Novel strategy for a bispecific antibody: induction of dual target internalization and degradation.

Activation of the extensive cross-talk among the receptor tyrosine kinases (RTKs), particularly ErbB family-Met cross-talk, has emerged as a likely source of drug resistance. Notwithstanding brilliant successes were attained while using small-molecule inhibitors or antibody therapeutics against specific RTKs in multiple cancers over recent decades, a high recurrence rate remains unsolved in patients treated with these targeted inhibitors. It is well aligned with multifaceted properties of cancer and cross-talk and convergence of signaling pathways of RTKs. Thereby many therapeutic interventions have been actively developed to overcome inherent or acquired resistance. To date, no bispecific antibody (BsAb) showed complete depletion of dual RTKs from the plasma membrane and efficient dual degradation. In this manuscript, we report the first findings of a target-specific dual internalization and degradation of membrane RTKs induced by designed BsAbs based on the internalizing monoclonal antibodies and the therapeutic values of these BsAbs. Leveraging the anti-Met mAb able to internalize and degrade by a unique mechanism, we generated the BsAbs for Met/epidermal growth factor receptor (EGFR) and Met/HER2 to induce an efficient EGFR or HER2 internalization and degradation in the presence of Met that is frequently overexpressed in the invasive tumors and involved in the resistance against EGFR- or HER2-targeted therapies. We found that Met/EGFR BsAb ME22S induces dissociation of the Met-EGFR complex from Hsp90, followed by significant degradation of Met and EGFR. By employing patient-derived tumor models we demonstrate therapeutic potential of the BsAb-mediated dual degradation in various cancers.

Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor with high invasive and metastatic potential. The hepatocyte growth factor (HGF)-Met signaling pathway has a critical role in mediating the invasive growth of many different types of cancer, including head and neck squamous cell carcinoma. HGF also stimulates NPC cell growth and invasion in the cell line model. In this study, we determined the inhibitory effect of Met, using a Met-targeting monoclonal antibody (SAIT301), on the invasive and growth potential of NPC cell lines. Met inhibition by SAIT301 resulted in highly significant inhibition of cell migration and invasion in both the HONE1 and HNE1 cell lines. In addition, we also found that co-treatment of SAIT301 and HGF decreased the anchorage-independent growth induced by HGF in HNE1 cell lines. After SAIT301 treatment, Met, together with its downstream signaling proteins, showed downregulation of p-Met and p-ERK, but not p-AKT, in both HONE1 and HNE1 cell lines. Interestingly, we found that HGF treatment of NPC cell lines induced early growth response protein (EGR-1) expression, which is involved in cell migration and invasion. In addition, co-treatment with SAIT301 and HGF inhibited the HGF-induced expression of EGR-1. Next, knockdown of EGR-1 using small-interfering RNA inhibited HGF-induced cell invasion in NPC cell lines, suggesting that the expression level of EGR-1 is important in HGF-induced cell invasion of NPC cells. Therefore, the results support that SAIT301 inhibited Met activation as well as the downstream EGR-1 expression and could have therapeutic potential in NPC. Taken together, we suggest that Met is an anticancer therapeutic target for NPC that warrants further investigation and clinical trials and SAIT301 may be a promising tool for NPC therapy.

Cbl-independent degradation of Met: ways to avoid agonism of bivalent Met-targeting antibody.

The Met receptor tyrosine kinase, found to be constitutively activated in many tumors, has become a leading target for cancer therapy. Disruptions in Met downregulation have been associated with aggressive tumor progression with several therapeutic strategies addressing this aspect of Met biology. Castias B-lineage lymphoma (Cbl) E3 ligase-mediated degradation, which attenuates Met signaling via ligand-dependent Met internalization, is a major negative regulator of Met expression. It is believed that one of the mechanisms by which the therapeutic anti-Met antibodies induce cancer cell death in Met overexpressing tumors is via internalization and subsequent degradation of Met from the cell surface. However, a previously reported Met-targeting antibody demonstrated intrinsic agonistic activity while being capable of inducing Cbl-mediated degradation of Met, suggesting that Cbl-mediated degradation requires receptor activation and impedes therapeutic application. We have developed a potent and selective bivalent Met-targeting antibody (SAIT301) that invokes Met degradation using an alternative regulator LRIG1. In this report, we demonstrate that LRIG1 mediates degradation of Met by SAIT301 and this degradation does not require Met activation. Furthermore, SAIT301 was able to downregulate Met and dramatically inhibit growth of tumors with low or no Cbl expression, as well as tumors with Met exon 14 deletion that prevents Met binding to Cbl. In summary, we demonstrate the enhanced therapeutic potential of a novel tumor-inhibiting anti-Met antibody, SAIT301, which utilizes a Cbl-independent, LRIG1-mediated Met degradation pathway and thereby avoids the agonism that limits the effectiveness of previously reported anti-Met antibodies.

Management of recurrent acute otitis media in children: systematic review of the effect of different interventions on otitis media recurrence, recurrence frequency and total recurrence time.

OBJECTIVE: To conduct a systematic review comparing the effect of three interventions (prophylactic antibiotics, tympanostomy tube insertion and adenoidectomy) on otitis media recurrence, recurrence frequency and total recurrence time. METHODS: Literature on recurrent otitis media was identified using the PubMed and Scopus search engines for the period January 1990 to March 2011. A hand search of the reference lists of relevant articles and textbooks was conducted to identify additional studies. Randomised, controlled trials with a minimum of 40 children and follow up of at least 12 months were included. RESULTS: Eighteen publications were identified. Each was assessed using preset inclusion criteria; seven publications met these criteria. CONCLUSION: Prophylactic antibiotics are effective in reducing otitis media recurrence, recurrence frequency and total recurrence time. Tympanostomy tube insertion failed to reduce the prevalence of otitis media recurrence, but reduced the recurrence frequency and total recurrence time. Adenoidectomy reduced otitis media recurrence; results on otitis media recurrence frequency differed but on average there was a reduction; however, the two studies with relevant data on total recurrence time had contradictory results.

VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells.

Apical proteins are sorted and delivered from the trans-Golgi network to the plasma membrane by a mechanism involving sphingolipid-cholesterol rafts. In this paper, we report the effects of changing the levels of VIP17/MAL, a tetraspan membrane protein localized to post-Golgi transport containers and the apical cell surface in MDCK cells. Overexpression of VIP17/MAL disturbed the morphology of the MDCK cell layers by increasing apical delivery and seemingly expanding the apical cell surface domains. On the other hand, expression of antisense RNA directed against VIP17/MAL caused accumulation in the Golgi and/or impaired apical transport of different apical protein markers, i.e., influenza virus hemagglutinin, the secretory protein clusterin (gp80), the transmembrane protein gp114, and a glycosylphosphatidylinositol-anchored protein. However, antisense RNA expression did not affect the distribution of E-cadherin to the basolateral surface. Because VIP17/MAL associates with sphingolipid-cholesterol rafts, these data provide functional evidence that this protein is involved in apical transport and might be a component of the machinery clustering lipid rafts with apical cargo to form apical transport carriers.

Immunocytochemical analysis of peptide hormone processing: importance of the positively charged N-terminal domain of signal peptide in correct ER targeting in yeast cells.

We used a morphological approach to determine the topogenic role of the signal peptide in mediating the ER translocation of yeast prepro-alpha-factor. In prepro-alpha-factor-somatostatin hybrids, changes in the N-terminal amino acid sequence from wild-type NH2-Met-Arg-Phe (MRF) to NH2-Met-Phe-Lys (MFK) caused a subtle difference in protein trafficking in yeast cells. Immunofluorescence microscopy on semithin cryosections and immunoelectron microscopy on ultrathin sections showed that the transposition of the charged amino acid at N-terminus caused the precursors to be associated with either nucleus or mitochondria. This suggests that the secretory proteins are mistargeted to the irrelevant organelles as the result of inefficient ER translocation. Structural aspects of nuclear or mitochondrial targeting proteins and common principles in membrane translocation systems account for the mistargeting of overexpressed mutant hybrid precursors that are not rapidly translocated into the ER. Based on our immunocytochemical study on individual cells, we propose here that the positively charged N-terminal domain of signal peptide is important not merely in the efficiency of ER translocation, but also in appropriate targeting of peptide hormone precursors in yeast cells where post-translational ER translocation is known to occur frequently.

Topogenic effect of positively charged N-terminal amino acid in ER translocation of yeast alpha-factor precursor.

Expression of fusion proteins between prepro-alpha-factor and somatostatin (SRIF) in yeast, resulted in the correct processing and secretion of the heterologous 14-amino acid SRIF peptide (1). When the chimeric genes were placed under the control of yeast acid phosphatase (PHO5) promoter, significant amount of an unglycosylated form of the fusion precursor molecule accumulated intracellularly, suggesting disruption of an endoplasmic reticulum-mediated function. We report here that the appearance of the precursor is due to an alteration in the three amino terminal residues of the chimera, i.e., Met-Arg-Phe in native prepro-alpha-factor is changed to Met-Phe-Lys in the hybrids. The unglycosylated precursor represents a population of molecules that are disrupted at an early stage of targeting to or translocation across the endoplasmic reticulum membrane. Our data demonstrate that the N-terminus plays an important role in topogenesis. Furthermore, these results show that translocation and glycosylation can be uncoupled from protein synthesis in vivo, and therefore can be posttranslational events in yeast.