Anti-Claudin-2 Antibody-Drug Conjugates for the Treatment of Colorectal Cancer Liver Metastasis.

We have demonstrated that Claudin-2 is required for colorectal cancer (CRC) liver metastasis. Expression of Claudin-2 in primary CRC is associated with poor survival and is highly expressed in liver metastases. Claudin-2 also promotes breast cancer liver metastasis by enabling seeding and cancer cell survival. These observations support Claudin-2 as a potential therapeutic target for managing patients with liver metastases. Antibody-drug conjugates (ADCs) are promising anti-tumor therapeutics that combine the specific targeting ability of monoclonal antibodies with the potent cell killing activity of cytotoxic drugs. Here we report the generation of twenty-eight anti-Claudin-2 antibodies for which the binding specificities, the cross-reactivity with Claudin family members and the cross-species reactivity were assessed by flow cytometry analysis. Multiple drug conjugates were tested and PNU was selected for conjugation with anti-Claudin-2 antibodies binding either extracellular loop 1 or extracellular loop 2. Anti-Claudin-2 ADCs were efficiently internalized and effective at killing Claudin-2-expressing CRC cancer cells in vitro. Importantly, PNU-conjugated-anti-Claudin-2 ADCs impaired the development of replacement type CRC liver metastases in vivo, using established CRC cell lines and patient-derived xenograft (PDX) models of CRC liver metastases. Our results suggest that the development of ADCs targeting Claudin-2 is a promising therapeutic strategy for managing CRC liver-metastatic patients that present with replacement type liver metastases.

Functional genomics of the rat neuromedin U receptor 1 reveals a naturally occurring deleterious allele.

Neuromedin U (NMU) plays an important role in a number of physiological processes, but the relative contribution of its two known receptors, NMUR1 and NMUR2, is still poorly understood. Here we report the existence of a SNP T(1022)→A (Val(341)→Glu) in the third exon of the rat Nmur1 gene that leads to an inactive receptor. This SNP is present within the coding region of the highly conserved NPXXY motif found within all class A type G protein-coupled receptors and translates to an NMUR1 receptor that is not expressed on the cell surface. Genetic analysis of the Nmur1 gene in a population of Sprague-Dawley rats revealed that this strain is highly heterogeneous for the inactivating polymorphism. The loss of functional NMUR1 receptors in Sprague-Dawley rats homozygous for the inactive allele was confirmed by radioligand binding studies on native tissue expressing NMUR1. The physiological relevance of this functional genomics finding was examined in two nociceptive response models. The pronociceptive effects of NMU were abolished in rats lacking functional NMUR1 receptors. The existence of naturally occurring NMUR1-deficient rats provides a novel and powerful tool to investigate the physiological role of NMU and its receptors. Furthermore, it highlights the importance of verifying the NMUR1 single nucleotide polymorphism status for rats used in physiological, pharmacological or toxicological studies conducted with NMUR1 modulators.

Biochemical characterization and in vitro activity of AZ513, a noncovalent, reversible, and noncompetitive inhibitor of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) hydrolyzes several bioactive lipids including the endocannabinoid anandamide. Synthetic FAAH inhibitors are being generated to help define the biological role(s) of this enzyme, the lipids it degrades in vivo, and the disease states that might benefit from its pharmacological modulation. AZ513 inhibits human FAAH (IC(50)=551 nM), is 20-fold more potent against rat FAAH (IC(50)=27 nM), and is inactive at 10 µM against the serine hydrolases acetylcholinesterase, thrombin, and trypsin. In contrast to most other potent FAAH inhibitors, AZ513 showed no evidence of covalently modifying the enzyme and displayed reversible inhibition. In an enzyme cross-competition assay, AZ513 did not compete with OL-135, an inhibitor that binds to the catalytic site in FAAH, which indicates that AZ513 does not bind to the catalytic site and is therefore noncompetitive with respect to substrate. AZ513 has good cell penetration as demonstrated by inhibition of anandamide hydrolysis in human FAAH-transfected HEK293 cells (IC(50)=360 nM). AZ513 was tested in a rat spinal cord slice preparation where CB(1) activation reduces excitatory post-synaptic currents (EPSCs). In this native tissue assay of synaptic activity, AZ513 reduced EPSCs, which is consistent with inhibiting endogenous FAAH and augmenting endocannabinoid tone. AZ513 has a unique biochemical profile compared with other published FAAH inhibitors and will be a useful tool compound to further explore the role of FAAH in various biological processes.

Distribution of the voltage-gated sodium channel Na(v)1.7 in the rat: expression in the autonomic and endocrine systems.

It is generally accepted that the voltage-gated, tetrodotoxin-sensitive sodium channel, Na(V)1.7, is selectively expressed in peripheral ganglia. However, global deletion in mice of Na(V)1.7 leads to death shortly after birth (Nassar et al. [2004] Proc. Natl. Acad. Sci. U. S. A. 101:12706-12711), suggesting that this ion channel might be more widely expressed. To understand better the potential physiological function of this ion channel, we examined Na(V)1.7 expression in the rat by in situ hybridization and immunohistochemistry. As expected, highest mRNA expression levels are found in peripheral ganglia, and the protein is expressed within these ganglion cells and on the projections of these neurons in the central nervous system. Importantly, we found that Na(V)1.7 is present in discrete rat brain regions, and the unique distribution pattern implies a central involvement in endocrine and autonomic systems as well as analgesia. In addition, Na(V)1.7 expression was detected in the pituitary and adrenal glands. These results indicate that Na(V)1.7 is not only involved in the processing of sensory information but also participates in the regulation of autonomic and endocrine systems; more specifically, it could be implicated in such vital functions as fluid homeostasis and cardiovascular control.

A stop codon mutation in SCN9A causes lack of pain sensation.

The general lack of pain experience is a rare occurrence in humans, and the molecular causes for this phenotype are not well understood. Here we have studied a Canadian family from Newfoundland with members who exhibit a congenital inability to experience pain. We have mapped the locus to a 13.7 Mb region on chromosome 2q (2q24.3-2q31.1). Screening of candidate genes in this region identified a protein-truncating mutation in SCN9A, which encodes for the voltage-gated sodium channel Na(v)1.7. The mutation is a C-A transversion at nucleotide 984 transforming the codon for tyrosine 328 to a stop codon. The predicted product lacks all pore-forming regions of Na(v)1.7. Indeed, expression of this altered gene in a cell line did not produce functional responses, nor did it cause compensatory effects on endogenous voltage-gated sodium currents when expressed in ND7/23 cells. Because a homozygous knockout of Na(v)1.7 in mice has been shown to be lethal, we explored why a deficiency of Na(v)1.7 is non-lethal in humans. Expression studies in monkey, human, mouse and rat tissue indicated species-differences in the Na(v)1.7 expression profile. Whereas in rodents the channel was strongly expressed in hypothalamic nuclei, only weak mRNA levels were detected in this area in primates. Furthermore, primate pituitary and adrenal glands were devoid of signal, whereas these two glands were mRNA-positive in rodents. This species difference may explain the non-lethality of the observed mutation in humans. Our data further establish Na(v)1.7 as a critical element of peripheral nociception in humans.