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Therapeutic inhibition of the immunosuppressive IDO1–TDO pathway in tumors is achieved in vivo with an enzyme. Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO)1,2,3,4,5. Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial5,6,7,8,9,10,11,12,13. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME. [ABSTRACT FROM AUTHOR]

The present study focuses on theformation of microcapsules containingcatalytically active l-asparaginase (L-ASNase), a proteindrug 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, theCaCO3template was generated by coprecipitation with theenzyme. After assembly of the polymer shell, the CaCO3corematerial was dissolved under mild conditions by dialysis against 20mM EDTA. Biochemical stability of the encapsulated l-asparaginasewas analyzed by treating the capsules with the proteases trypsin andthrombin, which are known to degrade and inactivate the enzyme duringleukemia treatment, allowing us to test for resistance against proteolysisby physiologically relevant proteases through measurement of residual l-asparaginase activities. In addition, the thermal stability,the stability at the physiological temperature, and the long-termstorage stability of the encapsulated enzyme were investigated. Weshow that encapsulation of l-asparaginase remarkably improvesboth proteolytic resistance and thermal inactivation at 37 °C,which could considerably prolong the enzyme’s in vivo half-lifeduring application in acute lymphoblastic leukemia (ALL). Importantly,the use of low EDTA concentrations for the dissolution of CaCO3by dialysis could be a general approach in cases where theactivity of sensitive biomacromolecules is inhibited, or even irreversiblydamaged, when standard protocols for fabrication of such LbL microcapsulesare used. Encapsulated and free enzyme showed similar efficacies indriving leukemic cells to apoptosis. [ABSTRACT FROM AUTHOR]

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (Tm) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti‐HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher Tm compared to the parental scFv and importantly to retain full antigen binding activity after 2 hr of incubation at 70°C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction. [ABSTRACT FROM AUTHOR]