custom buffy coat lrsc pbmc isolation kit  (Miltenyi Biotec)

Journal: Antibodies

Article Title: Structure-Guided Stapling of Dimeric Conformations and Linker Engineering Enhance Thermostability and Fine-Tune Activity of Bispecific VHH Cytokine Agonists

doi: 10.3390/antib14030074

Figure Lengend Snippet: Engagement of cytokine receptors via dual, bispecific VHHs results in various levels of SCA activity. ( A ) Cartoon representation of IL-18-mediated, IL-18 receptor assembly and intracellular signaling. IL-18 SCA is composed of an anti-IL-18Rα VHH (cyan) and an anti-IL-18Rβ VHH (red). ( B ) Cartoon representation of IL-2-mediated, IL-2 ternary receptor assembly and intracellular signaling. IL-2 SCA is composed of an anti-IL-2Rβ VHH (blue) and an anti-IL-2Rγ VHH (purple). ( C ) In vitro activity of a panel of 168 IL-18 SCAs at 100 nM. The NF-kB reporter activity in a HEK-Blue NF-kB reporter cell assay is plotted against secreted IFNγ of peripheral blood mononuclear cells (PBMCs) from healthy human donors. All values are normalized as a percentage of maximal signal obtained through stimulation with IL-18. ( D ) Three IL-18 SCAs induce PBMCs to secrete variable levels of IFNγ as a function of SCA concentration, shown as a fraction of maximum secretion induced by IL-18. ( E ) STAT5 phosphorylation and proliferation of NK and CD8+ T cells isolated from peripheral blood and treated with 300 nM IL-2 SCAs and 100 pM IL-2. In the multivariate plot, each dot represents an individual VHH dimer with a color gradient referencing the amount of IFNγ secretion as shown on the scale on the right. ( F ) Two IL-2 SCAs (DR638 and DR736) specific for IL-2Rβ and IL-2Rγ receptors induce varying levels of IL-2 partial agonism, as measured via phosphorylation of STAT5 normalized to IL-2 maximum signal. Alternative text: six-panel figure ( A – F ). ( A ) Cartoon representation showing IL-18Rα and IL-18Rβ extracellular domains bound by IL-18 and associated into a complex on the plasma membrane. Cartoons of signaling molecules on the intracellular side lead to NF-KB activation and secretion of IFNγ, which is indicated by an arrow across the plasma membrane. A cartoon of two VHHs targeting IL-18R subunits and connected via a linker is illustrated above the receptors. ( B ) Cartoon representation of IL-2Rβ and IL-2Ry extracellular domains bound by IL-2 and associated into a complex on the plasma membrane. Cartoons of signaling molecules on the intracellular side lead to STAT5 phosphorylation and activation. A carton of two VHHs targeting IL-2 R subunits and connected via a linker is illustrated above the receptors. ( C ) A graph plotting IFNγ secretion against NF-KB signaling, both as a percentage of IL-18 maximum signal, with all 168 SCAs represented by a circle. DR3087, DR3097, and DR3085 are highlighted as strong activators of both. ( D ) A graph plotting IFNγ secretion as a percentage of IL-18 maximum signal against concentration in log scale of 3 SCAs: DR3085, DR3097, and DR3087. All SCAs show sigmoidal dose–response curves with variable midpoints (potency, EC50) and the highest maximum signal for DR3085 (20% of IL-18 maximum signal). ( E ) Two multivariate graphs plot proliferation of NK cells at the top and CD8 cells at the bottom against STAT5 phosphorylation induced by IL-2 and by 120 IL-2 SCAs, which are shown as dots. Each dot is color-coded for concentration of IFNγ in the supernatant. Two arrows point at IL-2 and DR638 in the top right corners of the plot, as they are among the most active molecules on all three parameters, while another arrow points at DR736 in the middle of the plots, as this represents a milder mimic of IL-2. ( F ) A graph plotting STAT5 phosphorylation against concentrations in log scale of two IL-2 SCAs, DR638 and DR736, as well as IL-2. All three proteins show sigmoidal dose–response curves with different midpoints and maximum values. IL-2 displays the lowest midpoint concentration (EC50) and the highest maximum signal, while DR638 and DR736 display midpoints at higher concentrations and very different maximum levels: minimal for DR736 and intermediate for DR638 compared to IL-2.

Article Snippet: PBMC were isolated from human Buffy Coats or Leucocyte Reduction System Chambers (LRSC) obtained from the Stanford Blood Bank using the Custom Sedimentation Kit (Miltenyi, Bergisch-Gladbach, Germany, #130-126-357) and Custom Buffy Coat/LRSC PBMC Isolation kits (Miltenyi, 130-126-448) using protocol Cust5 on an autoMACS Pro Separator (Miltenyi) according to the manufacturer’s instructions.

Techniques: Activity Assay, In Vitro, Concentration Assay, Phospho-proteomics, Isolation, Clinical Proteomics, Membrane, Activation Assay

Journal: Antibodies

Article Title: Structure-Guided Stapling of Dimeric Conformations and Linker Engineering Enhance Thermostability and Fine-Tune Activity of Bispecific VHH Cytokine Agonists

doi: 10.3390/antib14030074

Figure Lengend Snippet: Stapling N- and C-terminal VHHs via different inter-VHH disulfide pairs results in modulation of thermostability and activity. ( A ) Residues mutated to cysteine at the interface between N- and C-terminal VHHs of DR3097 are shown and paired as outlined in purple. ( B ) Thermostability of the seven stapled SCAs measured by Nano DSF, turbidity, and particle radius as a function of temperature. ( C ) Binding of the seven stapled IL-18 SCAs to IL-18Rα and IL-18Rβ receptors measured via SPR. ( D ) Whole blood PBMC secretion of IFNγ driven by the stapled SCAs. ( E ) Summary table of data presented in this Figure. Alternative text: five-panel figure ( A – E ). ( A ) Residues chosen for disulfide stapling across the two VHHs are shown on the crystal structure of DR3097. Purple lines connecting two residues indicate the seven staple designs. ( B ) Graph showing results of thermal unfolding experiment of the stapled DR3097 variants. The graph has three sections showing (1) first derivative of the fluorescence signal at 350/330 nm, (2) turbidity, and (3) cumulant radius over a temperature ramp from 25 to 95 °C. Overall, the results show increased thermostability of disulfide-linked variants. ( C ) This panel shows sensograms obtained from an SPR experiment in which binding of DR3097 disulfide staple variants to IL-18Rα and IL-18Rβ was measured at different concentrations, ranging between 3.1 and 400 nM. Binding to IL-18Rα remained mostly constant, while binding to IL-18Rb was lost in three variants: S1, S6, and S7. ( D ) Graph showing dose–response curve of IFNγ secretion over SCA concentration on a log scale for DR3097 and the 7 stapled variants, highlighting strong variations in biological activity, both in terms of sigmoid midpoints and maximum values. Variants S1, S6, and S7 show the lowest maximum signals, S3 surpassing the parental molecule DR3097, while S2, S4, and S5 show variable maximum values approaching that of DR3097. ( E ) A summary table of data shown in the figure highlights S4 and S5 as stapled variants with increased thermostability and marginally reduced potency to DR3097.

Article Snippet: PBMC were isolated from human Buffy Coats or Leucocyte Reduction System Chambers (LRSC) obtained from the Stanford Blood Bank using the Custom Sedimentation Kit (Miltenyi, Bergisch-Gladbach, Germany, #130-126-357) and Custom Buffy Coat/LRSC PBMC Isolation kits (Miltenyi, 130-126-448) using protocol Cust5 on an autoMACS Pro Separator (Miltenyi) according to the manufacturer’s instructions.

Techniques: Activity Assay, Binding Assay, Fluorescence, Concentration Assay