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  1. Proteins involved in immune checkpoint pathways, such as CTLA4, PD1, and PD-L1, have become important targets for cancer immunotherapy; however, development of small molecule drugs targeting these pathways has proven difficult due to the nature of their protein–protein interfaces. Here, using a hierarchy of computational techniques, we design a cyclic peptide that binds CTLA4 and follow this with experimental verification of binding and biological activity, using bio-layer interferometry, cell culture, and a mouse tumor model. Beginning from a template excised from the X-ray structure of the CTLA4:B7-2 complex, we generate several peptide sequences using flexible docking and modeling steps. These peptides are cyclized head-to-tail to improve structural and proteolytic stability and screened using molecular dynamics simulation and MM-GBSA calculation. The standard binding free energies for shortlisted peptides are then calculated in explicit-solvent simulation using a rigorous multistep technique. The most promising peptide, cyc(EIDTVLTPTGWVAKRYS), yields the standard free energy −6.6 ± 3.5 kcal mol^−1, which corresponds to a dissociation constant of ∼15 μmol L^−1. The binding affinity of this peptide for CTLA4 is measured experimentally (31 ± 4 μmol L^−1) using bio-layer interferometry. Treatment with this peptide inhibited tumor growth in a co-culture of Lewis lung carcinoma (LLC) cells and antigen primed T cells, as well as in mice with an orthotropic Lewis lung carcinoma allograft model. 
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  2. The antitumor effects of a partially purified water extract from Euglena gracilis (EWE) and EWE treated by boiling (bEWE) were evaluated using orthotopic lung cancer syngeneic mouse models with Lewis lung carcinoma (LLC) cells. Daily oral administration of either EWE or bEWE started three weeks prior to the inoculation of LLC cells significantly attenuated tumor growth as compared to the phosphate buffered saline (PBS) control, and the attenuation was further enhanced by bEWE. The intestinal microbiota compositions in both extract-treated groups were more diverse than that in the PBS group. Particularly, a decrease in the ratio of Firmicutes to Bacteroidetes and significant increases in Akkermansia and Muribaculum were observed in two types of EWE-treated groups. Fecal microbiota transplantation (FMT) using bEWE-treated mouse feces attenuated tumor growth to an extent equivalent to bEWE treatment, while tumor growth attenuation by bEWE was abolished by treatment with an antibiotic cocktail. These studies strongly suggest that daily oral administration of partially purified water extracts from Euglena gracilis attenuates lung carcinoma growth via the alteration of the intestinal microbiota. 
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  3. null (Ed.)
    Beta glucans are known to have immunomodulatory effects that mediated by a variety of mechanisms. In this article, we describe experiments and simulations suggesting that beta-1,3 glucans may promote activation of T cells by a previously unknown mechanism. First, we find that treatment of a T lymphoblast cell line with beta-1,3 oligoglucan significantly increases mRNA levels of T cell activation-associated cytokines, especially in the presence of the agonistic anti-CD3 antibody. This immunostimulatory activity was observed in the absence of dectin-1, a known receptor for beta-1,3 glucans. To clarify the molecular mechanism underlying this activity, we performed a series of molecular dynamics simulations and free-energy calculations to explore the interaction of beta-1,3 oligoglucans with potential immune receptors. While the simulations reveal little association between beta-1,3 oligoglucan and the immune receptor CD3, we find that beta-1,3 oligoglucans bind to CD28 near the region identified as the binding site for its natural ligands CD80 and CD86. Using a rigorous absolute binding free-energy technique, we calculate a dissociation constant in the low millimolar range for binding of 8-mer beta-1,3 oligoglucan to this site on CD28. The simulations show this binding to be specific, as no such association is computed for alpha-1,4 oligoglucan. This study suggests that beta-1,3 glucans bind to CD28 and may stimulate T cell activation collaboratively with T cell receptor activation, thereby stimulating immune function. 
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