Targeting Adenosine with Adenosine Deaminase 2 to Inhibit Growth of Solid Tumors.

Extracellular adenosine in tumors can suppress immune responses and promote tumor growth. Adenosine deaminase 2 (ADA2) converts adenosine into inosine. The role of ADA2 in cancer and whether it can target adenosine for cancer therapy has not been investigated. Here we show that increased ADA2 expression is associated with increased patient survival and enrichment of adaptive immune response pathways in several solid tumor types. Several ADA2 variants were created to improve catalytic efficiency, and PEGylation was used to prolong systemic exposure. In mice, PEGylated ADA2 (PEGADA2) inhibited tumor growth by targeting adenosine in an enzyme activity-dependent manner and thereby modulating immune responses. These findings introduce endogenous ADA2 expression as a prognostic factor and PEGADA2 as a novel immunotherapy for cancer. SIGNIFICANCE: This study identifies ADA2 as a prognostic factor associated with prolonged cancer patient survival and introduces the potential of enzymatic removal of adenosine with engineered ADA2 for cancer immunotherapy.

PH20 is not expressed in murine CNS and oligodendrocyte precursor cells.

OBJECTIVE: Expression of Spam1/PH20 and its modulation of high/low molecular weight hyaluronan substrate have been proposed to play an important role in murine oligodendrocyte precursor cell (OPC) maturation in vitro and in normal and demyelinated central nervous system (CNS). We reexamined this using highly purified PH20. METHODS: Steady-state expression of mRNA in OPCs was evaluated by quantitative polymerase chain reaction; the role of PH20 in bovine testicular hyaluronidase (BTH) inhibition of OPC differentiation was explored by comparing BTH to a purified recombinant human PH20 (rHuPH20). Contaminants in commercial BTH were identified and their impact on OPC differentiation characterized. Spam1/PH20 expression in normal and demyelinated mouse CNS tissue was investigated using deep RNA sequencing and immunohistological methods with two antibodies directed against recombinant murine PH20. RESULTS: BTH, but not rHuPH20, inhibited OPC differentiation in vitro. Basic fibroblast growth factor (bFGF) was identified as a significant contaminant in BTH, and bFGF immunodepletion reversed the inhibitory effects of BTH on OPC differentiation. Spam1 mRNA was undetected in OPCs in vitro and in vivo; PH20 immunolabeling was undetected in normal and demyelinated CNS. INTERPRETATION: We were unable to detect Spam1/PH20 expression in OPCs or in normal or demyelinated CNS using the most sensitive methods currently available. Further, "BTH" effects on OPC differentiation are not due to PH20, but may be attributable to contaminating bFGF. Our data suggest that caution be exercised when using some commercially available hyaluronidases, and reports of Spam1/PH20 morphogenic activity in the CNS may be due to contaminants in reagents.

Mechanism of cataract formation in alphaA-crystallin Y118D mutation.

PURPOSE: The aim of this study was to elucidate the molecular mechanisms that lead to a dominant nuclear cataract in a mouse harboring the Y118D mutation in the alphaA-crystallin gene. METHODS: The physicochemical properties of alpha-crystallin obtained from mouse lenses with the Y118D mutation as well as a recombinant Y118D alphaA-crystallin were studied using gel filtration, two-dimensional (2D) gel electrophoresis, multi-angle light scattering, circular dichroism, fluorescence, and chaperone activities. RESULTS: Both native alpha-crystallin from mutant lens and recombinant alphaA-Y118D displayed higher molecular mass distribution than the wild-type. Circular dichroism spectra indicated changes in the secondary structures of alphaA-Y118D. The alphaA-Y118D protein prevented nonspecific protein aggregation more effectively than wild-type alphaA-crystallin. The gel filtration and 2D gel electrophoresis analysis showed a significant reduction of Y118D mutant protein in comparison with wild-type alphaA protein of heterozygous mutant lenses. Quantitative RT-PCR results confirmed a decrease in alphaA and alphaB transcripts in the homozygous mutant alpha A(Y118D/Y118D) lenses. CONCLUSIONS: The alphaA-Y118D mutant protein itself displays an increased chaperone-like activity. However, the dominant nuclear cataract is associated with a significant decrease in the amount of alphaA-crystallin, leading to a reduction in total chaperone capacity needed for maintaining lens transparency.