Yoshikazu Nakamura's research while affiliated with The University of Tokyo and other places

Objective To evaluate the efficacy and safety of a single-dose intravitreal umedaptanib pegol (anti-FGF2, investigational new drug) for the treatment of neovascular age-related macular degeneration (nAMD). Methods Nine participants who had a diagnosis of refractory nAMD were enrolled and received a single intravitreal injection of umedaptanib pegol at increasing doses of 0.2, 1.0 or 2.0 mg in the study eye. Results All three doses of umedaptanib pegol evaluated in the study were safe and well tolerated. No severe adverse event (AE) was observed in the study. There was an improvement in retinal fluid measured by central subfield thickness (CST) in most subjects. Remarkably, all three subjects who received 2.0 mg/eye showed improvement of more than 150 μm. Conclusions Intravitreal umedaptanib pegol was safe, well tolerated, and demonstrated an indication of bioactivity in participants that have persistent subretinal fluid refractory to the treatment with anti-VEGFs.

Background/Objective Intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents are the first-line treatment for exudative age-related macular degeneration (nAMD). Due to the limitations of these standard therapies, targeting alternative mechanisms of action may be helpful for treatment of this very common disease. Here, we investigated an anti-fibroblast growth factor-2 (FGF2) aptamer, umedaptanib pegol, a next generation therapeutic for the treatment of nAMD. Methods Three phase 2 studies were designed. First, a multicentre, randomized, double-masked TOFU study assessed the efficacy of intravitreal injections of umedaptanib pegol monotherapy or in combination with aflibercept, compared to aflibercept monotherapy in 86 subjects with anti-VEGF pretreated nAMD. Second, 22 subjects who had exited the TOFU study received 4 monthly intravitreal injections of umedaptanib pegol (extension, RAMEN study). Third, as an investigator-sponsored trial (TEMPURA study), a single-center, open-label, 4-month study was designed to evaluate the safety and treatment efficacy of umedaptanib pegol in five naïve nAMD patients who had not received any prior anti-VEGF treatment. Results The TOFU study demonstrated that umedaptanib pegol alone or in combination with aflibercept did not improve best-corrected visual acuity (BCVA) and central subfield thickness (CST) over aflibercept alone. However, the change in BCVA and CST at primary endpoint was marginal in all the three treatment groups, suggesting that umedaptanib pegol is effective to prevent the disease progression. The RAMEN study confirmed the cessation of disease progression. In the TEMPURA study, naïve nAMD patients showed improvement and no further macular degeneration, with striking improvement of visual acuity and central subfield thickness in some of the patients. Conclusions These results demonstrate, for the first time, clinical proof of concept for aptamer based anti-FGF2 therapy of nAMD.

Intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents are the first-line treatment for exudative/wet age-related macular degeneration (wet AMD). Alternative mechanisms of action may be helpful for treatment of this very common disease. Here we investigated an anti-fibroblast growth factor-2 (FGF2) aptamer, RBM-007, a next generation therapeutic for the treatment of wet AMD. First, a phase 1 (SUSHI) study confirmed the safety, tolerability and bioactivity of a single intravitreal injection of RBM-007. Second, a phase 2 (TOFU) study did not show any improvement in patients, who had previously undergone anti-VEGF treatment, however it did prevent disease progression. Third, in a phase 2 (TEMPURA) study patients treated with RBM-007, who had not received any prior anti-VEGF treatment, showed improvement and no further macular degeneration, with striking improvement of visual acuity and central subfield thickness in some of the patients. These results demonstrate clinical proof of concept for aptamer based anti-FGF2 therapy of wet AMD.

Transforming growth factor β (TGF-β) is a multifunctional cytokine that plays crucial pathophysiological roles in various diseases, such as cancer and fibrosis. However, the disease modulation by targeting TGF-β1 isoform remains to be established, regardless of several studies employed with limited antibodies. Here, we developed an RNA aptamer to human active TGF-β1, named APT-β1, and characterized its properties in vitro and in vivo. APT-β1 bound to human and mouse active TGF-β1 proteins with high affinity and specificity and strongly inhibited TGF-β1-induced downstream signaling and cell morphology with 50% inhibition concentration (IC50) values at picomolar concentrations. In a xenograft mouse model of non-small cell lung cancer, APT-β1 alone showed no appreciable effect on tumor growth, while it greatly enhanced the anti-tumor effect of gefitinib, an approved tyrosine kinase inhibitor. These findings strongly suggest that the anti-TGF-β1 medication may be a promising cancer therapy to suppress repopulation of lung cancer in combination with certain anti-cancer drugs, such as gefitinib.

Nucleic acid aptamers are generated by an in vitro molecular evolution method known as systematic evolution of ligands by exponential enrichment (SELEX). Various candidates are limited by actual sequencing data from an experiment. Here we developed RaptGen, which is a variational autoencoder for in silico aptamer generation. RaptGen exploits a profile hidden Markov model decoder to represent motif sequences effectively. We showed that RaptGen embedded simulation sequence data into low-dimensional latent space on the basis of motif information. We also performed sequence embedding using two independent SELEX datasets. RaptGen successfully generated aptamers from the latent space even though they were not included in high-throughput sequencing. RaptGen could also generate a truncated aptamer with a short learning model. We demonstrated that RaptGen could be applied to activity-guided aptamer generation according to Bayesian optimization. We concluded that a generative method by RaptGen and latent representation are useful for aptamer discovery.

Achondroplasia is the most prevalent genetic form of dwarfism in humans and is caused by activating mutations in FGFR3 tyrosine kinase. The clinical need for a safe and effective inhibitor of FGFR3 is unmet, leaving achondroplasia currently incurable. Here, we evaluated RBM-007, an RNA aptamer previously developed to neutralize the FGFR3 ligand FGF2, for its activity against FGFR3. In cultured rat chondrocytes or mouse embryonal tibia organ culture, RBM-007 rescued the proliferation arrest, degradation of cartilaginous extracellular matrix, premature senescence, and impaired hypertrophic differentiation induced by FGFR3 signaling. In cartilage xenografts derived from induced pluripotent stem cells from individuals with achondroplasia, RBM-007 rescued impaired chondrocyte differentiation and maturation. When delivered by subcutaneous injection, RBM-007 restored defective skeletal growth in a mouse model of achondroplasia. We thus demonstrate a ligand-trap concept of targeting the cartilage FGFR3 and delineate a potential therapeutic approach for achondroplasia and other FGFR3-related skeletal dysplasias.

Significance Discovery of ligands for G protein–coupled receptors (GPCRs) is of importance in receptor biology and pharmacology but is still a challenging issue. Here, we propose a method for the discovery of ligands against GPCRs by employing a virus-like particle (VLP) and show unique properties of identified nucleic acid aptamers for GPCR. One aptamer raised against purinergic receptor P2Y2 (P2RY2), a GPCR, behaves like a partial agonist to unliganded receptor, whereas it exhibits a positive allosteric modulator (PAM) activity to liganded receptor. We demonstrate the validity of our aptamer screening method targeting VLP-stabilized GPCR and a unique aptamer with dual function, agonist and PAM, for GPCR, depending on whether the intrinsic ligand is prebound to the receptor.

Nucleic acid aptamers are generated by an in vitro molecular evolution method known as systematic evolution of ligands by exponential enrichment (SELEX). A variety of candidates is limited by actual sequencing data from an experiment. Here, we developed RaptGen, which is a variational autoencoder for in silico aptamer generation. RaptGen exploits a profile hidden Markov model decoder to represent motif sequences effectively. We showed that RaptGen embedded simulation sequence data into low-dimension latent space dependent on motif information. We also performed sequence embedding using two independent SELEX datasets. RaptGen successfully generated aptamers from the latent space even though they were not included in high-throughput sequencing. RaptGen could also generate a truncated aptamer with a short learning model. We demonstrated that RaptGen could be applied to activity-guided aptamer generation according to Bayesian optimization. We concluded that a generative method by RaptGen and latent representation are useful for aptamer discovery. Codes are available at https://github.com/hmdlab/raptgen.

MTG8 (RUNX1T1) is a fusion partner of AML1 (RUNX1) in the leukemic chromosome translocation t(8;21). The AML1‐MTG8 fusion gene encodes a chimeric transcription factor. One of the highly conserved domains of MTG8 is TAFH which possesses homology with human TAF4 [TATA‐box binding protein (TBP)‐associated factor]. To obtain specific inhibitors of the AML1‐MTG8 fusion protein, we isolated RNA aptamers against the MTG8 TAFH domain using Systematic Evolution of Ligands by EXponential enrichment (SELEX). All TAF aptamers contained guanine‐rich sequences. Analyses of a TAF aptamer by NMR, CD and mutagenesis revealed that it forms a parallel G‐quadruplex structure in the presence of K+. Furthermore, the aptamer could bind to the AML1‐MTG8 fusion protein and dissociate the AML1‐MTG8/DNA complex, suggesting that it can inhibit the dominant negative effects of AML1‐MTG8 against normal AML1 function and serve as a potential therapeutic agent for leukemia.