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 T reg cells

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

4. IL-2 Receptor T reg and cytotoxic T both contain low levels of beta and gamma Only T reg 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

9. -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

10. 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

11. 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

12. 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

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

14. 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.

15. Dose response curves using flow cytometry to assay STAT5 phosphorylation Absence of CD25Presence of CD25 -Do IL-2 superkines demonstrate signal potencies? -Do they depend on cell surface expression of CD25?

16. 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.

17. 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.

18. Antitumor activities of IL-2 superkine IL-2 superkine H9, wildtype IL-2, and IL-2-anti-IL-2 mAb effects on CD25 low vs CD25 high 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 (T reg ) CD4 + cells –High levels of CD25

19. 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 mm 3 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

20. 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