Biology subjects

Yesiltepe, M.

Publications and source records attributed to Yesiltepe, M..

4 recordsLinked to original sources

bioRxiv2026

NN-800s: Brain-penetrant trimeric nanobodies enable potent TNFα inhibition via TfR1-mediated transcytosis

Tumor necrosis factor alpha (TNF) is a central mediator of neuroinflammation and synaptic dysfunction in multiple central nervous system (CNS) disorders, including Alzheimers disease. Although clinically approved TNF inhibitors are highly effective in peripheral inflammatory diseases, their therapeutic application in CNS disorders is severely limited by poor blood-brain barrier (BBB) penetration. Here, we report the development of NN-800s, a class of heterotrimeric nanobody-based biologics engineered to achieve both high TNF neutralization potency and efficient BBB transcytosis. NN-800s consist of two humanized anti-TNF nanobodies flanking a humanized anti-transferrin receptor 1 (TfR1) nanobody that mediates receptor-dependent transport across the BBB. These constructs exhibit picomolar TNF inhibitory activity and achieve cerebrospinal fluid (CSF)-to-serum ratios of up to ~0.4 following systemic administration. Importantly, NN-800s do not disrupt transferrin-TfR1 interactions and do not induce hematological toxicity in vivo in humanized Tf/TfR1 rat models, supporting a favorable safety profile. The constructs are efficiently produced in CHO cells with high purity, low endotoxin levels, and strong scalability, supporting their developability as therapeutic biologics. Together, these data establish NN-800s as a promising platform for CNS-targeted TNF inhibition and demonstrate a generalizable strategy for delivering biologics across the BBB with therapeutic-level exposure.

neuroscience↗

bioRxiv2025

Humanized TfR1 and transferrin gene-replacement rats for in vivo evaluation of BBB transport

The transferrin receptor 1 (TfR1)-transferrin (TF) axis plays a central role in iron homeostasis and has long been recognized as a promising route for delivering biologics across the blood-brain barrier (BBB). We have developed a class of human-specific anti-TfR1 nanobodies (NewroBus) that exploit this transport pathway. However, the lack of cross-reactivity with rodent TfR1 limits the utility of standard animal models for preclinical testing. To overcome this challenge, we generated knock-in (KI) rats in which the coding sequences of the endogenous Tfrc and Tf genes were replaced with human coding sequences, yielding animals that express human TfR1 and/or human TF under physiological control. Rats homozygous for both humanized alleles were viable and fertile, indicating that the human proteins can functionally replace their rodent equivalents. Nonetheless, these double homozygous rats exhibited erythropoietic abnormalities and tissue-specific alterations in iron distribution--characterized by decreased splenic and increased hepatic iron--suggesting incomplete functional compensation. In contrast, heterozygous rats showed only mild, subclinical hematologic changes (microcytosis and hypochromia). These findings demonstrate that the humanized TfR1-TF axis is compatible with life and iron regulation, albeit with varying degrees of compensation depending on gene dosage. Importantly, these KI rats provide a translationally relevant platform for evaluating pharmacokinetics, CNS penetration, and safety of human-specific BBB-targeting therapeutics, including NewroBus-based biologics and other TfR1-mediated delivery strategies.

neuroscience↗

bioRxiv2025

NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

Effective delivery of therapeutics to the brain is restricted by the blood-brain barrier (BBB). A strategy to overcome this limitation involves taking advantage of receptor-mediated transcytosis pathways, such as those mediated by transferrin receptor 1 (TfR1), which is highly expressed on brain endothelial cells and naturally transports iron-bound transferrin across the BBB. To exploit this mechanism, we immunized camelids with human TfR1 and cloned 470 VHH nanobody sequences from their B cells. From this repertoire, 24 nanobodies (TfR1b-Nbs) were identified that bind human TfR1 on the cell membrane. These nanobodies were screened for binding to human TfR1, lack of interference with transferrin binding and TfR1-mediated iron uptake, and the ability to cross the BBB via human TfR1-mediated transcytosis in newly generated humanized Tfr1h knock-in rats. To improve developability and reduce potential immunogenicity, selected TfR1b-Nbs were humanized and optimized with computational and artificial intelligence (AI) algorithms, enhancing humanness, solubility, and VHH-nativeness. Eight optimized TfR1b-Nbs retained BBB permeability and were fused to humanized anti-TNF nanobody inhibitors (TNFI- or TNFI-{beta}), generating 16 heterodimers. Fusion to these TNFIs served as a functional readout, confirming that TfR1b-Nbs can shuttle biologically active, BBB-impermeable payloads into the central nervous system (CNS). All heterodimers demonstrated CNS delivery after intravenous administration, and selected constructs also reached the brain via subcutaneous injection, maintaining high serum and cerebrospinal fluid (CSF) levels for up to 72 hours. A pilot study with one heterodimer showed that chronic administration in rats humanized for both transferrin and TfR1 caused no hematological toxicity or signs of anemia - a key safety concern when targeting TfR1. These results establish humanized TfR1b-Nbs - designated NewroBus - as promising BBB shuttles for the safe and effective therapeutic delivery of biologics to the brain.

bioengineering↗

bioRxiv2025

Pathological Mechanisms of Motor Dysfunction in Familial Danish Dementia: Insights from a Knock-In Rat Model

Familial Danish Dementia (FDD) is a rare autosomal dominant neurodegenerative disorder caused by a mutation in the integral membrane protein 2B (ITM2b) gene. Clinically, FDD is characterized by cerebral amyloid angiopathy (CAA), cerebellar ataxia, and dementia. Notably, FDD shares several neuropathological features with Alzheimers disease (AD), including CAA, neuroinflammation, and neurofibrillary tangles. In this study, we investigate the pathological mechanisms linking CAA, white matter damage, and motor dysfunction using a recently developed FDD knock-in (FDD-KI) rat model. This model harbors the Danish mutation in the endogenous rat Itm2b gene, along with an App gene encoding humanized amyloid-{beta} (A{beta}). Our analysis revealed substantial vascular Danish amyloid (ADan) deposition in the cerebellar subpial and leptomeningeal vessels of FDD-KI rats, showing an age-related increase comparable to that observed in human FDD patients. Additionally, vascular A{beta} deposits (A{beta}-CAA) were present in FDD-KI rats, but A{beta}-CAA patterns showed some differences between species. Motor function assessments in FDD-KI rats demonstrated age-accelerated motor deficits and gait abnormalities, mirroring the clinical characteristics of FDD patients. To further explore the mechanisms underlying these deficits, we examined cerebellar pathology and found age-related myelin disruption and axonal fiber loss, consistent with postmortem human FDD pathology. Cerebellar demyelination appeared to be driven by neuroinflammation, marked by increased microglial/macrophage activation in response to vascular amyloid deposition. Additionally, we observed extravascular fibrinogen leakage, indicating widespread vascular permeability in both white and gray matter, with fibrinogen deposits surrounding amyloid-positive vessels in aged FDD-KI rats and postmortem FDD cerebellum. These findings suggest that this FDD-KI rat model is the first animal model to recapitulate key neuropathological features of human FDD patients, including both ADan- and A{beta}-type CAA, neuroinflammation, and white matter lesions--pathologies that may underlie the motor and gait impairments seen in the disease.

neuroscience↗