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The tyrosine kinase receptor encoded by the MET oncogene has been extensively studied. Surprisingly, one extracellular domain, PSI, evolutionary conserved between plexins, semaphorins, and integrins, has no established function. The MET PSI sequence contains two CXXC motifs, usually found in protein disulfide isomerases (PDI). Using a scrambled oxidized RNAse enzymatic activity assay in vitro, we show, for the first time, that the MET extracellular domain displays disulfide isomerase activity, abolished by PSI domain antibodies. PSI domain deletion or mutations of CXXC sites to AXXA or SXXS result in a significant impairment of the cleavage of the MET 175 kDa precursor protein, abolishing the maturation of α and β chains, of, respectively, 50 kDa and 145 kDa, disulfide-linked. The uncleaved precursor is stuck in the Golgi apparatus and, interestingly, is constitutively phosphorylated. However, no signal transduction is observed as measured by AKT and MAPK phosphorylation. Consequently, biological responses to the MET ligand—hepatocyte growth factor (HGF)—such as growth and epithelial to mesenchymal transition, are hampered. These data show that the MET PSI domain is functional and is required for the maturation, surface expression, and biological functions of the MET oncogenic protein.

Background Antibody-drug conjugates (ADCs) targeting the RON receptor, a tumorigenic factor contributing to cancer malignancy, has been considered as a novel strategy for cancer therapy. Here we describe a humanized antibody recognizing the RON plexin-semaphorin-integrin (PSI) domain with increased drug delivery capability for potential clinical application. Method Monoclonal antibody PCM5B14 specific to the human and monkey RON PSI domain was generated and characterized by various immunological methods. Humanized antibody H5B14 was created by grafting PCM5B14 complementarity-determining regions into human IgG1/κ acceptor frameworks and conjugated with monomethyl auristatin E and duocarmycin to form two H5B14-based ADCs. Stability of H5B14-based ADCs in human plasma was measured using hydrophobic interaction chromatography. Various biochemical and biological assays were used to determine ADC- regulated RON internalization, cell viability, spheroid formation, and death of cancer stem-like cells. Efficacies of H5B14-based ADCs in vivo were validated using tumor xenograft models. Maximal tolerated doses of H5B14-based ADCs were established in mice. Results H5B14 was highly specific to the human RON PSI domain and superior over other anti-RON ADCs in induction of RON internalization in various cancer cell lines tested. H5B14-based ADCS had a drug to antibody ratio of ~ 3.70:1 and were stable in human plasma with a minimal dissociation within a 10-day period. Functionally, H5B14-mediated drug delivery decreased cell viability at early stages with an average IC50 at ~ 20 nM in multiple cancer cell lines examined. H5B14-based ADCs also inhibited spheroid formation and caused death of cancer stem-like cells with RON+/CD44+/ESA+ phenotypes. In vivo, H5B14-based ADCs in a single injection inhibited tumor xenograft growth mediated by multiple cancer cell lines. Tumoristatic concentrations calculated from xenograft tumor models were in the range of 0.63 to 2.0 mg/kg bodyweight. Significantly, H5B14-based ADCs were capable of eradicating tumors at variable levels across multiple xenograft models regardless their malignant statuses. Toxicologically, H5B14-based ADCs were well tolerated in mice up to 60 mg/kg. Conclusion H5B14-based ADCs targeting the RON PSI domain are superior in inducing RON internalization, leading to robust drug delivery and overall inhibition and eradication of tumors in multiple xenograft models. These findings warrant H5B14-based ADCs for clinical trials in the future.

The Andes Orthohantavirus (ANDV), which causes the hantavirus cardiopulmonary syndrome, enters cells via integrins, and a change from leucine to proline at residue 33 in the PSI domain (L33P), impairs ANDV recognition. We assessed the association between this human polymorphism and ANDV infection. We defined susceptible and protective genotypes as &ldquo, TT&rdquo, (coding leucine) and &ldquo, CC&rdquo, (coding proline), respectively. TT was present at a rate of 89.2% (66/74) among the first cohort of ANDV cases and at 60% (63/105) among exposed close-household contacts, who remained uninfected (p &lt, 0.05). The protective genotype (CC) was absent in all 85 ANDV cases, in both cohorts, and was present at 11.4% of the exposed close-household contacts who remained uninfected. Logistic regression modeling for risk of infection had an OR of 6.2&ndash, 12.6 (p &lt, 0.05) in the presence of TT and well-known ANDV risk activities. Moreover, an OR of 7.3 was obtained when the TT condition was analyzed for two groups exposed to the same environmental risk. Host genetic background was found to have an important role in ANDV infection susceptibility, in the studied population.

Hepatocyte growth factor receptor (HGFR, c-Met) is an essential member of the receptor tyrosine kinase (RTK) family that is often dysregulated during tumor progression, driving a malignant phenotypic state and modulating important cellular functions including tumor growth, invasion, metastasis, and angiogenesis, providing a strong rationale for targeting HGF/c-Met signaling axis in cancer therapy. Based on its protumorigenic potentials, we developed IRCR201, a potent antagonistic antibody targeting the plexin-semaphorin-integrin (PSI) domain of c-Met, using synthetic human antibody phage libraries. We characterized and evaluated the biochemical properties and tumor inhibitory effect of IRCR201 in vitro and in vivo. IRCR201 is a novel fully-human bivalent therapeutic antibody that exhibits cross-reactivity against both human and mouse c-Met proteins with high affinity and specificity. IRCR201 displayed low agonist activity and rapidly depleted total c-Met protein via the lysosomal degradation pathway, inhibiting c-Met-dependent downstream activation and attenuating cellular proliferation in various c-Met-expressing cancer cells. In vivo tumor xenograft models also demonstrated the superior tumor inhibitory responsiveness of IRCR201. Taken together, IRCR201 provides a promising therapeutic agent for c-Met-positive cancer patients through suppressing the c-Met signaling pathway and tumor growth.