Biology subjects

Galimidi, R. P.

Publications and source records attributed to Galimidi, R. P..

3 recordsLinked to original sources

bioRxiv2023

Engineering antigen-specific tolerance to an artificial protein hydrogel

Artificial protein hydrogels are an emerging class of biomaterials with numerous prospective applications in tissue engineering and regenerative medicine. These materials are likely to be immunogenic due their frequent incorporation of novel amino acid sequence domains, which often serve a functional role within the material itself. We engineered injectable "self" and "non-self" artificial protein hydrogels which were predicted to have divergent immune outcomes in vivo on the basis of their primary amino acid sequence. Following implantation in mouse, the non-self gels raised significantly higher anti-gel antibody titers than the corresponding self gels. Prophylactic administration of a fusion antibody targeting the non-self hydrogel epitopes to DEC-205, an endocytic receptor involved in Treg induction, fully suppressed the elevated antibody titer against the non-self gels. These results suggest that the clinical immune response to artificial protein biomaterials, including those that contain highly antigenic sequence domains, can be tuned through the induction of antigen-specific tolerance.

bioengineering↗

bioRxiv2021

Modular Fluorescent Nanoparticle DNA Probes for Detection of Peptides and Proteins

Fluorescently labeled antibody and aptamer probes are used in biological studies to characterize binding interactions, measure concentrations of analytes, and sort cells. Fluorescent nanoparticle labels offer an excellent alternative to standard fluorescent labeling strategies due to their enhanced brightness, stability and multivalency; however, challenges in functionalization and characterization have impeded their use. This work introduces a straightforward approach for preparation of fluorescent nanoparticle probes using commercially available reagents and common laboratory equipment. Fluorescent polystyrene nanoparticles, Thermo Fisher Scientific FluoSpheres, were used in proof-of-principle studies. Particle passivation was achieved by covalent attachment of amine-PEG-azide to carboxylated particles, neutralizing the surface charge from -43 to -15 mV. A conjugation-annealing handle and DNA aptamer probe was attached to the azide-PEG nanoparticle surface either through reaction of pre-annealed handle and probe or through a stepwise reaction of the nanoparticles with the handle followed by aptamer annealing. Nanoparticles functionalized with DNA aptamers targeting histidine tags and VEGF protein had high affinity (EC50s ranging from 3-12 nM) and specificity, and were more stable than conventional labels. This protocol for preparation of nanoparticle probes relies solely on commercially available reagents and common equipment, breaking down the barriers to use nanoparticles in biological experiments.

bioengineering↗

bioRxiv2018

Quantifying the Entropic and Energetic Effects of Linker Length and Rigidity within Synthetic HIV-1 Antibodies designed to Bind Bivalently to Env Spikes

Due to the low density of envelope (Env) spikes on the surface of HIV-1, neutralizing IgG antibodies rarely bind bivalently using both antigen-binding arms (Fabs) to crosslink between spikes (inter-spike crosslinking), instead resorting to weaker monovalent binding that is more sensitive to Env mutations. Synthetic antibodies designed to bivalently bind a single Env trimer (intra-spike crosslinking) were previously shown to exhibit increased neutralization potencies. In initial work, diFabs joined by varying lengths of rigid double-stranded DNA (dsDNA) were considered. Anticipating future experiments to improve synthetic antibodies, we investigate whether linkers with different rigidities could enhance diFab potency by modeling DNA-Fabs containing different combinations of rigid dsDNA and flexible single-stranded DNA (ssDNA) and characterizing their neutralization potential. Model predictions suggest that while a long flexible polymer may be capable of bivalent binding, it exhibits weak neutralization due to the large loss in entropic degrees of freedom when both Fabs are bound. In contrast, the strongest neutralization potencies are predicted to require a rigid linker that optimally spans the distance between two Fab binding sites on an Env trimer, and avidity can be further boosted by incorporating more Fabs into these constructs. These results inform the design of multivalent anti-HIV-1 therapeutics that utilize avidity effects to remain potent against HIV-1 in the face of the rapid mutation of Env spikes.\n\nSignificanceIgG antibodies utilize avidity to increase their apparent affinities through simultaneous binding of two antigen-binding Fabs - if one Fab dissociates from an antigen, the other Fab can remain attached, allowing rebinding. HIV-1 foils this strategy by having few, and highly-separated, Envelope spike targets for antibodies, forcing most IgGs to bind monovalently. Here we develop a statistical mechanics model of synthetic diFabs joined by DNA linkers of different lengths and flexibilities. This framework enables us to translate the energetic and entropic effects of the linker into the neutralization potency of a diFab. We demonstrate that the avidity of multivalent binding is enhanced by using rigid linkers or including additional Fabs capable of simultaneous binding, providing the means to quantitatively predict the potencies of other antibody designs.

immunology↗