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Background: CEACAM5 and CEACAM6 are glycosylphosphatidylinositol (GPI)- linked members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, which are frequently upregulated in epithelial cancers where they contribute to invasion, metastasis, immune evasion, and resistance to anoikis. CT109 is a novel antibody with dual specificity to both CEACAM5 and 6., Objectives: In this study, we aimed to perform the preclinical characterization of CT109 and antibody- drug conjugate (ADCs) derivatives of CT109, focusing on CT109-SN-38., Methods: CT109's cognate epitope was characterized by scanning mutagenesis. CT109 specificity and internalization kinetics were assessed by immunoblot and flow cytometry, respectively. Cognate antigen expression prevalence in colorectal cancer and normal tissue arrays was determined by immunohistochemistry. CT109 conjugations were generated by the reaction of reduced CT109 cysteines with maleimide-functionalized payload linkers. In vitro cytotoxic activity of CT109 ADCs was characterized on antigen-positive and negative pancreatic ductal adenocarcinoma cell (PDAC) lines using a luminometric viability assay. In vivo efficacy of CT109-SN-38 was assessed on a PDAC tumor xenograft model at 10 and 25 mg/kg concentrations., Results: CT109 was shown to bind a glycoepitope centered on N309. CT109 is internalized in the CEACAM5 + /CEACAM6 + double-positive PDAC line, BxPC-3, with a t 1/2 of 2.3 hours. CT109 ADCs elicit a dose and antigen-dependent cytotoxic effect, with CT109-SN-38 exhibiting an IC 50 value of 21 nM in BxPC-3 cells. In a BxPC-3 tumor xenograft model, CT109-SN-38 reduced tumor growth and induced regression in 3/10 mice at a concentration 25 mg/kg., Conclusion: These data suggest that further preclinical and clinical development of CT109-SN-38 is warranted., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

An N-linked glycan often increased during oncogenic transformation contains beta(1,6)-linked GlcNAc, synthesized by the N-acetylglucosaminyltransferase V (GnT-V). The progression of polyoma middle T-antigen oncoprotein-induced mammary carcinomas in GnT-V null mice was significantly retarded compared with that observed in wild-type mice. The matrix adhesion of mouse embryonic fibroblasts (MEF) from GnT-V null and wild-type mice was investigated to understand the mechanism by which deletion of GnT-V could retard tumor progression. GnT-V null MEF displayed enhanced adhesion to and spreading on fibronectin-coated plates with concomitant inhibition of cell migration. GnT-V null MEF also showed increased focal adhesion kinase tyrosine phosphorylation, consistent with decreased cell motility on fibronectin-coated plates. Expression of GnT-V cDNA in the null MEF reversed these abnormal characteristics, indicating the direct involvement of N-glycosylation events in these phenotypic changes. The alpha5beta1 fibronectin receptors exhibited increased clustering on the null MEF cell surfaces, consistent with previous studies that observed less integrin clustering in cells overexpressing GnT-V. Most surprisingly, GnT-V null MEF displayed increased expression levels of both alpha5 and beta1 subunits in lysates and on the cell surface. Increased alpha5beta1 expression in the null MEF was because of increased alpha5beta1 transcript levels that declined after re-expression of GnT-V cDNA, confirming that increased alpha5beta1 expression in null MEF was because of changes in GnT-V expression. The increased null MEF transcripts were shown to be caused at least in part by increased integrin promoter activity. Moreover, increased alpha5beta1 integrin transcripts in GnT-V null MEF were not due to a differential response to fibronectin; rather, they appeared to be mediated by activation of a protein kinase C signaling pathway. These results demonstrate that deletion of MEF GnT-V resulted in enhanced integrin clustering and activation of alpha5beta1 transcription by protein kinase C signaling, which in turn up-regulated levels of cell surface alpha5beta1 fibronectin receptors that resulted in increased matrix adhesion and inhibition of migration.

Vacuolar H(+)-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H(+)-ATPase via one of its transmembrane domains, TMD4, strongly associates with beta(1) integrin, affecting beta(1) integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of beta(1) integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose alpha(5)beta(1) integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of beta(1) integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of beta(1) integrin (premature form) and, consequently, the level of the 130-kDa form of beta(1) integrin (mature form). Further experiments showed that the normal levels of association between the premature beta(1) integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of beta(1) integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits beta(1) integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional beta(1) integrin.

Altered expression of cell surface N-linked oligosaccharides is associatedwith the oncogenic transformation of many types of animal cells. One of the most common forms of glycosylation in transformed cells and human tumors is the highly elevated beta1,6 branching of N-linked oligosaccharides caused by increased transcription of N-acetylglucosaminyltransferase V (GnT-V). To characterize the effects of increased beta1,6 branching on cell-matrix adhesion-mediated phenotypes, human fibrosarcoma HT1080 cells were transfected with retroviral systems encoding GnT-V that used both noninducible and tetracycline-inducible promoters. Increased GnT-V expression resulted in a >25% inhibition of cell attachment to and a >50% inhibition of cell spreading on fibronectin. Both cell adhesion and spreading were suppressed by function-blocking antibodies specific for the alpha(5) and beta(1) integrin subunits of the fibronectin receptor. Cell migration toward fibronectin and invasion through Matrigel were both substantially stimulated in cells with induced expression of GnT-V. Induction of GnT-V had no effect on the level of cell surface expression of alpha(5) and beta(1) integrin subunits but did result in a more diffuse staining of the alpha(5) and beta(1) integrin subunits on the cell surface, suggesting that inhibition of integrin clustering may be causing these cells to be less adhesive and more motile. Surprisingly, there was no detectable expression of N-linked beta1,6 branching on the alpha(5) subunit purified from HT1080 cells before and after induction of GnT-V; by contrast, however, the beta(1) subunit showed a basal level of beta1,6 branching that was greatly increased after induction of GnT-V. These results suggest that changes in N-linked beta1,6 branching that occur during oncogenesis alter cell-matrix adhesion and migration by modulating integrin clustering and subsequent signal transduction pathways. These effects most likely result from altered N-linked carbohydrate expression on the beta(1) integrin subunit.

The effects of the epidermal growth factor (EGF), a stimulator of tyrosine protein kinase (TPK), and phorblol-12-myristate-13-acetate (PMA), a stimulator of protein kinase C (PKC), on the activity of N-acetylglucosaminyltransferase V (GnT-V) were studied in human hepatocarcinoma cell line 7721 in order to elucidate the regulation of TPK and PKC on GnT-V. It was found that the GnT-V activity obviously increased after treatment of the cells with EGF or PMA for 48 h. A non-specific protein kinase inhibitor, quercetin, inhibited the activities of TPK and PKC(inhibited mainly the membranous TPK and PKC)as well as GnT-V simulatanously. Moreover, quercetin completely eliminated the stimulating effect of EGF or PMA on GnT-V. When Tyrohostin-25, a specific inhibitor of TPK, or sphingosine, the specific inhibitor of PKC, was used separately to substitute for quercetin, the induction effect of EGF of PMA on GnT-V was only partially eliminated. However, when both Tyrphostin-25 and sphingosine were added to the culture medium, the elevation of GnT-V caused by EGF or PMA was entirely blocked. Cycloheximide, a well-known inhibitor of protein synthesis, showed an effect similar to the inhibition of protein kinases;it not only inhibited the basal activity of GnT-V, but also abolished the inducing stimulation of GnT-V by EGF of PMA. These results indicate that EGF or PMA regulates the activity of GnT-V via protein kinases, and GnT-V is regulated by dual mechanism of membranous TPK and PKC. Membranous TPK is more important that membranous PKC in the regulation of GnT-V.

The essential groups of three N-acetylglucosaminyltransferases (GnTs) of rat kidney were studied by using chemical modification and substrate protection methods. It was found that the amino and indolyl groups were the common essential groups of GnT-III, GnT-IV and GnT-V. Amino group was proposed to participate in the binding of common donor substrate--UDP-GlcNAc. Indolyl group might be the binding group of acceptor substrate--Gn(2)M(3)Gn(2)-PA heptosaccharide glycan for GnT-III and GnT-IV, and it might be the essential group but not substrate-binding for GnT-V. Guanidino group was also supposed to be one of the donor substrate-binding groups of GnT-III, but was a non-substrate-binding essential group for GnT-IV and a non-essential group for GnT-V, respectively. Carboxyl group might be the binding group of acceptor substrate for GnT-V, but the non-substrate-binding essential group for GnT-III and non-essential for GnT-V, respectively. In contrast, sulfhydryl, hydroxyl and imidazolyl groups were found to be non-essential groups of the three GnTs. The effects of amino sugars on GnT-III and GnT-V also showed the difference in essential groups in the active centers of GnT-III and GnT-V.