Signal conversion as tumor micro environment (TME) specifically activated cytokine.

Targeted cytokine delivery has been gaining popularity in cancer immunotherapy since systemic recombinant cytokine treatment has not been successful due to low response rate and systemic toxicities in the clinical studies. In order to address these issues, we propose a new concept that cytokine signal is specifically activated at tumor-micro-environment (TME) by delivering two protein subunits of heterodimeric cytokine fused with a tumor targeting antibody respectively to TME and by bridging the two subunits into active heterodimeric form.Interleukin-12 (IL-12) is one of the major cytokines which can induce immune activation. IL-12 consists of two protein subunits which are p35 and p40. IL-12 signaling is initiated when it forms as the heterodimeric protein and binds to IL-12 receptor complex. We made fusion proteins of both IL-12p35 and IL-12p40 targeting specific tumor associated antigens (TAAs) and demonstrated the formation of bioactive IL12p70 with TME targeting antibody toward both p35 and p40 to form as the active molecule. We describe our concept validation in an in vitro based functional assay.

Effects of APRIL (TNFSF13) polymorphisms and splicing isoforms on the secretion of soluble APRIL.

Functional APRIL (TNFSF13) is a secreted trimer generated by furin protease cleavage. We previously reported the association of APRIL haplotypes formed by two nonsynonymous polymorphisms, Gly67Arg and Asn96Ser, with systemic lupus erythematosus. Here, we tested the hypothesis that polymorphisms and/or alternative splicing may influence the generation of soluble APRIL (sAPRIL). HEK 293T cells were transfected with plasmids containing one of the six combinations of splicing isoforms (α or ß) and haplotypes (susceptible, neutral, or protective). APRIL concentrations were quantitated in the cell lysates and supernatants using an enzyme-linked immunosorbent assay (ELISA). The association between splicing efficiency and polymorphisms was analyzed using quantitative reverse transcription polymerase chain reaction (RT-PCR). The efficiency of cleavage by furin protease was analyzed using western blotting. Although both splicing isoforms were cleaved by furin protease, sAPRIL was not detected in the supernatant of the cells transfected with the ß isoform, regardless of the haplotype. This suggested that, similarly to B-cell activating factor (BAFF), one of the major APRIL splicing isoforms may not be secreted as a functional molecule. Furthermore, the secretion of sAPRIL was decreased in the transfectants expressing the protective haplotype. An association between the polymorphisms and splicing efficiency or furin cleavage efficiency was not detected. In conclusion, these observations suggested that both alternative splicing and polymorphisms may affect the generation of functional sAPRIL.

Association of a functional polymorphism in the 3'-untranslated region of SPI1 with systemic lupus erythematosus.

OBJECTIVE: SPI1, also referred to as PU.1, is an Ets family transcription factor that interacts with IRF2, IRF4, and IRF8. In view of the significance of the type I interferon pathway in systemic lupus erythematosus (SLE), this study was undertaken to investigate a possible association between SPI1 polymorphisms and SLE. METHODS: A case-control association study was performed using 6 tag single-nucleotide polymorphisms (SNPs), as well as a SNP located upstream of SPI1 previously found to be associated with acute myelogenous leukemia, in 400 Japanese patients with SLE and 450 healthy controls. Resequencing of all exons and known regulatory regions was performed to identify functional polymorphisms. Association of genotype and SPI1 expression was examined using the GENEVAR database and reporter assays. RESULTS: A significant association was detected in 2 SNPs in intron 2 (rs10769258 and rs4752829) (P = 0.005 and P = 0.008, respectively, under the dominant model). The association was stronger in patients with nephropathy. Resequencing identified a potentially functional polymorphism in the 3'-untranslated region (3'-UTR), rs1057233, which was in strong linkage disequilibrium with the SNPs in intron 2. The number of risk alleles at rs1057233 was strongly correlated with SPI1 messenger RNA (mRNA) level in the database analysis (P = 0.0002), and was confirmed by a reporter assay. Interestingly, rs1057233 alters a target sequence for microRNA hsa-miR-569 (miR-569). Transfection experiments demonstrated that miR-569 inhibits expression of a reporter construct with the 3'-UTR sequence containing the nonrisk allele but not the risk allele. CONCLUSION: Our findings indicate that a SNP in the 3'-UTR of SPI1 is associated with elevated SPI1 mRNA level and with susceptibility to SLE.