Defining the epitope of a blood–brain barrier crossing single domain antibody specific for the type 1 insulin-like growth factor receptor

Sheff, Joey; Wang, Ping; Xu, Ping; Arbour, Mélanie; Masson, Luke; Faassen, Henk van; Hussack, Greg; Kemmerich, Kristin; Brunette, Eric; Stanimirovic, Danica; Hill, Jonathan D.; Kelly, John F.; Ni, Feng

doi:10.1038/s41598-021-83198-w

Cited by 17 publications

(21 citation statements)

References 53 publications

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“…IGF1R3, IGF1R4, and IGF1R5 sdAbs (V H H format) showed high‐affinity binding to human‐IGF1R with K D s of 1.3, 0.3, and 0.6 nM, respectively (Figure 1). The binding epitope of IGF1R5 sdAb on IGF1R has recently been characterized in detail 48 . Neither of the IGF1R V H Hs bound the insulin receptor ectodomain (data not shown).…”

Section: Resultsmentioning

confidence: 93%

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Targeting insulin‐like growth factor‐1 receptor (IGF1R) for brain delivery of biologics

et al. 2022

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The blood-brain barrier (BBB) prevents the majority of drugs from crossing into the brain and reaching neurons. To overcome this challenge, safe and non-invasive technologies targeting receptor-mediated pathways have been developed. In this study, three single-domain antibodies (sdAbs; IGF1R3, IGF1R4, and IGF1R5) targeting the extracellular domain of the human insulin-like growth factor-1 receptor (IGF1R), generated by llama immunization, showed enhanced transmigration across the rat BBB model (SV-ARBEC) in vitro. The rate of brain uptake of these sdAbs fused to mouse Fc (sdAb-mFc) in vivo was estimated using the fluorescent in situ brain perfusion (ISBP) technique followed by optical brain imaging and distribution volume evaluation.Compared to the brains perfused with the negative control A20.1-mFc, the brains perfused with anti-IGF1R sdAbs showed a significant increase of the total fluorescence intensity (~2-fold, p < .01) and the distribution volume (~4-fold, p < .01). The concentration curve for IGF1R4-mFc demonstrated a linear accumulation plateauing at approximately 400 µg (~1 µM), suggesting a saturable mechanism of transport. Capillary depletion and mass spectrometry analyses of brain parenchyma post-ISBP confirmed the IGF1R4-mFc brain uptake with ~25% of the total amount being accumulated in the parenchymal fraction in contrast to undetectable levels of A20.1-mFc after a 5-min perfusion protocol. Systemic administration of IGF1R4-mFc fused with the non-BBB crossing analgesic peptide galanin (2 and 5 mg/kg) induced a dose-dependent suppression of thermal hyperalgesia in the Hargreaves pain model. In conclusion, novel anti-IGF1R sdAbs showed receptor-mediated brain uptake with pharmacologically effective parenchymal delivery of non-permeable neuroactive peptides.

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Section: Resultsmentioning

confidence: 93%

“…The binding epitope of IGF1R5 sdAb on IGF1R has recently been characterized in detail. 48 Neither of the IGF1R V H Hs bound the insulin receptor ectodomain (data not shown).…”

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confidence: 90%

Targeting insulin‐like growth factor‐1 receptor (IGF1R) for brain delivery of biologics

et al. 2022

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“…Thus, IGFs can reach the CSF and specific regions of the brain. IGF1R has also emerged as a target to promote receptormediated transcytosis [100]. VHH-IR5, a nanobody targeting the type 1 insulin-like growth factor receptor 5 (IGF1R), does not appear to interfere with IGF1R kinase activity and its interactions with the endogenous ligand IGF-1.…”

Section: Receptor-mediated Transcytosismentioning

confidence: 99%

“…VHH-IR5, a nanobody targeting the type 1 insulin-like growth factor receptor 5 (IGF1R), does not appear to interfere with IGF1R kinase activity and its interactions with the endogenous ligand IGF-1. VHH-IR5 targets with high affinity a linear epitope at the α-helix in the C-terminal segment (α-CT) of IGF1R, sharing the binding site of IGF-1 in an independent and non-competitive manner [100,101], suggesting a potential role of the α-CT in VHH-IR5 transcytosis across the BBB.…”

Section: Receptor-mediated Transcytosismentioning

confidence: 99%

Transportation of Single-Domain Antibodies through the Blood–Brain Barrier

Ruiz-López

Schuhmacher

2021

Biomolecules

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Single-domain antibodies derive from the heavy-chain-only antibodies of Camelidae (camel, dromedary, llama, alpaca, vicuñas, and guananos; i.e., nanobodies) and cartilaginous fishes (i.e., VNARs). Their small size, antigen specificity, plasticity, and potential to recognize unique conformational epitopes represent a diagnostic and therapeutic opportunity for many central nervous system (CNS) pathologies. However, the blood–brain barrier (BBB) poses a challenge for their delivery into the brain parenchyma. Nevertheless, numerous neurological diseases and brain pathologies, including cancer, result in BBB leakiness favoring single-domain antibodies uptake into the CNS. Some single-domain antibodies have been reported to naturally cross the BBB. In addition, different strategies and methods to deliver both nanobodies and VNARs into the brain parenchyma can be exploited when the BBB is intact. These include device-based and physicochemical disruption of the BBB, receptor and adsorptive-mediated transcytosis, somatic gene transfer, and the use of carriers/shuttles such as cell-penetrating peptides, liposomes, extracellular vesicles, and nanoparticles. Approaches based on single-domain antibodies are reaching the clinic for other diseases. Several tailoring methods can be followed to favor the transport of nanobodies and VNARs to the CNS, avoiding the limitations imposed by the BBB to fulfill their therapeutic, diagnostic, and theragnostic promises for the benefit of patients suffering from CNS pathologies.

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“…The use of VHHs for BBB transcytosis and target engagement is promising. Specifically, Stanimirovic et al identified an insulin-like growth factor 1 receptor binding VHH which crosses the BBB by receptor mediated transcytosis [30, 31]. Danis et al identified and optimized VHHs to mitigate brain accumulation of pathological tau in a tauopathy mouse model [32].…”

Section: Introductionmentioning

confidence: 99%

Selection of single domain anti-transferrin receptor antibodies for blood-brain barrier transcytosis using a neurotensin based assay and histological assessment of target engagement in a mouse model of Alzheimer’s related amyloid-beta pathology

Esparza

Brody

2022

Preprint

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Background: The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti-mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Methods: Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and in vitro characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (Aβ) VHH dimer and labeled with fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related Aβ plaque pathology. Results: An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of <1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT induced hypothermia effects after intravenous injection of 600 nmol/kg body weight, ranging from undetectable for nonbinding mutants to -6°C for the best mutants. The anti-mTfR VHH variant M1P96H with the strongest hypothermia effect was fused to the anti-Aβ VHH dimer and labeled with Alexa647; the dye-labeled VHH fusion construct still bound both mTfR and Aβ plaques. However, after intravenous injection at 600 nmol/kg body weight into APP/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-induced hypothermia did not correlate with direct target engagement in cortex. Conclusions: There was a surprising dissociation between NT-induced hypothermia, presumably mediated by hypothalamus, and direct engagement with Aβ-plaques in cortex. Alternative methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular contrast agents.

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Defining the epitope of a blood–brain barrier crossing single domain antibody specific for the type 1 insulin-like growth factor receptor

Cited by 17 publications

References 53 publications

Targeting insulin‐like growth factor‐1 receptor (IGF1R) for brain delivery of biologics

Targeting insulin‐like growth factor‐1 receptor (IGF1R) for brain delivery of biologics

Transportation of Single-Domain Antibodies through the Blood–Brain Barrier

Selection of single domain anti-transferrin receptor antibodies for blood-brain barrier transcytosis using a neurotensin based assay and histological assessment of target engagement in a mouse model of Alzheimer’s related amyloid-beta pathology

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