A Partner Evokes Latent Differences between Hox Proteins

Hox transcription factors bind highly related DNA sequences in vitro, yet they regulate different genes and play distinct roles in anterior-posterior patterning in animals. Slattery et al. report that a common cofactor, Exd, accentuates latent sequence specificities of all eight Hox proteins and directs binding to relevant sites across the genome

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Residue-Based Preorganization of BH3-Derived α/β-Peptides: Modulating Affinity, Selectivity and Proteolytic Susceptibility in α‑Helix Mimics

We report progress toward a general strategy for mimicking the recognition properties of specific α-helices within natural proteins through the use of oligomers that are less susceptible than conventional peptides to proteolysis. The oligomers contain both α- and β-amino acid residues, with the density of the β subunits low enough that an α-helix-like conformation can be adopted but high enough to interfere with protease activity. Previous studies with a different protein-recognition system that suggested ring-constrained β residues can be superior to flexible β residues in terms of maximizing α/β-peptide affinity for a targeted protein surface. Here, we use mimicry of the 18-residue Bim BH3 domain to expand the scope of this strategy. Two significant advances have been achieved. First, we have developed and validated a new ring-constrained β residue that bears an acidic side chain, which complements previously known analogues that are either hydrophobic or basic. Second, we have discovered that placing cyclic β residues at sites that make direct contact with partner proteins can lead to substantial discrimination between structurally homologous binding partners, the proteins Bcl-x_L and Mcl-1. Overall, this study helps to establish that α/β-peptides containing ring-preorganized β residues can reliably provide proteolytically resistant ligands for proteins that naturally evolved to recognize α-helical partners

Extending Foldamer Design beyond α-Helix Mimicry: α/β-Peptide Inhibitors of Vascular Endothelial Growth Factor Signaling

Diverse strategies have been explored to mimic the surface displayed by an α-helical segment of a protein, with the goal of creating inhibitors of helix-mediated protein–protein interactions. Many recognition surfaces on proteins, however, are topologically more complex and less regular than a single α-helix. We describe efforts to develop peptidic foldamers that bind to the irregular receptor-recognition surface of vascular endothelial growth factor (VEGF). Our approach begins with a 19-residue α-peptide previously reported by Fairbrother et al. (<i>Biochemistry</i> <b>1998</b>, <i>37</i>, 17754) to bind to this surface on VEGF. Systematic evaluation of α→β replacements throughout this 19-mer sequence enabled us to identify homologues that contain up to ∼30% β residues, retain significant affinity for VEGF, and display substantial resistance to proteolysis. These α/β-peptides can block VEGF-stimulated proliferation of human umbilical vein endothelial cells

Evaluation of Diverse α/β-Backbone Patterns for Functional α-Helix Mimicry: Analogues of the Bim BH3 Domain

Peptidic oligomers that contain both α- and β-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of α-helix-forming conventional peptides (composed exclusively of α-amino acid residues). Here we describe a comprehensive evaluation of diverse α/β-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-x_L and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All α/β-peptide homologues retain the side-chain sequence of the Bim BH3 domain, but each homologue contains periodic α-residue → β³-residue substitutions. Previous work has shown that the ααβαααβ pattern, which aligns the β³-residues in a ’stripe’ along one side of the helix, can support functional α-helix mimicry, and the results reported here strengthen this conclusion. The present study provides the first evaluation of functional mimicry by ααβ and αααβ patterns, which cause the β³-residues to spiral around the helix periphery. We find that the αααβ pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an α/β-peptide bound to Bcl-x_L, affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best αααβ homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 α-peptide