9 research outputs found
Fluorescence-Activated Cell Sorting of Human l‑asparaginase Mutant Libraries for Detecting Enzyme Variants with Enhanced Activity
Immunogenicity is
one of the most common complications occurring
during therapy making use of protein drugs of nonhuman origin. A notable
example of such a case is bacterial l-asparaginases (L-ASNases)
used for the treatment of acute lymphoblastic leukemia (ALL). The
replacement of the bacterial enzymes by human ones is thought to set
the basis for a major improvement of antileukemic therapy. Recently,
we solved the crystal structure of a human enzyme possessing L-ASNase
activity, designated hASNase-3. This enzyme is expressed as an inactive
precursor protein and post-translationally undergoes intramolecular
processing leading to the generation of two subunits which remain
noncovalently, yet tightly associated and constitute the catalytically
active form of the enzyme. We discovered that this intramolecular
processing can be drastically and selectively accelerated by the free
amino acid glycine. In the present study, we report on the molecular
engineering of hASNase-3 aiming at the improvement of its catalytic
properties. We created a fluorescence-activated cell sorting (FACS)-based
high-throughput screening system for the characterization of rationally
designed mutant libraries, capitalizing on the finding that free glycine
promotes autoproteolytic cleavage, which activates the mutant proteins
expressed in an <i>E. coli</i> strain devoid of aspartate
biosynthesis. Successive screening rounds led to the isolation of
catalytically improved variants showing up to 6-fold better catalytic
efficiency as compared to the wild-type enzyme. Our work establishes
a powerful strategy for further exploitation of the human asparaginase
sequence space to facilitate the identification of <i>in vitro</i>-evolved enzyme species that will lay the basis for improved ALL
therapy
Preserving catalytic activity and enhancing biochemical stability of the therapeutic enzyme asparaginase by biocompatible multilayered polyelectrolyte microcapsules
The present study focuses on the formation of microcapsules containing catalytically active L-asparaginase (L-ASNase), a protein drug of high value in antileukemic therapy. We make use of the layer-by-layer (LbL) technique to coat protein-loaded calcium carbonate (CaCO3) particles with two or three poly dextran/poly-L-arginine-based bilayers. To achieve high loading efficiency, the CaCO3 template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO3 core material was dissolved under mild conditions by dialysis against 20 mM EDTA. Biochemical stability of the encapsulated L-asparaginase was analyzed by treating the capsules with the proteases trypsin and thrombin, which are known to degrade and inactivate the enzyme during leukemia treatment, allowing us to test for resistance against proteolysis by physiologically relevant proteases through measurement of residual L-asparaginase activities. In addition, the thermal stability, the stability at the physiological temperature, and the long-term storage stability of the encapsulated enzyme were investigated. We show that encapsulation of L-asparaginase remarkably improves both proteolytic resistance and thermal inactivation at 37 degrees C, which could considerably prolong the enzyme's in vivo half-life during application in acute lymphoblastic leukemia (ALL). Importantly, the use of low EDTA concentrations for the dissolution of CaCO3 by dialysis could be a general approach in cases where the activity of sensitive biomacromolecules is inhibited, or even irreversibly damaged, when standard protocols for fabrication of such LbL microcapsules are used. Encapsulated and free enzyme showed similar efficacies in driving leukemic cells to apoptosis
Preserving Catalytic Activity and Enhancing Biochemical Stability of the Therapeutic Enzyme Asparaginase by Biocompatible Multilayered Polyelectrolyte Microcapsules
The present study focuses on the
formation of microcapsules containing
catalytically active l-asparaginase (L-ASNase), a protein
drug of high value in antileukemic therapy. We make use of the layer-by-layer
(LbL) technique to coat protein-loaded calcium carbonate (CaCO<sub>3</sub>) particles with two or three poly dextran/poly-l-arginine-based bilayers. To achieve high loading efficiency, the
CaCO<sub>3</sub> template was generated by coprecipitation with the
enzyme. After assembly of the polymer shell, the CaCO<sub>3</sub> core
material was dissolved under mild conditions by dialysis against 20
mM EDTA. Biochemical stability of the encapsulated l-asparaginase
was analyzed by treating the capsules with the proteases trypsin and
thrombin, which are known to degrade and inactivate the enzyme during
leukemia treatment, allowing us to test for resistance against proteolysis
by physiologically relevant proteases through measurement of residual l-asparaginase activities. In addition, the thermal stability,
the stability at the physiological temperature, and the long-term
storage stability of the encapsulated enzyme were investigated. We
show that encapsulation of l-asparaginase remarkably improves
both proteolytic resistance and thermal inactivation at 37 °C,
which could considerably prolong the enzyme’s in vivo half-life
during application in acute lymphoblastic leukemia (ALL). Importantly,
the use of low EDTA concentrations for the dissolution of CaCO<sub>3</sub> by dialysis could be a general approach in cases where the
activity of sensitive biomacromolecules is inhibited, or even irreversibly
damaged, when standard protocols for fabrication of such LbL microcapsules
are used. Encapsulated and free enzyme showed similar efficacies in
driving leukemic cells to apoptosis
Unique Spatial Immune Profiling in Pancreatic Ductal Adenocarcinoma with Enrichment of Exhausted and Senescent T Cells and Diffused CD47-SIRP proportional to Expression
Background: Pancreatic ductal adenocarcinoma (PDAC) is resistant to single-agent immunotherapies. To understand the mechanisms leading to the poor response to this treatment, a better understanding of the PDAC immune landscape is required. The present work aims to study the immune profile in PDAC in relationship to spatial heterogeneity of the tissue microenvironment (TME) in intact tissues. Methods: Serial section and multiplex in situ analysis were performed in 42 PDAC samples to assess gene and protein expression at single-cell resolution in the: (a) tumor center (TC), (b) invasive front (IF), (c) normal parenchyma adjacent to the tumor, and (d) tumor positive and negative draining lymph nodes (LNs). Results: We observed: (a) enrichment of T cell subpopulations with exhausted and senescent phenotype in the TC, IF and tumor positive LNs; (b) a dominant type 2 immune response in the TME, which is more pronounced in the TC; (c) an emerging role of CD47-SIRP a axis; and (d) a similar immune cell topography independently of the neoadjuvant chemotherapy. Conclusion: This study reveals the existence of dysfunctional T lymphocytes with specific spatial distribution, thus opening a new dimension both conceptually and mechanistically in tumor-stroma interaction in PDAC with potential impact on the efficacy of immune-regulatory therapeutic modalities