606 SAR444245 (THOR-707), an engineered non-alpha IL-2, enhances NK mediated antibody-dependent cellular cytotoxicity

BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-natural amino acid serving as a bioconjugation site for a single PEG. The non-natural amino acid is positioned to enable the PEG bioconjugation to obscure block binding to the IL-2 alpha receptor, while retaining near-native affinity with the intermediate affinity βγ IL-2 receptor. The non-alpha features of SAR444245 minimize activation of immune suppressive regulatory CD4+ T cells, while retaining activity on CD8+ T cells and NK cells expressing the IL-2 βγ receptors. NK cells exert anti-tumor activity through antibody dependent cellular cytotoxicity (ADCC) of IgG antibodies as well as antibody independent mechanisms.MethodsHere, we utilized a panel of human primary PBMC based immunoassays and transcriptomic analysis to evaluate whether SAR444245 may improve ADCC function of IgG1 anti-tumor target antibodies.ResultsWe characterized the ability of SAR444245 to enhance the cytolytic function of NK cells towards the prototypic NK target cell K562 as well as to modulate NK cell ADCC in combination with EGFR or CD20-targeting antibodies. In vitro assays demonstrated that SAR444245 can activate NK cells, promote NK cell proliferation and improve cytotoxicity of NK cells against K562 cells and across a panel of human EGFR and CD20 positive cell lines. In PBMC based ADCC assays with 1ug/ml of antibody, SAR444245 improved ADCC function maximally by 9-fold for an anti-EGFR antibody and at 5-fold for an anti-CD20 antibody. SAR444245 exhibited dose-dependent enhancement of NK cell ADCC function. Notably, this activity was observed in cell lines expressing varying levels of EGFR and CD20. SAR444245 treatment was associated with dose dependent increases in NK cell degranulation and IFN-γ production. Transcriptomic profiling revealed that SAR444245 had broad effects on NK cell biology leading to changes in inhibitory and activating receptors.ConclusionsIn summary, these results indicate that SAR444245 can enhance the cytolytic activity of NK cells and enhance the ADCC effect of tumor-directed antibodies by activating NK cells.

Alteration in H-bond strength affects the stability of codon-anticodon interaction at in-frame UAG stop codon during in vitro translation

We have studied the decoding ability of a non-standard nucleobase modified tRNA for non-natural amino acid mutagenesis. The insertion of 2, 6-diaminopurine (D) base at the 3rd position of a tRNA anticodon enabled us to evaluate the effect of an additional hydrogen bond during translation. The presence of D at the tRNA anticodon led to stabilization of the codon-anticodon interaction due to an additional H-bond between the N2-exocyclic amine of D and the C2 carbonyl group of uracil during protein translation. While decoding UAG codons using stop codon suppression methodology, the enhanced codon-anticodon interaction improved codon readthrough and synthesis of modified protein with a non-natural amino acid at multiple sites. Our findings imply that the number of hydrogen bonds at the tRNA-mRNA duplex interface is an important criterion during mRNA decoding and improves protein translation at multiple UAG stop sites. This work provides valuable inputs towards improved non-natural amino acid mutagenesis for creating functional proteins.

Alteration in H-bond strength affects the stability of codon-anticodon interaction at in-frame UAG stop codon during in vitro translation

We have studied the decoding ability of a non-standard nucleobase modified tRNA for non-natural amino acid mutagenesis. The insertion of 2, 6-diaminopurine (D) base at the 3rd position of a tRNA anticodon enabled us to evaluate the effect of an additional hydrogen bond during translation. The presence of D at the tRNA anticodon led to stabilization of the codon-anticodon interaction due to an additional H-bond between the N2-exocyclic amine of D and the C2 carbonyl group of uracil during protein translation. While decoding UAG codons using stop codon suppression methodology, the enhanced codon-anticodon interaction improved codon readthrough and synthesis of modified protein with a non-natural amino acid at multiple sites. Our findings imply that the number of hydrogen bonds at the tRNA-mRNA duplex interface is an important criterion during mRNA decoding and improves protein translation at multiple UAG stop sites. This work provides valuable inputs towards improved non-natural amino acid mutagenesis for creating functional proteins.

Development of electronic modules for their integration with impedimetric transducers applied to sarcosine detection

The development of fast, simple, sensitive, and minimally invasive biosensors for detecting diseases, conventionally need specialized, expensive, and highly invasive instrumentation. Furthermore, such biosensors pertinently also, need the development of optoelectronic modules that are capable of implementing specific detection techniques while interacting with the user through a friendly interface. This work highlights the development of a system whose hardware and software contributes to the detection of analytes by impedimetric sensors, especially emphasizing on the detection of sarcosine, a natural amino acid associated with prostate cancer (PCa). Dummy circuits coupled with impedimetric transducers were used to perform precise measurements using a sinusoidal signal of 20 mV in the range from 0.1 Hz to 1 MHz.

Photodegradation of Brilliant Green Dye by a Zinc bioMOF and Crystallographic Visualization of Resulting CO2

We present a novel bio-friendly water-stable Zn-based MOF (1), derived from the natural amino acid L-serine, which was able to efficiently photodegrade water solutions of brilliant green dye in only 120 min. The total degradation was followed by UV-Vis spectroscopy and further confirmed by single-crystal X-ray crystallography, revealing the presence of CO2 within its channels. Reusability studies further demonstrate the structural and performance robustness of 1.

Identification and Characterization of an O-Succinyl-L-Homoserine Sulfhydrylase From Thioalkalivibrio sulfidiphilus

L-methionine is an important natural amino acid with broad application prospects. A novel gene encoding the enzyme with the ability to catalyze O-succinyl-L-homoserine (OSH) to L-methionine was screened and characterized. The recombinant O-succinyl-L-homoserine sulfhydrylase from Thioalkalivibrio sulfidiphilus (tsOSHS) exhibited maximum activity at 35°C and pH 6.5. OSHS displayed an excellent thermostability with a half-life of 21.72 h at 30°C. Furthermore, the activity of OSHS increased 115% after Fe2+ added. L-methionine was obtained with a total yield reaching 42.63 g/L under the concentration of O-succinyl-L-homoserine 400 mM (87.6 g/L). These results indicated that OSHS is a potential candidate for applying in the large-scale bioproduction of L-methionine.

Beneficial Impacts of Incorporating the Non-Natural Amino Acid Azulenyl-Alanine into the Trp-Rich Antimicrobial Peptide buCATHL4B

Antimicrobial peptides (AMPs) present a promising scaffold for the development of potent antimicrobial agents. Substitution of tryptophan by non-natural amino acid Azulenyl-Alanine (AzAla) would allow studying the mechanism of action of AMPs by using unique properties of this amino acid, such as ability to be excited separately from tryptophan in a multi-Trp AMPs and environmental insensitivity. In this work, we investigate the effect of Trp→AzAla substitution in antimicrobial peptide buCATHL4B (contains three Trp side chains). We found that antimicrobial and bactericidal activity of the original peptide was preserved, while cytocompatibility with human cells and proteolytic stability was improved. We envision that AzAla will find applications as a tool for studies of the mechanism of action of AMPs. In addition, incorporation of this non-natural amino acid into AMP sequences could enhance their application properties.