Engineering topics

Liu, Cassie

Publications and source records attributed to Liu, Cassie.

3 records

DOE OSTI2022

An anti-PD-1–GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR + PD-1 + T cells. The anti-PD-1–GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1–GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Enhanced 2'-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate

2'-Fucosyllactose (2'-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2'-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2'-FL, but accu- mulation of by-products due to overflow metabolism of glucose hampered efficient production of 2' -FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2'-FL by engi- neered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to pro- duce 2' -FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2'-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2'-FL production. The resulting engineered yeast produced 25.5 g/L of 2'-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2'-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2'-FL was secreted when a complex medium was used, 82% of the produced 2'-FL remained inside the cells when a minimal medium was used. In conclusion, as yeast extract is already used as food and animal feed ingredients, 2'-FL enriched yeast extract can be produced cost-effectively using the 2'-FL-accumu- lating yeast cells.

2′ -fucosyllactose↗

DOE OSTI2020

Data from Enhanced 2′-Fucosyllactose Production by Engineered Saccharomyces cerevisiae using Xylose as a Co-Substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli , Saccharomyces cerevisiae , Corynebacterium glutamicum , and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from E. coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.

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