Biology subjects

D'Adamio, L.

Publications and source records attributed to D'Adamio, L..

10 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↗

bioRxiv2025

Development of potent humanized TNFα inhibitory nanobodies for therapeutic applications in TNFα-mediated diseases

Tumor necrosis factor-alpha (TNF) is a key pro-inflammatory cytokine implicated in the pathogenesis of numerous inflammatory and autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease, and neurodegenerative disorders such as Alzheimers Disease. Effective inhibition of TNF is essential for mitigating disease progression and improving patient outcomes. In this study, we present the development and comprehensive characterization of potent humanized TNF inhibitory nanobodies (TNFINbs) derived from camelid single-domain antibodies. In silico analysis of the original camelid nanobodies revealed low immunogenicity, which was further reduced through machine-learning-guided humanization and developability optimization. The two humanized TNFI-Nb variants we developed demonstrated exceptional anti-TNF activity, achieving IC50 values in the picomolar range. Binding assays confirmed their high affinity for TNF, underscoring robust neutralization capabilities. These TNFI-Nbs present valid alternatives to conventional monoclonal antibodies currently used in human therapy, offering potential advantages in potency, specificity, and reduced immunogenicity. Our findings establish a solid foundation for further preclinical development and clinical translation of TNF-targeted nanobody therapies in TNF-mediated diseases.

biochemistry↗

bioRxiv2023

BRI2-mediated regulation of TREM2 processing in microglia and its potential implications for Alzheimer's disease and related dementias

ITM2B/BRI2 mutations cause familial forms of Alzheimers disease (AD)-related dementias by disrupting BRI2s protein function and leading to the accumulation of amyloidogenic peptides. Although typically studied in neurons, our findings show that BRI2 is highly expressed in microglia, which are crucial in AD pathogenesis due to the association of variants in the microglial gene TREM2 with increased AD risk. Our single-cell RNAseq (scRNAseq) analysis revealed a microglia cluster that depends on a Trem2 activity that is inhibited by Bri2, pointing to a functional interaction between Itm2b/Bri2 and Trem2. Given that the AD-related Amyloid-{beta} Precursor protein (APP) and TREM2 undergo similar proteolytic processing, and that BRI2 inhibits APP processing, we hypothesized that BRI2 may also regulate TREM2 processing. We found that BRI2 interacts with Trem2 and inhibits its processing by -secretase in transfected cells. In mice lacking Bri2 expression, we observed increased central nervous system (CNS) levels of Trem2-CTF and sTrem2, which are the products of -secretase processing of Trem2, indicating increased Trem2 processing by -secretase in vivo. Reducing Bri2 expression only in microglia resulted in increased sTrem2 levels, suggesting a cell-autonomous effect of Bri2 on -secretase processing of Trem2. Our study reveals a previously unknow role of BRI2 in regulating TREM2-related neurodegenerative mechanisms. The ability of BRI2 to regulate the processing of both APP and TREM2, combined with its cell-autonomous role in neurons and microglia, makes it a promising candidate for the development of AD and AD-related dementias therapeutics.

neuroscience↗

bioRxiv2022

Initial assessment of the spatial learning, reversal, and sequencing task capabilities of knock-in rats with humanizing mutations in the Ass-coding region of App

Model organisms mimicking the pathogenesis of human diseases are useful for identifying pathogenic mechanisms and testing therapeutic efficacy of compounds targeting them. Models of Alzheimers disease and related dementias aim to reproduce the brain pathology associated with these neurodegenerative disorders. Transgenic models, which involve random insertion of disease-causing genes under the control of artificial promoters, are efficient means of doing so. There are confounding factors associated with transgenic approaches, however, including target gene overexpression, dysregulation of endogenous gene expression at transgenes integration sites, and limitations in mimicking loss-of-function mechanisms. Furthermore, the choice of species is important, and there are anatomical, physiological, and cognitive reasons for favoring the rat over the mouse, which has been the standard for models of neurodegeneration and dementia. We report an initial assessment of the spatial learning, reversal, and sequencing task capabilities of knock-in Long-Evans rats with humanizing mutations in the A{beta}-coding region of App, which encodes amyloid precursor protein (Apph/h rats), using the IntelliCage, an automated operant social home cage system, at 6-8 weeks of age, then again at 4-5 months of age. These rats were previously generated as control organisms for studies on neurodegeneration involving other knock-in rat models from our lab. Apph/h rats of either sex can acquire place learning and reversal tasks. They can also acquire a diagonal sequencing task by 6-8 weeks of age, but not a more advanced serial reversal task involving alternating diagonals, even by 4-5 months of age.

neuroscience↗

bioRxiv2021

Familial Danish dementia young Knock-in rats expressing humanized APP and human Aβ show impaired pre and postsynaptic glutamatergic transmission

Familial British and Danish dementia (FBD and FDD) are two neurodegenerative disorders caused by mutations in the Integral membrane protein 2B (ITM2b). BRI2, the protein encoded by ITM2b, tunes excitatory synaptic transmission at both pre- and post-synaptic terminus. Too, BRI2 interacts with and modulates proteolytic processing of Amyloid-{beta} precursor Protein (APP), whose mutations cause familial forms of Alzheimer disease (FAD). To study pathogenic mechanism triggered by the Danish mutation we generated rats carrying the Danish mutation into the rat Itm2b gene (Itm2bD rats). Given the BRI2/APP interaction and the widely accepted relevance of human A{beta}, a proteolytic product of APP, to AD, Itm2bD rats were engineered to express two humanized App alleles, to produce human A{beta}. Here, we studied young Itm2bD rats to investigate early pathogenic changes. We found that peri-adolescent Itm2bD rats present subtle changes in human A{beta} levels along with decreased spontaneous glutamate release and AMPAR-mediated responses but increased short-term synaptic facilitation in the hippocampal Schaeffer-collateral pathway. These changes are like those observed in adult mice producing rodent A{beta} and carrying either the Danish or British mutations into the mouse Itm2b gene. Collectively, the data show that the pathogenic Danish mutation alters the physiological function of BRI2 at glutamatergic synapses; these functional alterations are detected across species and occur early in life. Future studies will be needed to determine whether this phenomenon represents an early pathogenic event in human dementia.

neuroscience↗

bioRxiv2020

Loss of CDC50A function drives Aβ/p3 production via increased β/α-secretase processing of APP

The Amyloid Precursor Protein (APP) undergoes extensive proteolytic processing to produce several biologically active metabolites which affect Alzheimers disease (AD) pathogenesis. Sequential cleavage of APP by {beta}- and {gamma}-secretases results in A{beta}, while cleavage by - and {gamma}-secretases produces the smaller p3 peptide. Here we report that in cells in which the P4-ATPase flippase subunit CDC50A has been knocked out, large increases in the products of {beta}- and -secretase cleavage of APP (sAPP{beta}/{beta}CTF and sAPP/CTF, respectively) and the downstream metabolites A{beta} and p3 are seen. These data indicate that APP cleavage by {beta}/-secretase are increased and suggest that phospholipid asymmetry plays an important role in APP metabolism and A{beta} production.

molecular biology↗

bioRxiv2020

TNFα reduces inhibitory transmission in young Trem2R47H Sporadic Alzheimer rats before observable Aβ and brain pathology

Trem2R47H rats, which carry the Alzheimers disease (AD) risk factor p.R47H variant of the microglia gene TREM2 and produce human A{beta}. Previously, we demonstrated that supraphysiological TNF- boost glutamatergic transmission and suppresses Long-term-Potentiation (LTP), a surrogate of learning and memory, in peri-adolescent Trem2R47H rats (Ren et al., 2020). Here we tested the effect of the p.R47H TREM2 variant on GABA transmission. We report that GABAergic transmission is decreased in Trem2R47H/R47H rats. This decrease is due to the acute and reversable action of TNF- and is not associated whit changes in human A{beta} levels and pathological brain lesions. Thus, the p.R47H TREM2 variant changes the excitatory/inhibitory balance between glutamate and GABA transmission, favoring excitation. This unbalance could potentiate glutamate excitotoxicity and, over time, contribute to neuronal dysfunction, enhanced neuronal cells death and neurodegeneration. Future studies will determine whether this unbalance represents an early, A{beta}-independent pathway leading to dementia.

neuroscience↗