Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy.

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-ß structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-ß structures gradually occur.

Single-molecule analyses of the dynamics of heat shock protein 104 (Hsp104) and protein aggregates.

Hsp104 solubilizes protein aggregates in cooperation with Hsp70/40. Although the framework of the disaggregase function has been elucidated, the actual process of aggregate solubilization by Hsp104-Hsp70/40 remains poorly understood. Here we developed several methods to investigate the functions of Hsp104 and Hsp70/40 from Saccharomyces cerevisiae, at single-molecule levels. The single-molecule methods, which provide the size distribution of the aggregates, revealed that Hsp70/40 prevented the formation of large aggregates from small aggregates and that the solubilization of the small aggregates required both Hsp104 and Hsp70/40. We directly visualized the individual association-dissociation dynamics of Hsp104 on immobilized aggregates and found that the lifetimes of the Hsp104-aggregate complex are divided into two groups: short (∼4 s) and long (∼30 s). Hsp70/40 stimulated the association of Hsp104 with aggregates and increased the duration of this association. The single-molecule data provide novel insights into the functional mechanism of the Hsp104 disaggregation machine.

A cross-sectional survey to investigate the prevalence of pain in Japanese patients with major depressive disorder and schizophrenia.

We conducted a cross-sectional survey to assess the prevalence of physical pain in Japanese major depressive disorder (MDD) and schizophrenia (SZ) patients as well as in healthy controls (HCs). We also examined the association between their psychopathology and characteristics of pain according to a face-to-face survey by an experienced psychiatrist and psychologist. We analyzed 233 HCs, 94 MDD patients, and 75 SZ patients using the McGill Pain Questionnaire (MPQ) and SF-8 (all participants), the Hamilton Depression Rating Scale 21 items (MDD patients), and the Positive and Negative Symptom Scale (SZ patients). Although MDD patients experienced more pain than HCs, there was no difference in the prevalence of pain between SZ patients and HCs. Moreover, HCs with pain did not have higher SF-8 total scores than those without pain, whereas both MDD and SZ patients with pain had higher SF-8 total scores than those without pain. The severity of psychopathology in MDD and SZ patients was also positively associated with both the prevalence of pain and MPQ scores. MPQ scores were also associated with positive symptoms in SZ patients. Considering these results, physicians need to query MDD patients about physical pain during examination if they are to ensure a favorable and quick response to treatment. The severity of positive symptoms (i.e., clinical status) in SZ patients might also be associated with pain sensitivity, and warrants further investigation.

A bispecific antibody NXT007 exerts a hemostatic activity in hemophilia A monkeys enough to keep a nonhemophilic state.

BACKGROUND: Emicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients. OBJECTIVES: To identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation. METHODS: New pairs of light chains for emicizumab's heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A. RESULTS: NXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab's historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well. CONCLUSION: NXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab.

SAIL66, a next generation CLDN6-targeting T-cell engager, demonstrates potent antitumor efficacy through dual binding to CD3/CD137.

BACKGROUND: Ovarian cancer remains a formidable challenge in oncology, necessitating innovative therapeutic approaches. Claudin-6 (CLDN6), a member of the tight junction molecule CLDN family, exhibits negligible expression in healthy tissues but displays aberrant upregulation in various malignancies, including ovarian cancer. Although several therapeutic modalities targeting CLDN6 are currently under investigation, there is still a need for more potent therapeutic options. While T-cell engagers (TCEs) hold substantial promise as potent immunotherapeutic agents, their current efficacy and safety in terms of target antigen selection and T-cell exhaustion due to only CD3 stimulation without co-stimulation must be improved, particularly against solid tumors. To provide an efficacious treatment option for ovarian cancer, we generated SAIL66, a tri-specific antibody against CLDN6/CD3/CD137. METHODS: Using our proprietary next-generation TCE technology (Dual-Ig), SAIL66 was designed to bind to CLDN6 with one Fab and CD3/CD137 with the other, thereby activating T cells through CD3 activation and CD137 co-stimulation. The preclinical characterization of SAIL66 was performed in a series of in vitro and in vivo studies which included comparisons to a conventional TCE targeting CLDN6 and CD3. RESULTS: Despite the high similarity between CLDN6 and other CLDN family members, SAIL66 demonstrated high specificity for CLDN6, reducing the risk of off-target toxicity. In an in vitro co-culture assay with CLDN6-positive cancer cells, we confirmed that SAIL66 strongly activated the CD137 signal in the Jurkat reporter system, and preferentially induced activation of both CD4+ and CD8+ T cells isolated from human peripheral blood mononuclear cells compared to conventional TCEs. In vivo studies demonstrated that SAIL66 led to a more pronounced increase in intratumor T-cell infiltration and a decrease in exhausted T cells compared with conventional CLDN6 TCE by contribution of CD137 co-stimulation, resulting in better antitumor efficacy in tumor-bearing mouse models. CONCLUSION: Our data demonstrate that SAIL66, designed to engage CLDN6, CD3, and CD137, has the potential to enhance antitumor activity and provide a potent therapeutic option for patients with ovarian and other solid tumors expressing CLDN6. Clinical trials are currently underway to evaluate the safety and efficacy of SAIL66.

Preclinical in vitro evaluation of immune suppression induced by GYM329, Fc-engineered sweeping antibody.

Fc-engineering is commonly used to improve the therapeutic potency of antibody (Ab) treatments. Because FcγRIIb is the only inhibitory FcγR that contains an immunoreceptor tyrosine-based inhibition motif (ITIM), Fc-engineered Abs with enhanced binding affinity to FcγRIIb might provide immune suppression in clinical contexts. GYM329 is an anti-latent myostatin Fc-engineered Ab with increased affinity to FcγRIIb which is expected to improve muscle strength in patients with muscular disorders. Cross-linking of FcγRIIb by immune complex (IC) results in phosphorylation of ITIM to inhibit immune activation and apoptosis in B cells. We examined whether the IC of Fc-engineered Abs with enhanced binding affinity to FcγRIIb causes phosphorylation of ITIM or B cell apoptosis using GYM329 and its Fc variant Abs in human and cynomolgus-monkey (cyno) immune cells in vitro. IC of GYM329 with enhanced binding affinity to human FcγRIIb (×5) induced neither ITIM phosphorylation nor B cell apoptosis. As for GYM329, FcγRIIb should work as an endocytic receptor of small IC to sweep latent myostatin, so it is preferable that GYM329 induces neither ITIM phosphorylation nor B cell apoptosis to prevent immune suppression. In contrast, IC of myo-HuCy2b, the Ab with enhanced binding affinity to human FcγRIIb (×4), induced ITIM phosphorylation and B cell apoptosis. The result of the present study demonstrated that Fc-engineered Abs with similar binding affinity to FcγRIIb had different effects. Thus, it is important to also investigate FcγR-mediated immune functions other than binding to fully understand the biological effects of Fc-engineered Abs.

Characterizations of a neutralizing antibody broadly reactive to multiple gluten peptide:HLA-DQ2.5 complexes in the context of celiac disease.

In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.

Novel myostatin-specific antibody enhances muscle strength in muscle disease models.

Myostatin, a member of the transforming growth factor-ß superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via "sweeping antibody technology", GYM329 reduces or "sweeps" myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.

Escitalopram versus paroxetine controlled release in major depressive disorder: a randomized trial.

OBJECTIVE: There are no direct comparisons between escitalopram and paroxetine controlled release in patients with major depressive disorder (MDD). METHODS: We conducted a 24-week, rater-masked, randomized trial of escitalopram (5-20 mg/day) versus paroxetine controlled release (12.5-50 mg/day) in patients with MDD (UMIN000011191). Patients with the diagnosis of moderate-to-severe MDD (a 17-item Hamilton Rating Scale for Depression [HAMD-17], with total score at baseline being ≥20) were recruited to participate in a parallel, randomized, controlled trial. The primary outcome for efficacy was an improvement in the 21-item HAMD (HAMD-21) total score at 24 weeks. The secondary outcomes were the response, remission, and discontinuation rates and the incidence of individual adverse events. RESULTS: A total of 88 patients with MDD (males, 61.4%; mean age, 40.8±13.4 years) were recruited. The discontinuation rate was 58.0% (escitalopram, 55.8%; paroxetine controlled release, 60.0%). Both escitalopram and paroxetine controlled-release treatment groups exhibited significant reduction in the HAMD-21 total score at 2, 4, 8, 12, and 24 weeks from the baseline. However, there were no significant differences in the HAMD-21 total score, response rate, remission rate, and discontinuation rate at any time point between the groups. In addition, there were no significant differences in the incidence of any individual adverse events (eg, nausea, vomiting, and somnolence) between the treatment groups. CONCLUSION: Our results suggest that escitalopram and paroxetine controlled release had similar efficacy and safety profiles in patients with MDD. One of the primary limitations of this study is the small sample size.

Peptide sequences converting polyglutamine into a prion in yeast.

Amyloids are ordered protein aggregates composed of cross-ß sheet structures. Amyloids include prions, defined as infectious proteins, which are responsible for mammalian transmissible spongiform encephalopathies, and fungal prions. Although the conventional view is that typical amyloids are associated with nontransmissible mammalian neurodegenerative diseases such as Alzheimer's disease, increasing evidence suggests that the boundary between transmissible and nontransmissible amyloids is ambiguous. To clarify the mechanism underlying the difference in transmissibility, we investigated the dynamics and the properties of polyglutamine (polyQ) amyloids in yeast cells, in which the polyQ aggregates are not transmissible but can be converted into transmissible amyloids. We found that polyQ had an increased tendency to form aggregates compared to the yeast prion Sup35. In addition, we screened dozens of peptides that converted the nontransmissible polyQ to transmissible aggregates when they flanked the polyQ stretch, and also investigated their cellular dynamics aiming to understand the mechanism of transmission.