Efficient therapeutic strategies that concurrently target both Aβ aggregation and oxidative stress in the Alzheimer's disease (AD) microenvironment emerge as a cutting‐edge tool to combat the ...intricate pathogenesis of AD. Here, a multivalent nanobody conjugate with rigid, reactive oxygen species (ROS) scavenging scaffold is developed to achieve simultaneous Aβ amyloidogenesis mitigation, ROS elimination, and Aβ plaque clearance. Grafting Aβ segment (33‐GLMVGGVVIA‐42) into the third complementary‐determining region of a parent nanobody generates an engineered nanobody NB that can recognize Aβ and inhibit its aggregation through homotypic interactions. NB is further genetically modified with a fragment of human interleukin‐1β (163‐VQGEESNDK‐171), so that the obtained fusion nanobody NBIL can also facilitate the Aβ clearance by microglia. Linking NBIL covalently onto a rigid, ROS scavenging scaffold poly(deca‐4,6‐diynedioic acid) (PDDA) creates the multivalent nanobody conjugate PNBIL, which not only boosts the binding affinity between NBIL and Aβ aggregates for nearly 100 times but also possesses a long‐term capability of oxidative stress alleviation, inflammation reduction, and neuron protection. PNBIL has significantly attenuated symptoms on two AD mouse models through amyloidogenesis inhibition and AD microenvironment modulation, validating that the multivalent nanobody conjugate design based on combinatory nanobody and molecular engineering is a promising approach of multi‐target therapeutic strategies.
A multivalent nanobody conjugate PNBIL incorporating a rigid, reactive oxygen species (ROS) scavenging scaffold PDDA and engineered nanobody NBIL, whose third complementary‐determining region is grafted by Aβ 33–42 and C‐terminus is fused with human IL‐1β 163–171, realizes the multi‐target therapy of Alzheimer's disease through effectively suppressing Aβ aggregation, promoting Aβ plaque clearance, and eliminating ROS in the AD microenvironment.
The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their ...biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1. Its sequence was identified as an instability hot spot by the PROSS program, and the available structural information indicated that four CDR1 residues bound directly to the antigen. We therefore modified the loop flexibility with the addition of an extra glycine rather than by mutating single amino acids. This approach significantly increased the nanobody yields but traded-off with moderate affinity loss. Accurate modeling coupled with atomistic molecular dynamics simulations enabled the modifications induced by the glycine insertion and the rationale behind the engineering design to be described in detail.
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