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Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’

Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. May 22, 2012 Joseph Argus, Pardeep Singh, Uland Lau. IL-2. IL = interleukin = cytokine of immune system 15.5 kD, variably glycosylated Necessary for growth and function of T cells

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Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’

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  1. Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’ May 22, 2012 Joseph Argus, Pardeep Singh, Uland Lau

  2. IL-2 • IL = interleukin = cytokine of immune system • 15.5 kD, variably glycosylated • Necessary for growth and function of T cells • Promotes differentiation and proliferation of natural killer cells • Used in clinic to upregulate immune system (chronic viral infection, adjuvant for vaccines, cancer therapy) • Also adverse effects, at least partially due to upregulation of Treg cells

  3. Goal: Create modified IL-2 that stimulates cytotoxic T cells and natural killer cells with less Treg activation (fewer side effects)

  4. IL-2 Receptor • Treg and cytotoxic T both contain low levels of beta and gamma • Only Treg contain high levels of alpha (in resting state) • Locking IL-2 in the active (purple) conformation will bypass the need for alpha and increase the relative proportion of cytotoxic:regulatory T cell activation

  5. Summary: • Developed versions of IL-2 (“superkines”) that bypass the need for the alpha subunit of receptor using directed evolution • Verified nature of mutations using physical biochemistry, crystallography • Verified biological significance using: • in vitro assays (pSTAT5) • in vivo assays (splenic lymphocyte number, tumor volume, and lung metastases)

  6. Directed Evolution

  7. -Five of the six mutations clustered on the B-C loop and within the C helix core.-V85, F80, andV86 substitutions appeared to collapse into a hydrophobic cluster to stabilize the loop by fixing helix C into the core of the molecule. Crystallization of D10 IL-2 superkine

  8. Low-resolution structure of D10 ternary complex -Is this heterodimeric architecture the same when D10 binds as compared with wild type IL-2? Answer-Found to be essentially identical r.m.s.d.=0.43 angstoms

  9. Conformation of unliganded IL-2/D10 and ligand bound CD25 -Unliganded D10 is conformationally similar to the IL-2Ralpha[CD25] as compared to the unliganded IL-2

  10. Molecular Dynamics simulations of IL-2 and D10 -Analysis of anatomically detailed Markov state models showed that D10 was more stable than IL-2 -B/B-C/and C all had lower visible deviations compared to wild type IL-2

  11. Comparison of average IL-2 wt vs.D10

  12. Conclusion from set of experiments The reduced flexibility of helix C in the IL-2 superkine is due to improved core packing with helix B. Structural and molecular dynamics results show that evolved mutations cause a conformational stabilization of the cytokine, reducing the energetic penalties for binding to IL-2Rβ.

  13. Dose response curves using flow cytometry to assay STAT5 phosphorylation -Do IL-2 superkines demonstrate signal potencies? -Do they depend on cell surface expression of CD25? Absence of CD25 Presence of CD25

  14. Probing CD25-independence with a mutation of IL-2 F42A= Phe 42 replaced with Ala. Reduces binding to CD25 by 220-fold for H9 and 120-fold for IL-2.

  15. Dose response curves on T cells from mice with absent CD25. Flow cytometry fluorescence assay Superkines=spread throughout/low density. IL-2=concentrated/ lacks replication/ high density.

  16. Antitumor activities of IL-2 superkine • IL-2 superkine H9, wildtype IL-2, and IL-2-anti-IL-2 mAb effects on CD25low vs CD25high T cells • IL-2-anti-IL-2 mAb • Shown to reduce pulmonary edema and have potent antitumor responses in vivo • Memory-phenotype (MP) CD8+ T cells • Low levels of CD25 • High levels of IL-2Rβγ • Regulatory T (Treg) CD4+ cells • High levels of CD25

  17. Different tumor models • Mice injected subcutaneously with B16F10 melanoma cells, murine colon carcinoma, and Lewis lung carcinoma • Treatments: • PBS-control • High-dose IL-2 • IL-2-anti-IL-2 mAb complexes • H9 IL-2 superkine • PBS-control : tumor reached 1500 mm3 at day 18 • IL-2 treatment: delayed as much as 39% at day 18 • Similar effects between IL-2-anti-IL-2 mAb and H9 IL-2 superkine • Reduced tumor growth by more than 80% • Compared to IL-2, >70% reduction

  18. Conclusions Engineered IL-2 superkine via in vitro directed evolution Eliminated CD25 dependency of IL-2 Increased binding infinity towards IL-2Rβ IL-2 superkine elicited proliferation of T cells irrespective of CD25 expression Improved antitumor responses in vivo (reduced pulmonary edema) Showed activation of cytotoxic CD8+ T cells and NK cells – antitumor immune response Minimal toxicity

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