Development of ASG-15ME, a Novel Antibody-Drug Conjugate Targeting SLITRK6, a New Urothelial Cancer Biomarker.

SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody-drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301-10. ©2016 AACR.

AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody-Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML.

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody-drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34(+)CD38(-) leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML.

Differential temporal changes in brain and gut substance P mRNA expression throughout the time-course of cisplatin-induced vomiting in the least shrew (Cryptotis parva).

Cisplatin and related chemotherapeutics are potent emetogens in humans and least shrews, a small animal emesis model which also vomits in response to substance P (SP). The SP-producing preprotachykinin-1 (PPT1) mRNA is transcribed from the Tac1 gene, which has been sequenced from several animal species and humans and is highly conserved. Despite its prominent role in chemotherapy-induced vomiting, the tachykininergic system is not well-characterized in emesis-competent species. This study was undertaken to further establish Cryptotis parva as an emesis model, by sequencing and characterizing SP mRNA, and then comparing the least shrew tachykininergic system to other mammalian species (vomiting and non-vomiting). The cDNA for least shrew beta-PPT1 was successfully cloned and partially sequenced, and found to be 90% homologous to the human sequence, with the SP-producing portion identical to humans. Initial in situ hybridization results demonstrated induction of beta-PPT1 mRNA in the gut following cisplatin administration. These were followed up with mRNA quantification (via QPCR) at multiple time points following cisplatin injection. PPT1 mRNA levels in the brain spiked at 4 h (19-fold increase) and 24 h (20-fold increase) in correlation with cisplatin-induced emesis. PPT1 mRNA in the gut spiked at 28 h (approximately 6.5-fold increase), correlated with the later phase of vomiting. These results validate the least shrew as a tachykinin model at the molecular level.

A re-evaluation of the neurotransmitter basis of chemotherapy-induced immediate and delayed vomiting: evidence from the least shrew.

Although the neurotransmitter basis of chemotherapy-induced vomiting (CIV) is thought to be multifactorial, it is generally accepted that acute (immediate) CIV is mainly due to the release of serotonin (5-HT) within the gastrointestinal tract, while the delayed phase occurs following substance P (SP) release in the brainstem. The aim of the current study was to test this dogma in the least shrew model of vomiting. Thus, we initially investigated the temporal development of cisplatin's immediate and delayed emetic effects in the least shrew and subsequently determined the concomitant changes in the turnover of major emetic neurotransmitters both in the central and peripheral loci associated with CIV. Cisplatin (0, 5, 10 and 20 mg/kg, i.p.) caused dose- and time-dependent emetic effects. A 10 mg/kg dose of cisplatin produced both phases of emesis with corresponding peak mean frequencies occurring at 2-3 and 33 h post-treatment, at 5 mg/kg it failed to cause significant emesis in either phase, while its 20 mg/kg dose induced both phases earlier but toxicity restricted the full 47 hour observation. Cisplatin (10 mg/kg, i.p.)-induced peak immediate and delayed phases were associated with concomitant increases in the turnover of 5-HT, dopamine and SP in both the shrew brainstem and jejunum. The discussed increases during both phases appear to be site specific since neurotransmitter release was not persistently altered in the shrew frontal cortex or duodenum, although occasionally increases or decreases did occur. Our findings suggest that the least shrew appears to be a sensitive and rapid emesis model for both phases of CIV, and both emetic phases are associated with specific increases in the release of all of the cited neurotransmitters in both the brainstem and jejunum. Thus, the generally accepted neurotransmitter dogma needs to be updated since more recent neurochemical studies in humans as well as other clinical findings support the current basic results obtained in the least shrew.

Utilization of the least shrew as a rapid and selective screening model for the antiemetic potential and brain penetration of substance P and NK1 receptor antagonists.

Substance P (SP) is thought to play a cardinal role in emesis via the activation of central tachykinin NK1 receptors during the delayed phase of vomiting produced by chemotherapeutics. Although the existing supportive evidence is significant, due to lack of an appropriate animal model, the evidence is indirect. As yet, no study has confirmed that emesis produced by SP or a selective NK1 receptor agonist is sensitive to brain penetrating antagonists of either NK1, NK2, or NK3 receptors. The goals of this investigation were to demonstrate: 1) whether intraperitoneal (i.p.) administration of either SP, a brain penetrating (GR73632) or non-penetrating (e.g. SarMet-SP) NK1 receptor agonist, an NK2 receptor agonist (GR64349), or an NK3 receptor agonist (Pro7-NKB), would induce vomiting and/or scratching in the least shrew (Cryptotis parva) in a dose-dependent manner; and whether these effects are sensitive to the above selective receptor antagonists; 2) whether an exogenous emetic dose of SP (50 mg/kg, i.p.) can penetrate into the shrew brain stem and frontal cortex; 3) whether GR73632 (2.5 mg/kg, i.p.)-induced activation of NK1 receptors increases Fos-measured neuronal activity in the neurons of both brain stem emetic nuclei and the enteric nervous system of the gut; and 4) whether selective ablation of peripheral NK1 receptors can affect emesis produced by GR73632. The results clearly demonstrated that while SP produced vomiting only, GR73632 caused both emesis and scratching behavior dose-dependently in shrews, and these effects were sensitive to NK1-, but not NK2- or NK3-receptor antagonists. Neither the selective, non-penetrating NK1 receptor agonists, nor the selective NK2- or NK3-receptor agonists, caused a significant dose-dependent behavioral effect. An emetic dose of SP selectively and rapidly penetrated the brain stem but not the frontal cortex. Systemic GR73632 increased Fos expression in the enteric nerve plexi, the medial subnucleus of nucleus tractus solitarius, and the dorsal motor nucleus of the vagus, but not the area postrema. Ablation of peripheral NK1 receptors attenuated the ability of GR73632 to induce a maximal frequency of emesis and shifted its percent animals vomiting dose-response curve to the right. The NK1-ablated shrews exhibited scratching behavior after systemic GR73632-injection. These results, for the first time, affirm a cardinal role for central NK1 receptors in SP-induced vomiting, and a facilitatory role for gastrointestinal NK1 receptors. In addition, these data support the validation of the least shrew as a specific and rapid behavioral animal model to screen concomitantly both the CNS penetration and the antiemetic potential of tachykinin NK1 receptor antagonists.

New tissue microarray technology for analyses of gene expression in frozen pathological samples.

Tissue microarrays (TMAs) are widely used to analyze gene expression in multiple pathological samples on a single slide. Currently, most TMA slides are made by coring paraffin-embedded tissues and arraying them into a paraffin block, from which TMA sections are cut. However paraffin-based TMA technology may not be compatible with frozen clinical tissue samples, which have a higher quality of RNAs and proteins for preparing TMAs than paraffin-embedded tissue samples. In this study, we developed an alternative TMA technology that is applicable to a broader range of frozen tissue samples. Our method takes advantage of a newly developed array recipient block that can be used to array small tissue cores. After arraying tissue cores, the tissue block can be immediately sectioned on a cryostat microtome to make TMA slides. TMAs made using this method have well-defined array configurations and good tissue/cell morphology. Immunohistochemistry and in situ hybridization study also revealed well-preserved proteins and mRNAs on TMA slides. Our method significantly simplifies TMA preparation and assembly when frozen pathological tissues are used. Our technology provides an alternative tool for creating high-quality TMAs for the general research community to study gene expressions in pathological samples.

Recurrent inguinal hernia: a ten-year review.

BACKGROUND: This study evaluates the results obtained in recurrent inguinal hernia repair over the past ten years in a general hospital using laparoscopic (LAP) and open tension-free mesh (Lichtenstein) procedures. METHODS: A prospective controlled study with 258 recurrent inguinal hernias in 235 patients over a ten-year period. The main outcome measurements were recurrence rate, operating time, hospital stay, postoperative complications, and cost. RESULTS: There were 10 recurrences (4.3%): 7 in the Lichtenstein group (5.7%) and 3 (2.2%) in the LAP group (P = nonsignificant [NS]). There were 15 (12.2%) postoperative complications in the Lichtenstein group and 6 (4.4%) in the LAP group (P =0.04). The operating room costs were higher in the LAP group, but this difference was offset by a significantly shorter hospital stay, shorter operating time, and earlier return to work. CONCLUSION: Laparoscopic repair is an effective option for the treatment of recurrent inguinal hernia. The TEP approach combines the advantages of minimal invasive surgery and those of tension-free mesh repair, reducing operating time, postoperative morbidity, and recurrence rate.