A Polymer-Based Antibody-Vinca Drug Conjugate Platform: Characterization and Preclinical Efficacy.

Antibody-drug conjugates (ADC) are an emerging drug class that uses antibodies to improve cytotoxic drug targeting for cancer treatment. ADCs in current clinical trials achieve a compromise between potency and physicochemical/pharmacokinetic properties by conjugating potent cytotoxins directly to an antibody at a 4:1 or less stoichiometric ratio. Herein, we report a novel, polyacetal polymer-based platform for creating ADC that use poly-1-hydroxymethylethylene hydroxymethyl-formal (PHF), also known as Fleximer. The high hydrophilicity and polyvalency properties of the Fleximer polymer can be used to produce ADC with high drug loading without compromising physicochemical and pharmacokinetic properties. Using trastuzumab and a vinca drug derivative to demonstrate the utility of this platform, a novel Fleximer-based ADC was prepared and characterized in vivo. The ADC prepared had a vinca-antibody ratio of 20:1. It exhibited a high antigen-binding affinity, an excellent pharmacokinetic profile and antigen-dependent efficacy, and tumor accumulation in multiple tumor xenograft models. Our findings illustrate the robust utility of the Fleximer platform as a highly differentiated alternative to the conjugation platforms used to create ADC currently in clinical development.

Helicobacter pylori virulence factors affecting gastric proton pump expression and acid secretion.

Acute Helicobacter pylori infection of gastric epithelial cells and human gastric biopsies represses H,K-ATPase α subunit (HKα) gene expression and inhibits acid secretion, causing transient hypochlorhydria and supporting gastric H. pylori colonization. Infection by H. pylori strains deficient in the cag pathogenicity island (cag PAI) genes cagL, cagE, or cagM, which do not transfer CagA into host cells or induce interleukin-8 secretion, does not inhibit HKα expression, nor does a cagA-deficient strain that induces IL-8. To test the hypothesis that virulence factors other than those mediating CagA translocation or IL-8 induction participate in HKα repression by activating NF-κB, AGS cells transfected with HKα promoter-Luc reporter constructs containing an intact or mutated NF-κB binding site were infected with wild-type H. pylori strain 7.13, isogenic mutants lacking cag PAI genes responsible for CagA translocation and/or IL-8 induction (cagA, cagζ, cagε, cagZ, and cagß), or deficient in genes encoding two peptidoglycan hydrolases (slt and cagγ). H. pylori-induced AGS cell HKα promoter activities, translocated CagA, and IL-8 secretion were measured by luminometry, immunoblotting, and ELISA, respectively. Human gastric biopsy acid secretion was measured by microphysiometry. Taken together, the data showed that HKα repression is independent of IL-8 expression, and that CagA translocation together with H. pylori transglycosylases encoded by slt and cagγ participate in NF-κB-dependent HKα repression and acid inhibition. The findings are significant because H. pylori factors other than CagA and IL-8 secretion are now implicated in transient hypochlorhydria which facilitates gastric colonization and potential triggering of epithelial progression to neoplasia.

Discrimination of normal and esophageal cancer plasma proteomes by MALDI-TOF mass spectrometry.

BACKGROUND: Most patients presenting with symptoms of esophageal cancer (EC) have advanced disease. Even with resection, the cure rate is extremely low due to local recurrence and metastatic disease. Early detection and effective therapeutic intervention are essential to improve survival. AIMS: This study tested the hypothesis that the presence of EC modulates concentrations of specific plasma proteins and peptides, potentially allowing discrimination between EC and controls based on mass spectrometric analysis of the respective plasma proteomes. METHODS: Blood samples from 79 esophageal cancer patients and 40 age-matched normal subjects were processed to plasma, and protein/peptide sub-fractions were isolated using HIC8 or WAX-derivatized superparamagnetic beads. Triplicate matrix-assisted laser desorption time-of-flight mass spectra were acquired for specific plasma fractions from each subject. RESULTS: HIC8 and WAX-derivatized plasma eluates yielded 79 and 77 candidate features, respectively, and a Random Forest algorithm identified a subset of features whose peak intensities allowed discrimination between cancer patients and controls. Areas under the curve in receiver operating characteristic curves for HIC8 spectra were 0.88 and 0.83 for WAX spectra. The combined feature set discriminated EC from control plasma with 79 % sensitivity and 79 % specificity, with positive and negative test likelihood ratios of >14 and 0.17, respectively. CONCLUSIONS: These data lay the foundation for the development of a clinically useful test for esophageal cancer based on statistical analysis of proteomic spectra of patient plasma samples. This approach will be validated by analysis of larger patient cohorts, development of cancer-specific classifiers, and assessment of racial origin imbalances.

Helicobacter pylori-induced posttranscriptional regulation of H-K-ATPase α-subunit gene expression by miRNA.

Acute Helicobacter pylori infection of gastric epithelial cells induces CagA oncoprotein- and peptidoglycan (SLT)-dependent mobilization of NF-κB p50 homodimers that bind to H-K-ATPase α-subunit (HKα) promoter and repress HKα gene transcription. This process may facilitate gastric H. pylori colonization by induction of transient hypochlorhydria. We hypothesized that H. pylori also regulates HKα expression posttranscriptionally by miRNA interaction with HKα mRNA. In silico analysis of the HKα 3' untranslated region (UTR) identified miR-1289 as a highly conserved putative HKα-regulatory miRNA. H. pylori infection of AGS cells transfected with HKα 3' UTR-Luc reporter construct repressed luciferase activity by 70%, whereas ΔcagA or Δslt H. pylori infections partially abrogated repression. Transfection of AGS cells expressing HKα 3' UTR-Luc construct with an oligoribonucleotide mimetic of miR-1289 induced maximal repression (54%) of UTR activity within 30 min; UTR activity was unchanged by nontargeting siRNA transfection. Gastric biopsies from patients infected with cagA(+) H. pylori showed a significant increase in miR-1289 expression compared with uninfected patients or those infected with cagA(-) H. pylori. Finally, miR-1289 expression was necessary and sufficient to attenuate biopsy HKα protein expression in the absence of infection. Taken together, these data indicate that miR-1289 is upregulated by H. pylori in a CagA- and SLT-dependent manner and targets HKα 3' UTR, affecting HKα mRNA translation. The sensitivity of HKα mRNA 3' UTR to binding of miR-1289 identifies a novel regulatory mechanism of gastric acid secretion and offers new insights into mechanisms underlying transient H. pylori-induced hypochlorhydria.

Helicobacter pylori represses proton pump expression and inhibits acid secretion in human gastric mucosa.

BACKGROUND AND AIMS: Helicobacter pylori infection of gastric mucosa causes gastritis and transient hypochlorhydria, which may provoke emergence of a mucosal precancer phenotype; H pylori strains containing a cag pathogenicity island (PAI) augment cancer risk. Acid secretion is mediated by the catalytic alpha subunit of parietal cell H,K-ATPase (HKalpha). In AGS gastric epithelial cells, H pylori induces nuclear factor-kappaB (NF-kappaB) binding to and repression of transfected HKalpha promoter activity. This study sought to identify bacterial genes involved in HKalpha repression and to assess their impact on acid secretion. METHODS AND RESULTS: AGS cells transfected with an HKalpha promoter construct or human gastric body biopsies were infected with wild-type (wt) or isogenic mutant (IM) H pylori strains. AGS cell HKalpha promoter activity, and biopsy HKalpha mRNA, protein and H(+) secretory activity were measured by luminometry, reverse transcription-PCR, immunoblotting and extracellular acidification, respectively. Wt H pylori and DeltavacA, DeltaureA, Deltaslt and DeltaflaA IM strains repressed HKalpha promoter activity by approximately 50%, a DeltacagA IM strain repressed HKalpha by approximately 33%, and DeltacagE, DeltacagM and DeltacagL IM strains elicited no HKalpha repression. Wt H pylori-infected biopsies had markedly reduced HKalpha mRNA and protein compared with IM strain infections or mock-infected controls. Histamine-stimulated, SCH28080-sensitive biopsy acid secretion was significantly inhibited by wt but not by DeltacagL IM H pylori infection compared with vehicle-only controls. CONCLUSIONS: It is concluded that H pylori cag PAI gene products CagE, CagM, CagL and, possibly, CagA are mechanistically involved in repression of HKalpha transcription. Further, acute H pylori infection of human gastric mucosa downregulates parietal cell H,K-ATPase expression, significantly inhibiting acid secretion.

Helicobacter pylori CagL activates ADAM17 to induce repression of the gastric H, K-ATPase alpha subunit.

BACKGROUND & AIMS: Infection with Helicobacter pylori represses expression of the gastric H, K-adenosine triphosphatase alpha-subunit (HKalpha), which could contribute to transient hypochlorhydria. CagL, a pilus protein component of the H pylori type IV secretion system, binds to the integrin alpha(5)beta1 to mediate translocation of virulence factors into the host cell and initiate signaling. alpha(5)beta1 binds a disintegrin and metalloprotease (ADAM) 17, a metalloenzyme that catalyzes ectodomain shedding of receptor tyrosine kinase ligands. We investigated whether H pylori-induced repression of HKalpha is mediated by CagL activation of ADAM17 and release of heparin-binding epidermal growth factor (HB-EGF). METHODS: HKalpha promoter and ADAM17 activity were measured in AGS gastric epithelial cells transfected with HKalpha promoter-reporter constructs or ADAM17-specific small interfering RNAs and infected with H pylori. HB-EGF secretion was measured by enzyme-linked immunosorbent assay analysis, and ADAM17 interaction with integrins was investigated by coimmunoprecipitation analyses. RESULTS: Infection of AGS cells with wild-type H pylori or an H pylori cagL-deficient isogenic mutant that also contained a wild-type version of cagL (P12DeltacagL/cagL) repressed HKalpha promoter-Luc reporter activity and stimulated ADAM17 activity. Both responses were inhibited by point mutations in the nuclear factor-kappaB binding site of HKalpha or by infection with P12DeltacagL. Small interfering RNA-mediated silencing of ADAM17 in AGS cells inhibited the repression of wild-type HKalpha promoter and reduced ADAM17 activity and HB-EGF production, compared to controls. Coimmunoprecipitation studies of AGS lysates showed that wild-type H pylori disrupted ADAM17-alpha5beta1 complexes. CONCLUSIONS: During acute H pylori infection, CagL dissociates ADAM17 from the integrin alpha(5)beta1 and activates ADAM17-dependent, nuclear factor-kappaB-mediated repression of HKalpha. This might contribute to transient hypochlorhydria in patients with H pylori infection.

The role of Sp1 in IL-1beta and H. pylori-mediated regulation of H,K-ATPase gene transcription.

Helicobacter pylori infection of the gastric body induces transient hypochlorhydria and contributes to mucosal progression toward gastric carcinoma. Acid secretion is mediated by parietal cell H,K-ATPase, in which the catalytic alpha-subunit (HKalpha) promoter activity in transfected gastric epithelial [gastric adenocarcinoma (AGS)] cells is repressed by H. pylori through NF-kappaB p50 homodimer binding to the promoter. IL-1beta, an acid secretory inhibitor whose mucosal level is increased by H. pylori, upregulates HKalpha promoter activity in AGS cells. Because IL-1beta also activates NF-kappaB signaling, we investigated disparate HKalpha regulation by H. pylori and IL-1beta, testing the hypothesis that IL-1beta-induced HKalpha promoter activation is mediated by the transcription factor Sp1. DNase I footprinting revealed Sp1 binding to the HKalpha promoter at -56 to -39 bp. IL-1beta stimulated the activity of three HKalpha promoter constructs containing NF-kappaB and Sp1 sites transfected into AGS cells and also stimulated a construct containing only an Sp1 site. This stimulation was abrogated by mutating the HKalpha promoter Sp1 binding site. Gelshift assays showed that IL-1beta increased Sp1 but not p50 binding to cognate HKalpha probes and that Sp1 also interacts with an HKalpha NF-kappaB site when bound to its cognate HKalpha cis-response element. H. pylori did not augment Sp1 binding to an HKalpha Sp1 probe, and small interfering RNA-mediated knockdown of Sp1 expression abrogated IL-1beta-induced HKalpha promoter stimulation. We conclude that IL-1beta upregulates HKalpha gene transcription by inducing Sp1 binding to HKalpha Sp1 and NF-kappaB sites and that the H. pylori perturbation of HKalpha gene expression is independent of Sp1-mediated basal HKalpha transcription.

Helicobacter pylori-induced H,K-ATPase alpha-subunit gene repression is mediated by NF-kappaB p50 homodimer promoter binding.

Infection of human gastric body mucosa by the gram-negative, microaerophilic bacterium Helicobacter pylori induces an inflammatory response and a transitory hypochlorhydria that progresses in approximately 2% of patients to atrophic gastritis, dysplasia, and gastric adenocarcinoma. We have previously shown that H. pylori infection of cultured gastric epithelial cells (AGS) represses the activity of the transfected alpha-subunit (HKalpha) promoter of H,K-ATPase, the parietal cell enzyme mediating acid secretion. However, the mechanistic details of H. pylori-mediated repression of HKalpha and ensuing hypochlorhydria are unknown. H. pylori is known to upregulate the transcription factor NF-kappaB through the ERK1/2 MAPK pathway. We identified NF-kappaB-binding regions in the HKalpha promoter and found that H. pylori inoculation of AGS cells increased NF-kappaB p50 binding to the transfected HKalpha promoter and repressed its transcriptional activity. Immunoblot and DNA-protein interaction studies showed that although active phosphorylated NF-kappaB p65 is present in H. pylori-infected AGS cells, an NF-kappaB p50/p65 heterodimeric complex fails to bind to the HKalpha promoter. Point mutations at -159 and -161 bp in the HKalpha promoter NF-kappaB binding sequence prevented binding of NF-kappaB p50 and prevented H. pylori repression of point-mutated HKalpha promoter activity in transfected AGS cells. Small interfering RNA-mediated knockdown of NF-kappaB p50 in H. pylori-infected AGS cells also abrogated H. pylori-induced HKalpha repression, whereas NF-kappaB p65 knockdown did not. We conclude that H. pylori inhibits HKalpha gene expression by ERK1/2-mediated NF-kappaB p50 homodimer binding to the HKalpha promoter. This study identifies a novel pathogen-dependent mechanism of H,K-ATPase inhibition and contributes to understanding of H. pylori pathophysiology.

IL-1beta modulation of H,K-ATPase alpha-subunit gene transcription in Helicobacter pylori infection.

Helicobacter pylori infection of the human gastric body induces hypochlorhydria by perturbing acid secretion. H. pylori inhibits parietal cell H,K-ATPase alpha-subunit (HKalpha) gene and protein expression, providing a mechanistic basis for clinical hypochlorhydria. Given that H. pylori infection increases gastric mucosal IL-1beta, an acid secretory inhibitor, we investigated the role of IL-1beta in H. pylori-mediated inhibition of HKalpha transcription. Human gastric adenocarcinoma (AGS) cells were transfected with promoter-reporter constructs containing human HKalpha 5'-flanking sequence deletions. IL-1beta (10 ng/ml) had no effect on the transcriptional activity of six progressively shorter deletion constructs of the HKalpha promoter (HKalpha2179-HKalpha340) and significantly stimulated the activity of HKalpha206, HKalpha177, HKalpha165, and HKalpha102 deletion constructs (80%, 100%, 46%, and 35%, respectively). H. pylori inhibited the transcriptional activity of HKalpha2179, HKalpha206, HKalpha177, and HKalpha165; IL-1beta relieved the H. pylori inhibition of HKalpha2179 and HKalpha206 activity but not HKalpha177 and HKalpha165 activity. AGS cell pretreatment with a MEK1/2 inhibitor prevented the IL-1beta-mediated stimulation, but p38 and JNK pathway inhibitors did not. IL-1beta mRNA levels in AGS cells were low and unaffected by H. pylori, and ELISAs of H. pylori-conditioned AGS culture media showed no measurable IL-1beta secretion. These data indicate that an IL-1beta-dependent cis-response element lies downstream of -206 nt in the HKalpha promoter and that IL-1beta-mediated upregulation of HKalpha transcription is affected by an ERK1/2 kinase signal pathway. We conclude that an IL-1beta-responsive HKalpha cis element positively regulates HKalpha gene transcription in shortened deletion constructs and that H. pylori-induced inhibition of HKalpha transcription is not mediated by IL-1beta.

Inhibition of gastric H,K-ATPase activity and gastric epithelial cell IL-8 secretion by the pyrrolizine derivative ML 3000.

BACKGROUND: ML 3000 ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid) is an inhibitor of both cyclooxygenase and 5-lipoxygenase in vitro, and shows promise as a novel non-steroidal anti-inflammatory drug (NSAID). Unlike conventional NSAIDs which are associated with gastric ulcerogenic effects, ML 3000 causes little or no damage to the gastric mucosa, even though it significantly depresses gastric prostaglandin synthesis. METHODS: As part of an effort to clarify mechanisms underlying the gastric sparing properties of ML 3000, we studied the effects of ML 3000 on H,K-ATPase activity in vitro, on acid accumulation in isolated gastric parietal cells, and on IL-8 secretion by gastric epithelial cells in culture. RESULTS: SCH28080-sensitive H,K-ATPase activity in highly-purified pig gastric microsomes was dose-dependently inhibited by ML 3000 (IC50 = 16.4 microM). Inhibition was reversible, and insensitive to ML 3000 acidification in the pH range 2.0-8.0. In rabbit gastric parietal cells,ML 3000 dose-dependently inhibited histamine-stimulated acid accumulation (IC50 = 40 microM) and forskolin-stimulated acid accumulation (IC50 = 45 microM). Lastly, in human gastric adenocarcinoma (AGS) cells, ML 3000 dose-dependently inhibited both baseline and IL-1beta-stimulated (20 ng/ml) IL-8 secretion with IC50s of 0.46 microM and 1.1 microM respectively. CONCLUSION: The data indicate that ML 3000 affects acid-secretory mechanisms downstream of cAMP mobilization induced by histamine H2 receptor activation, that it directly inhibits H,K-ATPase specific activity, and that baseline gastric epithelial cell IL-8 secretory inhibition may be mediated by ML 3000 inhibition of 5-lipoxygenase activity. We conclude that these gastric function inhibitory data may underlie the gastric sparing properties of ML 3000.