Biology subjects

Kedzior, M.

Publications and source records attributed to Kedzior, M..

6 recordsLinked to original sources

bioRxiv2025

Engineering HIV antibodies with enhanced breadth and potency of neutralization through multistate affinity maturation

Broadly neutralizing antibodies (bnAbs) against HIV hold promise as therapeutic and prophylactic agents, but realizing this potential requires antibodies that function across the antigenic heterogeneity of the HIV envelope glycoprotein (Env). Although numerous bnAbs have been isolated from infected individuals, their breadth and potency may not be sufficient to tackle global viral diversity, motivating efforts to further improve their neutralization capacity. Here, we address this challenge using a multistate antibody engineering approach integrating deep mutational scanning with combinatorial library screening across diverse Env variants. This strategy enables identification of mutation patterns that confer improved binding across antigenically distinct targets. Starting from one of the best-in-class CD4-binding site bnAbs, we performed iterative optimization to increase binding affinity across diverse Env variants. The resulting lead candidate exhibited improved breadth and up to 100-fold higher potency against pseudoviruses from large cross-clade historical and contemporary panels while maintaining biophysical and pharmacokinetic profiles conducive to clinical development. Structural and molecular dynamics analyses revealed a unique tri-tyrosine aromatic triad and reinforced electrostatic contacts that stabilized the bnAb/Env interface. These findings demonstrate that systematic in vitro engineering can generate bnAbs with enhanced breadth and potency, providing a generalizable strategy for developing therapeutic antibodies against highly diverse pathogens.

bioengineering↗

bioRxiv2025

Improvement of affinity and potency of a monoclonal antibody against Shigella flexneri 3a O-antigen via phage display and whole-cell in-solution panning

As rates of antimicrobial resistance (AMR) among bacterial pathogens continue to rise, the discovery and development of novel classes of therapeutics that can serve as alternatives or adjuncts to traditional small-molecule antibiotics, such as monoclonal antibodies (mAbs), is a public health priority. Some of the most promising antigen targets for antibacterial mAbs are surface polysaccharides such as O-antigen (O-Ag), a component of the lipopolysaccharide found on the outer membrane of gram-negative bacteria. However, developing mAbs against bacterial surface polysaccharides with sufficient breadth and potency to be clinically viable is difficult in part because antibodies against polysaccharides are generally low affinity, and the challenging biochemistry of polysaccharides often precludes further affinity maturation of mAbs against these targets in vitro. Here, we use a phage display library and a whole-cell in-solution panning strategy to successfully improve the affinity of a mAb against Shigella flexneri 3a O-Ag in vitro without requiring the purification of the target antigen. We demonstrate that a single mutation can improve apparent affinity approximately 10-fold without detectably increasing polyreactivity, and increased affinity correlates with enhanced potency in antibacterial effector function and anti-virulence assays. In addition, the most potent variants also gained increased breadth, successfully coordinating complement deposition and complement-independent opsonophagocytosis against S. flexneri 3b, a serotype weakly recognized by the parent mAb. Altogether, this work represents an important first step towards expanding the antibody engineering toolkit for bacterial surface polysaccharides, which will aid the development of novel mAb therapeutics against AMR bacterial pathogens.

bioengineering↗

bioRxiv2024

A Comprehensive Engineering Strategy Improves Potency and Manufacturability of a Near Pan-neutralizing Antibody Against HIV

Anti-HIV envelope broadly neutralizing antibodies (bnAbs) are alternatives to conventional antiretrovirals with the potential to prevent and treat infection, reduce latent reservoirs, and/or mediate a functional cure. Clinical trials with "first generation" bnAbs used alone or in combination show promising antiviral effects but also highlight that additional engineering of "enhanced" antibodies will be required for optimal clinical utility, while preserving or enhancing cGMP manufacturing capability. Here we report the engineering of an anti-CD4 binding-site (CD4bs) bnAb, N49P9.3, purified from the plasma of an HIV elite-neutralizer. Through a series of rational modifications we produced a variant that demonstrates: enhanced potency; superior antiviral activity in combination with other bnAbs; low polyreactivity; and longer circulating half-life. Additional engineering for manufacturing produced a final variant, eN49P9, with properties conducive to cGMP production. Overall, these efforts demonstrate the feasibility of developing enhanced anti-CD4bs bnAbs with greatly improved antiviral properties as well as potential translational value.

bioengineering↗

bioRxiv2023

The acetylase activity of Cdu1 protects Chlamydia effectors from degradation to regulate bacterial exit from infected cells

Many cellular processes are regulated by ubiquitin-mediated proteasomal degradation. Pathogens can regulate eukaryotic proteolysis through the delivery of proteins with de-ubiquitinating (DUB) activities. The obligate intracellular pathogen Chlamydia trachomatis secretes Cdu1 (ChlaDUB1), a dual deubiquitinase and Lys-acetyltransferase, that promotes Golgi remodeling and survival of infected host cells presumably by regulating the ubiquitination of host and bacterial proteins. Here we determined that Cdu1s acetylase but not its DUB activity is important to protect Cdu1 from ubiquitin-mediated degradation. We further identified three C. trachomatis proteins on the pathogen-containing vacuole (InaC, IpaM, and CTL0480) that required Cdu1 s acetylase activity for protection from degradation and determined that Cdu1 and these Cdu1-protected proteins are required for optimal egress of Chlamydia from host cells. These findings highlight a non-canonical mechanism of pathogen-mediated protection of virulence factors from degradation after their delivery into host cells and the coordinated regulation of secreted effector proteins.

microbiology↗

bioRxiv2021

Molecular foundations of Precambrian uniformitarianism

The earliest geochemical indicators of microbes--and the enzymes that powered them--extend back almost 3.8 billion years on our planet. Paleobiologists often attempt to understand these indicators by assuming that the behaviors of modern microbes and enzymes are consistent (uniform) with those of their predecessors. A uniformitarian assumption (i.e., the idea that fundamental geobiological processes have occurred in much the same manner over Earth history) seems at odds with our understanding of the inherent variability of living systems. Here, we examine whether a uniformitarian assumption for an enzyme thought to generate carbon isotope indicators of biological activity, RuBisCO, can be corroborated by independently studying the history of changes recorded within RuBisCOs genetic sequences. Specifically, we resurrected a Precambrian-age, ancient RuBisCO by engineering its ancient DNA inside a modern cyanobacterium genome and measured the engineered organisms fitness and carbon-isotope-discrimination profile. The envelope of ancestral RuBisCO isotopic fractionation observed here indicates that uniformitarian assumptions may be warranted, but with important caveats. Our results suggest that further inquiries that link molecule-level evolutionary changes with planet-level geochemical conditions are needed to discern whether enzyme-affected isotope fractionation trends extend deeper into the early Precambrian. Experimental studies illuminating lifes early molecular innovations are crucial to explore the foundations of Precambrian uniformitarian assumptions.

evolutionary biology↗

bioRxiv2021

System-level effects of CO2 and RuBisCO concentration on carbon isotope fractionation

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO2 concentrations), molecular, and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a range of genetic and environmental factors that may have impacted ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO and characterized the resultant physiological and isotope fractionation effects. We specifically investigated how both increased atmospheric CO2 concentrations and RuBisCO regulation influence cell growth, oxygen evolution rate, and carbon isotope fractionation in cyanobacteria. We found that elevated CO2 increases the growth rate of wild-type and mutant strains, and that the pool of active RuBisCO enzyme increases with increased expression. RuBisCO overexpression in our engineered strain does not significantly affect isotopic discrimination at all tested CO2 concentrations, yielding cellular 13C/12C isotope discrimination ({varepsilon}p) of [~]24{per thousand} for both wild-type and mutant strains at elevated CO2. Understanding the environmental factors that impact gene regulation, physiology, and evolution is crucial for reconciling microbially driven carbon isotope fractionation with the geologic record carbon biosignatures.

microbiology↗