Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug‐to‐Antibody Ratios

ChemSusChem ◽  
2021 ◽  
Author(s):  
Antonio Angelastro ◽  
Alexey Barkhanskiy ◽  
Ashley P. Mattey ◽  
Edward G. Pallister ◽  
Reynard Spiess ◽  
...  
Keyword(s):  
Galactose Oxidase ◽  
Modular Assembly ◽  
High Drug

scholarly journals Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios

2021 ◽  
Author(s):  
Antonio Angelastro ◽  
Alexey Barkhanskiy ◽  
Ashley P. Mattey ◽  
Edward G. Pallister ◽  
Reynard Spiess ◽  
...  
Keyword(s):  
Drug Loading ◽  
Anticancer Therapy ◽  
Galactose Oxidase ◽  
Reaction Conditions ◽  
Modular Assembly ◽  
High Drug ◽  
Drug Conjugates ◽  
Antibody Conjugates ◽  
High Drug Loading ◽  
Antibody Drug

The potential of antibody conjugates with high drug loading in anticancer therapy has recently been highlighted by the approval of Trastuzumab deruxtecan and Sacituzumab govitecan. These biopharmaceutical approaches have spurred interest in bioconjugation strategies with high and defined degrees antibody-to-drug (DAR) ratios, in particular on native antibodies. Here we report a glycoengineering methodology to generate antibody drug conjugates with DAR of up to eight, by combining highly selective enzymatic galactosylation and oxidation with biorthogonal tandem Knoevenagel-Michael addition chemistry. This three step approach offers a selective route to conjugates from native antibodies with high drug loading, and thus illustrates how biocatalysis can be used for the generation of biopharmaceuticals using mild reaction conditions.

Functional Fractionation of Platelets: Aggregation Kinetics and Glycoprotein Labeling of Differing Platelet Populations

1982 ◽  
Vol 48 (02) ◽  
pp. 211-216 ◽  
Author(s):  
V M Haver ◽  
A R L Gear
Keyword(s):  
Galactose Oxidase ◽  
Aggregation Kinetics ◽  
Maximal Rate ◽  
Membrane Glycoproteins ◽  
Whole Cells ◽  
Reversible Aggregation ◽  
Significant Difference ◽  
Small Doses ◽  
Major Loss ◽  
Kinetics Of

SummaryPlatelet heterogeneity has been studied with a technique called functional fractionation which employs gentle centrifugation to yield subpopulations (“reactive” and “less-reactive” platelets) after exposure to small doses of aggregating agent. Aggregation kinetics of the different platelet populations were investigated by quenched-flow aggregometry. The large, “reactive” platelets were more sensitive to ADP (Ka = 1.74 μM) than the smaller “less-reactive” platelets (Ka = 4.08 μM). However, their maximal rate of aggregation (Vmax, % of platelets aggregating per sec) of 23.3 was significantly lower than the “less-reactive” platelets (Vmax = 34.7). The “reactive” platelets had a 2.2 fold higher level of cyclic AMP.Platelet glycoproteins were labeled using the neuraminidase-galactose oxidase – [H3]-NaBH4 technique. When platelets were labeled after reversible aggregation, the “reactive” platelets showed a two-fold decrease in labeling efficiency (versus control platelets). However, examination of whole cells or membrane preparations from reversibly aggregated platelets revealed no significant difference in Coomassie or PAS (Schiff) staining.These results suggest that the large, “reactive” platelets are more sensitive to ADP but are not hyperaggregable in a kinetic sense. Reversible aggregation may cause a re-orientation of membrane glycoproteins that is apparently not characterized by a major loss of glycoprotein material.

Investigating the influence of aromatic moieties on the formulation of hydrophobic natural products and drugs in poly(2-oxazoline) based amphiphiles

2018 ◽  
Author(s):  
Robert Luxenhofer ◽  
Michael M Lübtow ◽  
Lukas Hahn ◽  
Thomas Lorson ◽  
Rainer Schobert
Keyword(s):  
Natural Products ◽  
Small Molecules ◽  
Water Solubility ◽  
Drug Loading ◽  
High Drug ◽  
Long Term Stability ◽  
Aromatic Content ◽  
Important Interaction ◽  
Amorphous Structures ◽  
High Drug Loading

Many natural compounds with interesting biomedical properties share one physicochemical property, namely a low water solubility. Polymer micelles are, among others, a popular means to solubilize hydrophobic compounds. The specific molecular interactions between the polymers and the hydrophobic drugs are diverse and recently it has been discussed that macromolecular engineering can be used to optimize drug loaded micelles. Specifically, π-π stacking between small molecules and polymers has been discussed as an important interaction that can be employed to increase drug loading and formulation stability. Here, we test this hypothesis using four different polymer amphiphiles with varying aromatic content and various natural products that also contain different relative amounts of aromatic moieties. While in the case of paclitaxel, having the lowest relative content of aromatic moieties, the drug loading decreases with increasing relative aromatic amount in the polymer, the drug loading of curcumin, having a much higher relative aromatic content, is increased. Interestingly, the loading using schizandrin A, a dibenzo[a,c]cyclooctadiene lignan with intermediate relative aromatic content is not influenced significantly by the aromatic content of the polymers employed. The very high drug loading, long term stability, the ability to form stable highly loaded binary coformulations in different drug combinations, small sized formulations and amorphous structures in all cases, corroborate earlier reports that poly(2-oxazoline) based micelles exhibit an extraordinarily high drug loading and are promising candidates for further biomedical applications. The presented results underline that the interaction between the polymers and the incorporated small molecules are complex and must be investigated in every specific case.<br>

scholarly journals Influence of protein (human galectin-3) design on aspects of lectin activity

2020 ◽  
Vol 154 (2) ◽  
pp. 135-153 ◽  
Author(s):  
Gabriel García Caballero ◽  
Donella Beckwith ◽  
Nadezhda V. Shilova ◽  
Adele Gabba ◽  
Tanja J. Kutzner ◽  
...  
Keyword(s):  
Protein Design ◽  
Full Range ◽  
Biological Information ◽  
The Novel ◽  
Modular Assembly ◽  
Binding Partners ◽  
Galectin 3 ◽  
Potential Applications ◽  
Similar Affinity

Abstract The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure–activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms.

Design of dual-responsive nanocarries with high drug loading capacity based on hollow mesoporous organosilica nanoparticles

2019 ◽  
Vol 233 ◽  
pp. 230-235 ◽  
Author(s):  
Li-li Lu ◽  
Wen-ya Xiong ◽  
Jun-bin Ma ◽  
Tian-fang Gao ◽  
Si-yuan Peng ◽  
...  
Keyword(s):  
Drug Loading ◽  
Loading Capacity ◽  
High Drug ◽  
Dual Responsive ◽  
Mesoporous Organosilica ◽  
High Drug Loading
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