Implicit attitudes towards dementia after education: Preliminary trial results from Japan.

OBJECTIVE: This study aimed to investigate the feasibility and validity of measuring implicit attitudes towards dementia in adults and older adults and evaluate the impact of dementia-friendly education using virtual reality (VR) on implicit attitudes. METHODS: This study was a secondary analysis of data from a randomised controlled trial. Community members in Tokyo aged 20-90 years participated in dementia-friendly education with or without VR. At the end of the dementia-friendly education programs, implicit attitudes towards dementia were measured using the Implicit Relational Assessment Procedure (IRAP). RESULTS: Of the 145 participants, 89 (61%) started the IRAP, and 21 (15%) completed it. Lower age was significantly associated with the start/completion of the IRAP, and the age thresholds at which 50% of participants would not start/complete it were estimated to be 72.3/44.8 years, respectively. Those who had experience interacting with people with dementia other than family members had lower IRAP scores than those who had no such experience. The intervention group participating in the VR program had lower IRAP scores than the control group (p = .09). CONCLUSIONS: Although measuring implicit attitudes using IRAP is deemed not feasible for people in their 70s and older, the differences in interaction experience would be evidence supporting the validity of the measurements of implicit attitudes towards dementia. The results suggest that dementia-friendly education, using VR, improves implicit attitudes towards dementia.

Biological Evaluation of Antibody-Drug Conjugates Produced by Tag-Free Lipoate Ligase A Modification.

The concept of tag-free protein modification has attracted considerable interest in chemical biology because of its flexible and straightforward reaction process. In 2021, a groundbreaking approach using lipoate ligase A (LplA) for tag-free enzymatic modification of antibodies was unveiled, demonstrating its potential for the generation of precise antibody conjugates. In this study, to further explore LplA-mediated antibody-drug conjugate (ADC) synthesis, we performed initial biological evaluations of ADCs synthesized using LplA. Using the anti-HER2 antibody trastuzumab, we introduced octanoic acid azide using LplA and subsequently obtained an ADC using click chemistry with the drug DBCO-VC-PAB-MMAE. The bioactivity of the synthesized anti-HER2-ADC was evaluated using HER2-positive SKBR-3 and HER2-negative MCF7 cells. Its toxicity and selectivity were found to be comparable to those of the FDA-approved Kadcyla. In addition, a stability study involving rat and human plasma demonstrated the stability of the LplA-mediated ADC. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. These preliminary in vitro evaluations suggested that LplA-derived ADCs can have considerable pharmaceutical potential. Our results can set the stage for further in vivo evaluations and safety assessments. We suggest that the integration of tag-free LplA methods into the production of ADCs can offer a novel and promising approach for biopharmaceutical manufacturing.

Durvalumab after chemoradiotherapy in non-small cell lung cancer with EGFR mutation: A real-world study (HOT2101).

Durvalumab has been administered to patients with unresectable stage III non-small cell lung cancer (NSCLC). However, it remains unclear whether durvalumab benefits these patients with epidermal growth factor receptor (EGFR) mutation. We conducted a retrospective, multicenter study of patients with EGFR mutation who received chemoradiotherapy (CRT) between June 2018 and March 2021. We assessed patient characteristics, efficacy of durvalumab, and durvalumab safety before and after targeted therapy. We collected data on a total of 673 patients, of whom 401 (59.6%) underwent EGFR mutation testing. Fifty-one patients were EGFR positive and 311 were EGFR negative. In the EGFR-positive group, there were higher proportions of females, never-smokers, and patients with adenocarcinoma histology. Of the 51 patients in the positive group and 311 in the negative group who received CRT, 45 (88.2%) and 247 (79.4%) received durvalumab, with median progression-free survival of 23.0 and 24.2 months in the positive and negative groups, respectively (hazard ratio 1.03; 95% confidence interval: 0.64-1.67). The main adverse event was pneumonitis (positive group: 62.2%; 4.4% grade 3; negative group: 62.3%; 6.9% grade 3). No treatment-related deaths were observed. Of the 45 patients in the positive group who received durvalumab, 14 (31.1%) received targeted therapy after durvalumab at the data cutoff. One patient discontinued targeted therapy after developing pneumonitis. In patients with unresectable stage III NSCLC with EGFR mutation, durvalumab after CRT is potentially safe and effective. This may be a suitable treatment sequence for these patients.

Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation.

Bispecific antibodies (BisAbs) are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule, however, their engineering requires genetic modification and remains time-consuming. Therefore, we used AJICAP second-generation technology, which drives the production of site-specific conjugation without genetic modification requirements, to generate BisAbs. Using haloketone chemistry as an alternative to maleimide chemistry, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant BisAbs were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that the antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated BisAbs. This novel approach introduces a fully chemical, site-specific strategy capable of producing BisAbs, heralding a new era in the field of biotherapeutics.

Real-world data on the efficacy and safety of immune-checkpoint inhibitors in elderly patients with non-small cell lung cancer.

PURPOSE: Immune-checkpoint inhibitors (ICIs) are effective against advanced non-small cell lung cancer (NSCLC). However, whether the efficacy and safety of ICI treatment in elderly patients are similar to those in younger patients is unclear. This study was designed to address this question. METHODS: We enrolled patients who received ICI monotherapy in Japan between December 2015 and December 2017; those ≥75 years of age comprised the elderly group. We compared the efficacy and safety of ICI monotherapy in elderly patients with those in younger patients and explored prognostic factors in elderly patients. RESULTS: We enrolled 676 patients; 137 (20.3%) were assigned to the elderly group. The median age of the elderly and younger groups was 78 (range, 75-85) and 66 (range, 34-74) years. The median progression-free survival (4.8 months vs. 3.3 months, p = 0.1589) and median overall survival (12.3 months vs. 13.0 months, p = 0.5587) were similar between the elderly and younger groups. Multivariate analysis revealed that a significantly better OS in the elderly group was associated with better responses to first- or second-line ICI treatment (p = 0.011) and more immune-related adverse events (irAEs) (p = 0.02). IrAEs that led to ICI discontinuation occurred in 34 of 137 patients (24.8%) in the elderly group, and their survival was significantly higher than that in those who did not have irAEs. CONCLUSION: ICI is also effective in elderly NSCLC patients, and treatment discontinuation due to irAEs may be a good prognostic marker.

Bacterially Secretable Single-Chain Tandem Macrocyclic Peptides for High Affinity and Inhibitory Activity.

The inhibition of protein-protein interactions (PPIs) is an effective approach for therapy. Owing to their large binding surface areas to target proteins, macrocyclic peptides are suitable molecules for PPI inhibition. In this study, we developed single-chain tandem macrocyclic peptides (STaMPtides) that inhibits the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2). They were artificially designed to comprise two different VEGFR2-binding macrocyclic peptides linked in tandem by peptide linkers and secreted by Corynebacterium glutamicum. Most potent VEGFR2-inhibitory STaMPtides with length-optimized linkers exhibited >1000 times stronger inhibitory activity than their parental monomeric peptides, possibly due to the avidity effect of heterodimerization. Our approach of using STaMPtides for PPI inhibition may be used to inhibit other extracellular factors, such as growth factors and cytokines.

Real-world outcomes of chemotherapy for lung cancer patients undergoing hemodialysis: A multicenter retrospective cohort study (NEJ-042).

INTRODUCTION: Malignant tumors are the major cause of death in hemodialysis patients. Management of these patients remains challenging as there is no evidence that chemotherapy is beneficial, and a lack of information about actual clinical practice. METHODS: This multicenter retrospective study included hemodialysis patients who were diagnosed with lung cancer from January 2002 to June 2018. We reviewed their clinical information including patient characteristics associated with lung cancer and end-stage renal disease, regimen, efficacy and safety of chemotherapy, and outcomes. RESULTS: A total of 162 patients from 22 institutions in Japan were registered. Of 158 eligible patients, 91 received chemotherapy (80 as palliative chemotherapy and 11 as chemoradiotherapy) and 67 received best supportive care only regardless of cancer stage. In small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) patients who received cytotoxic chemotherapy, the objective response rates (ORR) and median overall survival (OS) were 68.1 %, 12.3 months and 37.0 %, 8.5 months, respectively. The ORR and median OS in patients with EGFR-mutant NSCLC treated with EGFR-tyrosine kinase inhibitors (TKI) were 44.4 % and 38.6 months. The treatment-related adverse events (Grade 3 or higher) induced by cytotoxic chemotherapy were myelosuppression and febrile neutropenia; treatment-related death (TRD) was observed in one patient. TRD occurred in 3 of 18 patients who received EGFR-TKI. CONCLUSION: Chemotherapy should be considered for hemodialysis patients with EGFR-mutant NSCLC and SCLC. However, the survival benefits of chemotherapy for NSCLC patients with EGFR-wild type are unclear; physicians should carefully consider whether to offer chemotherapy to this patient subset.

A Two-Step Model for Encouraging the General Public to Exhibit Helping Behaviors Toward People Living With Dementia.

Background and Objectives: Dementia awareness initiatives aim to promote helping behaviors toward people living with dementia. We applied the bystander intervention process model in the context of the general public's helping behaviors toward people living with dementia, and we sought to identify the mechanisms underlying the association between dementia knowledge and helping behaviors. Research Design and Methods: In a survey featuring vignettes for the general public in Japan (N = 904), we presented four situations in which people could exhibit helping behaviors toward a person with dementia. Guttman scale analysis was used to test this sequential ordering of the bystander intervention process model: (a) interpreting the need to help, (b) perceiving personal responsibility, and (c) intention to provide help. Mediation analysis was used to examine whether the effects of knowledge on helping behaviors were mediated by attitude toward people living with dementia and the bystander intervention process. Results: The results support the two-step model in which interpreting the situation as one where assistance is required is a prerequisite of helping behavior. Dementia knowledge had a significant total effect on intention to provide help (ß = 0.136, p < .001). Interpretation (indirect effect: ß = 0.092, p < .001), as well as attitude (indirect effect: ß = 0.044, p < .001), was found to completely mediate the effect of dementia knowledge on intention to provide help. Discussion and Implications: Dementia awareness initiatives designed to promote helping behaviors should focus on knowledge transfer, improving the general public's attitudes toward people living with dementia, and their ability to interpret when such people need assistance.

Single-chain tandem macrocyclic peptides as a scaffold for growth factor and cytokine mimetics.

Mimetics of growth factors and cytokines are promising tools for culturing large numbers of cells and manufacturing regenerative medicine products. In this study, we report single-chain tandem macrocyclic peptides (STaMPtides) as mimetics in a new multivalent peptide format. STaMPtides, which contain two or more macrocyclic peptides with a disulfide-closed backbone and peptide linkers, are successfully secreted into the supernatant by Corynebacterium glutamicum-based secretion technology. Without post-secretion modification steps, such as macrocyclization or enzymatic treatment, bacterially secreted STaMPtides form disulfide bonds, as designed; are biologically active; and show agonistic activities against respective target receptors. We also demonstrate, by cell-based assays, the potential of STaMPtides, which mimic growth factors and cytokines, in cell culture. The STaMPtide technology can be applied to the design, screening, and production of growth factor and cytokine mimetics.

First-line osimertinib in elderly patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer: a retrospective multicenter study (HOT2002).

Osimertinib is a standard of care therapy for previously untreated epidermal growth factor receptor mutation-positive non-small cell lung cancer. However, limited data exist regarding the efficacy and safety of osimertinib as a first-line therapy for elderly patients aged 75 years or older. To assess the potential clinical benefits of osimertinib in this population, this retrospective multi-institutional observational study included 132 patients with non-small cell lung cancer (age ≥ 75 years), who received osimertinib as first-line treatment. The proportion of patients with 1-year progression-free survival was 65.8% (95% confidence interval 57.1-73.5). The median progression-free survival was 19.4 (95% confidence interval 15.9-23.9) months. The median overall survival was not reached (95% confidence interval 24.6-not reached). The frequency of pneumonitis was 17.4%, with a grade 3 or higher rate of 9.1%. More than two-thirds of treatment discontinuations due to pneumonitis occurred within 3 months of starting osimertinib, and the prognosis of patients with pneumonitis was unsatisfactory. Osimertinib is one of the effective first-line therapeutic options for patients aged 75 years or older; however, special caution should be exercised due to the potential development of pneumonitis.

Prognostic factors in patients with advanced non-small cell lung cancer after long-term Anti-PD-1 therapy (HOT1902).

OBJECTIVES: Limited information is available on the appropriate treatment duration of immune checkpoint inhibitors (ICIs). We aimed to identify candidates who would benefit from ICI discontinuation after one year of treatment for metastatic non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: This retrospective multi-institutional observational study examined medical records of all consecutive patients with advanced or recurrent NSCLC, who started ICI monotherapy at 15 institutions in Japan between December 2015 and December 2017. Patients who received initial ICI therapy for >1 year without progressive disease were defined as the long-term treatment (LT) group; others were defined as the non-long-term treatment (NLT) group. Primary outcomes included the prognostic factors in the LT group, whereas secondary outcomes included efficacy of ICI rechallenge, safety, and survival outcomes in the overall population. RESULTS: In total, 676 patients were enrolled, and 114 (16.9 %) were assigned to the LT group. The median time interval from the start of initial ICI administration to data cutoff was 34.3 months (range, 24.1-47.8); thus, all surviving patients were followed-up for at least 2 years from the start of initial ICI. Median progression-free survival (PFS) was longer in the LT than in the NLT group (33.6 months vs. 2.7 months; p < 0.001). On multivariate analysis, significantly better PFS was associated with smoking (hazard ratio [HR]=0.36, p = 0.04), and complete response (CR; HR=uncomputable, p < 0.001) in the LT group. Thirty-seven patients (5.5 %) received ICI rechallenge, including 10 in the LT group. Among patients receiving rechallenge treatment, the median PFS was 2.2 months, with no difference between the LT and NLT groups. CONCLUSIONS: In the LT group, smoking and achieving CR were significantly associated with better PFS. Since rechallenge treatment was not effective, careful consideration is required for discontinuing ICI. However, these prognostic factors are helpful in considering candidates for ICI discontinuation. TRIAL REGISTRATION: UMIN ID, UMIN000041403.

One-step construction of ferritin encapsulation drugs for cancer chemotherapy.

Conventionally, a disassembly and reassembly method has been used for encapsulation of drug molecules in ferritin protein nano-cages. However, clinical applications of ferritin have been greatly restricted by its limited drug-loading capacity and process complexity. Here, we establish a simple high yield process for preparing high drug-loaded ferritin nanomedicine for industrial production. A complex of ferritin and a target drug was obtained by incubating the mixture at an appropriate pH. An electrostatic charge potential and small ferritin cavity facilitates the passage of drug molecules through the pores, traversing the ferritin shell and enabling deposition of the drug in the ferritin cavity. Compared to the disassembly/reassembly method, the loading capacity of a doxorubicin-loaded ferritin heavy chain (DOX-FTH), constructed by our novel method, was over 3-fold higher, while doxorubicin recovery was 10-fold higher. Results of transmission electron microscopy, size exclusion chromatography, dynamic light scattering, and zeta potential indicate that DOX-FTH exhibits the same physicochemical characteristics of natural apo-ferritin. Moreover, DOX-FTH can be taken up and induce apoptosis of cancer cells overexpressing TfR1. Here, we have demonstrated the successful introduction of more than ten drug molecule types into ferritin nano-cages using a novel method. These results demonstrate that this one-step method is a powerful production process to construct a drug-loading ferritin drug delivery system carrier.

Incorporation of Halogenated Amino Acids into Antibody Fragments at Multiple Specific Sites Enhances Antigen Binding.

Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059-152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17-fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.

A randomized phase II trial of cisplatin plus gemcitabine versus carboplatin plus gemcitabine in patients with completely resected non-small cell lung cancer: Hokkaido Lung Cancer Clinical Study Group Trial (HOT0703).

PURPOSE: This study evaluated the efficacy and safety of platinum plus gemcitabine (P/G) combinations as postoperative adjuvant chemotherapies for non-small cell lung cancer. METHODS: Patients with postoperative stage IB-IIIA non-small cell lung cancer were randomly assigned to receive either cisplatin plus gemcitabine (GP arm) or carboplatin plus gemcitabine (GC arm) every 3 weeks for four cycles. The primary endpoint was 2-year disease-free survival (DFS). Secondary endpoints were safety, feasibility, overall survival (OS), and biomarker analyses. RESULTS: A total of 102 patients were randomized (stage IB, 22%; II, 36%; IIIA, 42%; histology: 74% adenocarcinoma). Of the 51 patients in each arm, 37 (73%) completed 4 cycles. During follow-up (median 5.8 years; range 0.1-9.7 years), estimated DFS and OS rates at 2 years were 59.6% and 86.3% with GP and 68.0% and 86.3% with GC, respectively. No significant difference in DFS was noted between arms (P = 0.163), although 3-, 4-, and 5-year DFS rates were higher with GC. Hematological toxic effects were comparable and non-hematological toxic effects were infrequent. DFS was significantly higher in the excision repair cross-complementation group 1 (ERCC1)-low group than in the ERCC1-high group for the GP arm (P = 0.045). CONCLUSION: Both P/G combination regimens were feasible and well-tolerated, and thus may represent valid options for postoperative adjuvant treatment of non-small cell lung cancer. Although no significant differences in DFS were evident between regimens, the present data favor the adoption of GC for further evaluation. CLINICAL TRIAL REGISTRATION: UMIN-CTR ( https://www.umin.ac.jp/ctr/ ) identifier: UMIN000000913.

Improved Stability of siRNA-Loaded Lipid Nanoparticles Prepared with a PEG-Monoacyl Fatty Acid Facilitates Ligand-Mediated siRNA Delivery.

Peptide modification is a popular strategy for developing an active targeting lipid nanoparticle (LNP). In modifying the surface of an LNP with a peptide, the sequence and structure of the peptide strongly affects the formation of the LNP. Specifically, a peptide with a high hydrophobicity can induce coarsening and aggregation of the LNP. In an attempt to prevent this from occurring, we incorporated monoacyl and diacyl group-conjugated poly(ethylene glycol) (PEG) into a LNP. We previously developed an original LNP, a multifunctional envelope type nanodevice (MEND) modified with an Epi-1 peptide, a ligand with a high affinity for the epithelial cell adhesion molecule (EpCAM). Using this peptide-modified MEND, the efficiency of delivery of a small interfering RNA (siRNA) encapsulated in the MEND was significantly improved. Although increasing the ratio of modification enhanced cellular uptake, the increase also induced aggregation of the LNP, particularly in the case of a large scale preparation. Our results indicate that a monoacyl PEG-lipid can prevent aggregation, even when the LNP is modified with higher molar ratios of peptide, but that this also results in a decrease in delivery efficiency. Moreover, the Epi-1-modified MEND exhibited a strong silencing effect in an ovarian cancer peritoneal dissemination model. Our results suggest that the simple incorporation of a monoacyl derivative into the PEG-lipid resulted in the formation of a peptide-modified LNP with improved characteristics.

Pathotypes and Drug Susceptibility of Escherichia coli Isolated from Companion Dogs in Japan.

Considering the possibility that Escherichia coli carried by companion dogs could infect owners and human society, we investigated their pathogenicity and drug resistance. E. coli was isolated from stool samples of companion dogs (n = 90) to examine the O-serogroup, virulence genes, and drug susceptibility. The age of dogs ranged from 4 months to 16 years, and they were mainly treated with cefalexin, enrofloxacin, or amoxicillin. A total of 69 samples were positive for E. coli (76% of examined dogs), and the most common O-serogroup was O18 (n = 13). Nine diarrheagenic E. coli, including enteropathogenic E. coli (n = 3), enteroaggregative E. coli (n = 1), and astA-carrying E. coli (n = 5), were isolated. In addition, we isolated 28 E. coli strains resistant to at least one of six antimicrobials, including cephalothin (CET), ceftazidime (CAZ), cefotaxime (CTX), chloramphenicol (CP), fosfomycin (FOM), and norfloxacin (NLFX). The resistance pattern was as follows: CET, n = 16; NLFX, n = 3; CET/CP (resistance to both CET and CP), n = 1; CET/NLFX, n = 1; CET/CAZ/CTX, n = 3; CET/CTX/NLFX, n = 2; CET/CP/NLFX, n = 1; and CET/CAZ/CTX/NLFX, n = 1. Moreover, ten E. coli isolates were found to produce extended-spectrum ß-lactamase (ESBL), including AmpC (n = 4; OUT, O18, O74, and O166), CTX-M-1 (n = 1; O25), CTX-M-9 (n = 4; OUT, O18, O18, and O125), and AmpC/CTX-M-9 (n = 1; OUT) groups. The AmpC-producing E. coli strains included enteropathogenic and astA-carrying E. coli. Our results showed that the human-infectious diarrheagenic E. coli was isolated from some dogs, and some strains exhibited ESBL. Therefore, future studies are needed to investigate the possibility of transmission of these E. coli strains to humans.

Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor.

Activation of hepatocyte growth factor (HGF) by proteolytic processing is triggered in cancer microenvironments, and subsequent signaling through the MET receptor is involved in cancer progression. However, the structure of HGF remains elusive, and few small/medium-sized molecules can modulate HGF. Here, we identified HiP-8, a macrocyclic peptide consisting of 12 amino acids, which selectively recognizes active HGF. Biochemical analysis and real-time single-molecule imaging by high-speed atomic force microscopy demonstrated that HiP-8 restricted the dynamic domains of HGF into static closed conformations, resulting in allosteric inhibition. Positron emission tomography using HiP-8 as a radiotracer enabled noninvasive visualization and simultaneous inhibition of HGF-MET activation status in tumors in a mouse model. Our results illustrate the conformational change in proteolytic activation of HGF and its detection and inhibition by a macrocyclic peptide, which may be useful for diagnosis and treatment of cancers.

Cellular signaling and gene expression profiles evoked by a bivalent macrocyclic peptide that serves as an artificial MET receptor agonist.

Non-native ligands for growth factor receptors that are generated by chemical synthesis are applicable to therapeutics. However, non-native ligands often regulate cellular signaling and biological responses in a different manner than native ligands. Generation of surrogate ligands comparable to native ligands is a challenging need. Here we investigated changes in signal transduction and gene expression evoked by a bivalent macrocyclic peptide (aMD5-PEG11) capable of high-affinity binding to the MET/hepatocyte growth factor (HGF) receptor. Binding of aMD5-PEG11 to the MET extracellular region was abolished by deletion of the IPT3-IPT4 domain, indicating the involvement of IPT3-IPT4 in the binding of aMD5-PEG11 to the MET receptor. aMD5-PEG11 induced dimerization and activation of the MET receptor and promoted cell migration that was comparable to induction of these activities by HGF. Signal activation profiles indicated that aMD5-PEG11 induced phosphorylation of intracellular signaling molecules, with a similar intensity and time dependency as HGF. In 3-D culture, aMD5-PEG11 as well as HGF induced epithelial tubulogenesis and up-regulated the same sets of functionally classified genes involved in multicellular organism development. Thus, a non-native surrogate ligand that consisted of a bivalent macrocyclic peptide can serve as an artificial MET receptor agonist that functionally substitutes for the native ligand, HGF.

MET Activation by a Macrocyclic Peptide Agonist that Couples to Biological Responses Differently from HGF in a Context-Dependent Manner.

Non-native ligands for growth factor receptors with distinct chemical properties and different biological activities have the potential to become therapeutic applications. We previously generated MET/hepatocyte growth factor (HGF) receptor agonists using bivalent macrocyclic peptides. The highest MET-activating agonists exhibited biological activity that was indistinguishable from the effects of HGF. In this study, we investigated MET activation, signal characteristics, and biological responses induced by a macrocyclic peptide partial agonist known as aML5-PEG11. aML5-PEG11 induced weak tyrosine phosphorylation of MET while enhancing cell migration with potency comparable to HGF. aML5-PEG11 induced marked AKT (protein kinase B) and ERK (extracellular signal-regulated kinase) activation at a comparable potency and time-dependency to HGF, which suggests that enhancement of cell motility is attributable to activation of these molecules. In a 3-D culture of bile duct cancer cells in collagen gel, HGF induced robust activation of MET, ERK, and AKT, which was associated with enhanced expression of genes involved in bile duct development and subsequent branching of tubulogenesis. In contrast, aML5-PEG11 induced marginal activation of MET, ERK, and AKT (levels near the detection limits), which was associated with failure to enhance the expression of genes involved in bile duct development and a lack of tubulogenic response. Thus, MET activation by aML5-PEG11 couples to biological responses differently from HGF in an extracellular context-dependent manner.

Globo-A Binds to the Recombinant Natural Cytotoxicity Receptor NKp44.

Natural killer (NK) cells play an important role in tumor immunity and infection control. The natural cytotoxicity receptors (NCRs) NKp46, NKp44 and NKp30 are involved in the control of the activation of NK cells. Few reports have investigated the ligands of NCRs. We previously reported the NCRs binding affinity to heparin and glycosaminoglycans. We also showed that multimeric sialyl Lewis X-expressing transferrin, secreted by human hepatoma HepG2 cells, binds to NKp46 and NKp44, but not to NKp30. In this study, we investigated the binding between NCRs and glycolipids. The possible binding of glycolipids to NCRs was screened by microarray, using the recombinant extracellular domain of NKp46, NKp44 and NKp30 tagged with 6×His (rNKp46, rNKp44 and rNKp30). We found that rNKp44 binds to Globo-A. However, we did not detect the interaction between rNKp46 or rNKp30 and any of the glycolipids investigated. Direct binding assays supported the results of the microarray screening. Therefore, we concluded that Globo-A is a novel ligand for NKp44 but not NKp46 and NKp30, and showed differences in the ligand selectivity of NCRs.