcome. Lapatinib, a dual HER2 and EGFR tyrosine kinase inhibitor, has shown promising in-vitro results in treating HER2+ cancer cells. However, several studies have shown that activation of alternative receptor tyrosine kinases can mediate resistance to HER-targeted therapy. Here we investigated whether activated MET can confer resistance to lapatinib inhibition of GC cells. A panel of gastric cancer cell lines were treated with lapatinib and we observed that cell proliferation was reduced by 70% and that the degree of HER2 amplification corresponds to sensitivity to lapatinib. Immunoblotting analysis indicated that phosphorylation of HER2, EGFR, MET, AKT and ERK were inhibited by lapatinib and presumably led to cell cycle arrest as observed using flow cytometry. HGF activation of MET receptors rescued cells from lapatinib-induced growth inhibition by re-stimulating the downstream pathways and restoring normal cell cycle progression. This rescue effect could be abrogated by inhibiting MET using PHA-665752 (a highly specific MET inhibitor), or downregulating MET expression with siRNA. No synergy in growth inhibition was observed when cells were treated with a combination of lapatinib and PHA-665752. Repeat studies using insulin-like growth factor 1 and fibroblast growth factor 3 could not uniformly rescue the lapatinib treated GC cells. In conclusion, HGF/MET mediated resistance to lapatinib is a novel mechanism of resistance to HER2-targeted agents in GC cells. Development of inhibitors targeting multiple receptors or common downstream signaling proteins merits further investigation.

microRNA-9 Targets Matrix Metalloproteinase 14 to Inhibit Invasion, Metastasis, and Angiogenesis of Neuroblastoma Cells

Abstract: Matrix metalloproteinase (MMP)-14 is the only membrane-anchored MMP that plays a critical role in tumor metastasis and angiogenesis. However, the mechanisms underlying MMP-14 expression in tumors still remain largely unknown. In this study, MMP-14 immunostaining was identified in 29/42 neuroblastoma tissues, which was correlated with clinicopathologic features and shorter patients' survival. In subtotal 20 neuroblastoma cases, microRNA 9 (miR-9) was downregulated and inversely correlated with MMP-14 expression. Bioinformatics analysis revealed a putative miR-9-binding site in the 3'-untranslated region (3'-UTR) of MMP-14 mRNA. Overexpression or knockdown of miR-9 responsively altered both the mRNA and protein levels of MMP-14 and its downstream gene, vascular endothelial growth factor, in cultured neuroblastoma cell lines SH-SY5Y and SK-N-SH. In an MMP-14 3'-UTR luciferase reporter system, miR-9 downregulated the luciferase activity, and these effects were abolished by a mutation in the putative miR-9-binding site. Overexpression of miR-9 suppressed the invasion, metastasis, and angiogenesis of SH-SY5Y and SK-N-SH cells in vitro and in vivo. In addition, the effects of miR-9 on MMP-14 expression, adhesion, migration, invasion, and angiogenesis were rescued by overexpression of MMP-14 in these cells. Furthermore, anti-miR-9 inhibitor or knockdown of MMP-14 respectively increased or inhibited the migration, invasion, and angiogenesis of neuroblastoma cells. These data indicate that miR-9 suppresses MMP-14 expression via the binding site in the 3'-UTR, thus inhibiting the invasion, metastasis, and angiogenesis of neuroblastoma.

DNA Damage Repair Pathways in Cancer Stem Cells

Abstract: The discovery of tumor-initiating cells endowed with stem-like features has added a further level of complexity to the pathobiology of neoplastic diseases. In the attempt of dissecting the functional properties of this uncommon cellular subpopulation, investigators are taking full advantage of a body of knowledge about adult stem cells, as the "cancer stem cell model" implies that tissue-resident stem cells are the target of the oncogenic process. It is emerging that a plethora of molecular mechanisms protect cancer stem cells (CSC) against chemotherapy- and radiotherapy-induced death stimuli. The ability of CSCs to survive stressful conditions is correlated, among others, with a multifaceted protection of genome integrity by a prompt activation of the DNA damage sensor and repair machinery. Nevertheless, many molecular-targeted agents directed against DNA repair effectors are in late preclinical or clinical development while the identification of predictive biomarkers of response coupled with the validation of robust assays for assessing biomarkers is paving the way for biology-driven clinical trials.

Garcinol Regulates EMT and Wnt Signaling Pathways In Vitro and In Vivo, Leading to Anticancer Activity against Breast Cancer Cells

Abstract: Anticancer properties of Garcinia indica-derived garcinol are just beginning to be elucidated. We have earlier reported its cancer cell-specific induction of apoptosis in breast cancer cells, which was mediated through the downregulation of NF-κB signaling pathway. To gain further mechanistic insight, here, we show for the first time that garcinol effectively reverses epithelial-to-mesenchymal transition (EMT), that is, it induces mesenchymal-to-epithelial transition (MET) in aggressive triple-negative MDA-MB-231 and BT-549 breast cancer cells. This was associated with upregulation of epithelial marker E-cadherin and downregulation of mesenchymal markers vimentin, ZEB-1, and ZEB-2. We also found that garcinol upregulates the expression of miR-200 and let-7 family microRNAs (miRNAs), which provides a molecular mechanism for the observed reversal of EMT to MET. Transfection of cells with NF-κB p65 subunit attenuated the effect of garcinol on apoptosis induction through reversal of MET to EMT. Forced transfection of p65 and anti-miR-200s could also reverse the inhibitory effect of garcinol on breast cancer cell invasion. Moreover, treatment with garcinol resulted in increased phosphorylation of β-catenin concomitant with its reduced nuclear localization. The results were also validated in vivo in a xenograft mouse model where garcinol was found to inhibit NF-κB, miRNAs, vimentin, and nuclear β-catenin. These novel findings suggest that the anticancer activity of garcinol against aggressive breast cancer cells is, in part, due to reversal of EMT phenotype, which is mechanistically linked with the deregulation of miR-200s, let-7s, NF-κB, and Wnt signaling pathways.

Safety, Pharmacokinetics, and Activity of GRN1005, a Novel Conjugate of Angiopep-2, a Peptide Facilitating Brain Penetration, and Paclitaxel, in Patients with Advanced Solid Tumors

Abstract: GRN1005 is a novel peptide-drug conjugate composed of paclitaxel covalently linked to a peptide, angiopep-2, that targets the low-density lipoprotein receptor-related protein 1. This first-in-human study evaluated the safety, tolerability, pharmacokinetics, and efficacy of GRN1005 in patients with advanced solid tumors. Patients in sequential cohorts (one patient per cohort until grade 2 toxicity, then 3 + 3 design) received intravenous GRN1005 at escalating doses between 30 and 700 mg/m(2) once in every 21 days. In the maximum tolerated dose (MTD) expansion group, patients were required to have brain metastases. Fifty-six patients received GRN1005, including 41 with brain metastases (median number of prior therapies = 4). MTD was 650 mg/m(2); the main dose-limiting toxicity was myelosuppression. Sixteen of 20 patients dosed at the MTD had brain metastases. Pharmacokinetics was dose linear and the mean terminal-phase elimination half-life was 3.6 hours. No evidence of accumulation was observed after repeat dosing. No anti-GRN1005 antibodies were detected. Five of the 20 patients (25%) dosed at 650 mg/m(2) (MTD), three of whom had previous taxane therapy, achieved an overall partial response (breast, n = 2; non-small cell lung cancer, n = 2; and ovarian cancer, n = 1); responses in all five patients were also accompanied by shrinkage of brain lesions (-17% to -50%). In addition, six patients (11%; doses 30-700 mg/m(2)) experienced stable disease that lasted 4 months or more. GRN1005 was well tolerated and showed activity in heavily pretreated patients with advanced solid tumors, including those who had brain metastases and/or failed prior taxane therapy.

Cyclin E2 overexpression is associated with endocrine resistance but not insensitivity to CDK2 inhibition in human breast cancer cells

Abstract: Cyclin E2, but not cyclin E1, is included in several gene signatures that predict disease progression in either tamoxifen-resistant or metastatic breast cancer. We therefore examined the role of cyclin E2 in antiestrogen resistance in vitro and its potential for therapeutic targeting through cyclin-dependent kinase (CDK) inhibition. High expression of CCNE2, but not CCNE1, was characteristic of the luminal B and HER2 subtypes of breast cancer and was strongly predictive of shorter distant metastasis-free survival following endocrine therapy. After antiestrogen treatment of MCF-7 breast cancer cells, cyclin E2 mRNA and protein were downregulated and cyclin E2–CDK2 activity decreased. However, this regulation was lost in tamoxifen-resistant (MCF-7 TAMR) cells, which overexpressed cyclin E2. Expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression contributes to the antiestrogen resistance of tamoxifen-resistant cells. Ectopic expression of cyclin E1 or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, CDK2 inhibition of E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition. Cyclin E2 overexpression is therefore a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition. CDK2 inhibitors hold promise as a component of combination therapies in endocrine-resistant disease as they effectively inhibit cyclin E1 and E2 overexpressing cells and enhance the efficacy of other therapeutics.

Regression of Human Prostate Cancer Xenografts in Mice by AMG 212/BAY2010112, a Novel PSMA/CD3-Bispecific BiTE Antibody Cross-Reactive with Non-Human Primate Antigens

Abstract: For treatment of patients with prostate cancer (PCa), we developed a novel T cell-engaging (BiTE) antibody designated AMG 212 or BAY2010112 that is bispecific for prostate-specific membrane antigen (PSMA) and the CD3 epsilon subunit of the T cell receptor complex. AMG 212/BAY2010112 induced target cell-dependent activation and cytokine release of T cells, and efficiently redirected T cells for lysis of target cells. In addition to Chinese hamster ovary cells stably expressing human or cynomolgus monkey PSMA, T cells redirected by AMG 212/BAY2010112 also lysed human PCa cell lines VCaP, 22Rv1, MDA PCa 2b, C4-2, PC-3-huPSMA, and LnCaP at half maximal BiTE concentrations between 0.1 and 4 ng/mL (1.8-72 pmol/L). No lysis of PSMA-negative human PCa cell lines PC-3 and DU145 was observed. The subcutaneous (s.c.) formation of tumors from PC-3-huPSMA cells in NOD/SCID mice was significantly prevented by once daily intravenous (i.v.) injection of AMG 212/BAY2010112 at a dose level as low as 0.005 mg/kg/d. Rapid tumor shrinkage with complete remissions were observed in NOD/SCID mice bearing established s.c. 22Rv1 xenografts after repeated daily treatment with AMG 212/BAY2010112 by either the i.v. or s.c. route. Of note, 22Rv1 tumors were grown in the absence of human T cells followed by intraperitoneal injection of T cells 3 days before BiTE treatment. No effects on tumor growth were observed in the absence of human T cells or AMG 212/BAY2010112. On the basis of these preclinical results, AMG 212/BAY2010112 appears as a promising new BiTE antibody for the treatment of patients with PSMA-expressing PCa.

Inhibition of STAT3 Signaling Pathway by Nitidine Chloride Suppressed the Angiogenesis and Growth of Human Gastric Cancer

Abstract: STAT3 has been strongly implicated in human malignancies, and constitutive activation of STAT3 serves a crucial role in cell survival, angiogenesis, immune evasion, and inflammation. In this study, we showed that nitidine chloride, a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb) DC, exerts potent anticancer activity through STAT3 signaling cascade. Nitidine chloride dose dependently suppressed VEGF-induced endothelial cell proliferation, migration, and tubular structure formation in vitro and dramatically reduced VEGF-triggered neovascularization in mouse cornea and Matrigel plugs in vivo. This angiogenesis inhibition mediated by nitidine chloride was well interpreted by the suppression of Janus kinase 2/STAT3 signaling and STAT3 DNA-binding activity in endothelial cells. Furthermore, nitidine chloride suppressed the constitutively activated STAT3 protein, its DNA-binding activity, and the expression of STAT3-dependent target genes, including cyclin D1, Bcl-xL, and VEGF in human gastric cancer cells. Consistent with the earlier findings, nitidine chloride inhibited gastric tumor cell growth and induced tumor cell apoptosis in vitro and effectively suppressed the volume, weight, and microvessel density of human SGC-7901 gastric solid tumors (n = 8) at a dosage of 7 mg/kg/d (intraperitoneal injection). Immunohistochemistry and Western blot analysis further revealed that the expression of STAT3, CD31, and VEGF protein in xenografts was remarkably decreased by the alkaloid. Taken together, we propose that nitidine chloride is a promising anticancer drug candidate as a potent STAT3 signaling inhibitor.

Nanobodies Targeting the Hepatocyte Growth Factor: Potential New Drugs for Molecular Cancer Therapy

Abstract: Hepatocyte growth factor (HGF) and its receptor c-Met are associated with increased aggressiveness of tumors and poor prognostic outcome of patients with cancer. Here, we report the development and characterization of therapeutic anti-HGF (αHGF)-Nanobodies and their potential for positron emission tomographic (PET) imaging to assess HGF expression in vivo. Two αHGF-Nanobodies designated 1E2 and 6E10 were identified, characterized, and molecularly fused to an albumin-binding Nanobody unit (Alb8) to obtain serum half-life extension. The resulting Nanobody formats were radiolabeled with the positron emitter zirconium-89 ((89)Zr, t(1/2;) = 78 hours), administered to nude mice bearing U87 MG glioblastoma xenografts, and their biodistribution was assessed. In addition, their therapeutic effect was evaluated in the same animal model at doses of 10, 30, or 100 μg per mouse. The (89)Zr-Nanobodies showed similar biodistribution with selective tumor targeting. For example, 1E2-Alb8 showed decreased blood levels of 12.6%ID/g ± 0.6%ID/g, 7.2%ID/g ± 1.0%ID/g, 3.4%ID/g ± 0.3%ID/g, and 0.3%ID/g ± 0.1%ID/g at 1, 2, 3, and 7 days after injection, whereas tumor uptake levels remained relatively stable at these time points: 7.8%ID/g ± 1.1%ID/g, 8.9%ID/g ± 1.0%ID/g, 8.7%ID/g ± 1.5%ID/g, and 7.2%ID/g ±1.6%ID/g. Uptake in normal tissues was lower than in tumor, except for kidneys. In a therapy study, all Nanobody-treated mice showed tumor growth delay compared with the control saline group. In the 100-μg group, four of six mice were cured after treatment with 1E2-Alb8 and 73 days follow-up, and three of six mice when treated with 6E10-Alb8. These results provide evidence that Nanobodies 1E2-Alb8 and 6E10-Alb8 have potential for therapy and PET imaging of HGF-expressing tumors.