Expression of the Cytokine IL-7 in E. coli and Pichia pastoris for the Identification of IL-7 Mutants that Modulate T-Cell Signaling

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1 Expression of the Cytokine IL-7 in E. coli and Pichia pastoris for the Identification of IL-7 Mutants that Modulate T-Cell Signaling Purification Expression of USP14 and for the Development purification of Small of USP14 Molecule for Compunds the development for Cancer of Therapy small molecule inhibitors for 12th P4EU meeting Prague, cancer therapy Improving life for cancer patients through transformative drugs

2 Expression of the Cytokine IL-7 in E. coli and Pichia pastoris for the Identification of IL-7 Mutants that Modulate T-Cell Signaling Purification Expression of USP14 and for the development purification of small of USP14 molecule for compunds the development for cancer of therapy small molecule inhibitors for cancer therapy Introduction to the Protein and Antibody Facility at the CRTD/ Biotec ( Dresden, Germany) Type 1 Diabetes related projects with Prof. Ezio Bonifacio Comparison of the purification of human IL-7 cytokine expressed in Escherichia coli and Pichia pastoris Mutagenesis of IL-7 to obtain a competitor for the human IL-7 / common gamma chain interaction

3 The Protein and Antibody Facility at the CRTD Aim: Express and purify proteins that are in the native fold and that are biologically active, and use them for immunizations in order to generate antibodies that recognize the protein antigen in its native, folded state. Protein expression and purification services (and advice upon): Vectors for the expression of proteins with various affinity tags and in various expression hosts Protein expression in E. coli, Pichia pastoris, insect and mammalian cells Protein purification by standard chromatography methods Antibody services (and advice upon): Immunization of rabbits / cultivation of hybridoma cells Antibody affinity purification and labeling

4 Adapted from the late George Eisenbarth Type 1 Diabetes related projects with Prof. Ezio Bonifacio (Preclinical approaches to stem cell therapy/ Diabetes) Autoimmune disease in which a T-cell response leads to beta-cell destruction in the islets of Langerhans Genetic risk factors for T1D were identified, but molecular mechanisms and possible environmental events for triggering disease progression are currently unknown Antibodies against beta-cell autoantigens (e.g. proinsulin/ insulin, Gad65, IA-2, ZnT8) are diagnostic markers prior to disease onset: Autoantibody assay development : Autoantigen were expressed and purified either in fusion with a luciferase for luciferase immunoprecipitation assays, or with a tag suitable for surface attachment An Interleukin-7 mutant as a competitive inhibitor: Il-7 plays a role in the recurrence of autoimmunity against beta-cells after islet transplantation Onset of b-cell destruction Overt Diabetes Asymptomatic phase

5 Adapted from the late George Eisenbarth Type 1 Diabetes related projects with Prof. Ezio Bonifacio (Preclinical approaches to stem cell therapy/ Diabetes) Autoimmune disease in which a T-cell response leads to beta-cell destruction in the islets of Langerhans Genetic risk factors for T1D were identified, but molecular mechanisms and possible environmental events for triggering disease progression are currently unknown Antibodies against beta-cell autoantigens (e.g. proinsulin/ insulin, Gad65, IA-2, ZnT8) are diagnostic markers prior to disease onset: Autoantibody assay development : Autoantigen were expressed and purified either in fusion with a luciferase for luciferase immunoprecipitation assays, or with a tag suitable for surface attachment An Interleukin-7 mutant as a competitive inhibitor: Il-7 plays a role in the recurrence of autoimmunity against beta-cells after islet transplantation Onset of b-cell destruction Overt Diabetes Asymptomatic phase

6 Cation exchange chromatography of Pichia pastoris secreted IL-7 Try to reproduce expression and purification of human IL-7 from Pichia pastoris from a previous publication (Luo et al., Protein Expression and Purification 63, 2009, 1-4). Cation exchange chromatography: Proliferation assay with Il-7 dependent mouse cell line Tib-293: Biologically active IL-7 was expressed and secreted by Pichia pastoris, with expression level higher in full medium compared to minimal medium. After cation exchange chromatography IL-7 purified from medium not pure, according to SDS page. Zaremba-Czogalla et al., Protein Expression and Purification 110 (2015) 65 71, 2015

7 Pichia pastoris -secreted IL-7 is N-linked glycosylated Luo et al., 2009: Our result: Luo et al.; Protein Expression and Purification 63 (2009) 1-4 Zaremba-Czogalla et al., 2015 human IL-7 has 3 potential N-linked glycosylation sites Pichia pastoris secreted IL-7 by can be de-glycosylated by PNGaseF under denaturating conditions, under native conditions de-glycosylation is uncomplete and interferes with the biological activity of the protein.

8 Purification of IL-7 solubilized from E.coli inclusion bodies increases the refolding yield (1) Cation exchange under denaturation conditions Refolding of IL-7wt: Proliferation assay with IL-7 dependent cell line: IL-7 biological activity is confined in the SEC fractions that correspond to monomeric protein Zaremba-Czogalla et al., 2015

9 Purification of IL-7 solubilized from E.coli inclusion bodies increases the refolding yield (2) IL-7 IL-7 C-His Yield E.coli crude lysate solubilized IB CEX crude lysate solubilized IB IMAC Total protein (mg/l)* Purity (%) Not only cation exchange chromatography of untagged IL-7, but also IMAC purification of Histagged IL-7 after solubilization from inclusion bodies increases refolding yields.

10 Effect of human and mouse IL-7 with and without His-tag on the proliferation of the IL-7 dependent mouse cell line Tib-293 human IL-7 with a C-terminal His-tag is slightly less active compared to mouse IL-7 in supporting survival and proliferation of the IL-7 dependent mouse cell line Tib-293. Endotoxin concentrations of His-tagged IL-7 3 to 5 orders of magnitude lower than an endotoxin concentration that had no apparent effect on survival and proliferation of the Tib-293 cell line. Zaremba-Czogalla et al., 2015

11 Mutagenesis of IL-7 to obtain a competitor for the IL-7 / common gamma (cγ) chain interaction Based upon the IL-7 R alpha and IL-7 cocrytal, and of the cγ chain structure ((Laporte et al., 2008; Wang et al., 2005), a model for the interaction of IL-7 with the common gamma chain was build. Residues of IL-7 helix D interact with the cγ chain receptor subunit. Mc Elroy et al., Structure 17, 54 65, 2009

12 Glycine scan mutagenesis of IL-7 D-helix residues supposedly important for the interaction between IL-7 and the c receptor subunit Mc Elroy et al., Structure 17, 54 65, 2009

13 Activity tests of Il-7 C His D-helix mutants by proliferation of the IL-7 dependent mouse cell line Tib-293 0,6 0,5 0,4 0,3 0,2 1ng 5ng 10ng 50ng 100ng 0,1 0 Il7 wt His tag R133G Q136G E137G K139G T140G W142G N143G K144G Several D-helix residues are less efficient than the IL-7 wt in supporting survival and proliferation of the mouse Tib-293 cell line. The W142G as one of the worst refolding IL-7 mutants is, at high concentrations, still able to support Tib-293 survival and proliferation. Manuscript in preparation

14 Re-evaluate the model of the IL-7 / common gamma chain interaction mini helix C B A D helices A-D and mini helix 1 of IL-7 are marked Sergey Samsonov, M. Teresa Pisabarro group, based on Mc Elroy et al., 2009

15 Calculation of energy contribution of IL-7 residues to the IL-7/ IL-7 receptor ternary complex mini helix v v v v mini helix D helix.residues calculated with high binding energy contribution to interaction between IL-7 and the common gamma chain are located in a mini helix (R44, H45, I46), and in the D-helix (R133, T140, M147). Calculation by Sergey Samsonov, M. Teresa Pisabarro group, based on Mc Elroy et al., 2009

16 mau Purification and refolding of 2 nd round Il-7 C-His mutants 1. IL-7 C-His wt 2. R44Q 3. R44E 4. I46A 5. R133Q 6. E137K 7. E137Q 8. N143D 9. M147A soluble aggregates monomers IL7wt R44Q R44E I46A R133Q E137K E137Q N143D M147A

17 Activity tests of 2 nd round IL-7 C His mutants by proliferation of the IL-7 dependent mouse cell line Tib-293 0,6 0,5 0,4 0,3 0,2 1ng 5ng 10ng 50ng 100ng 0,1 0 The IL-7 mutants D21G, K139E, N143R, N143D are deficient in supporting survival and proliferation of the IL-7 dependent cell line Tib-293 (measured in triplicate).

18 Summary of IL-7 mutants activity and binding to the IL-7 R alpha receptor subunit proliferation of IL-7 dependent cell line pstat5 K D IL-7 R alpha (M) IL-7 C-his wt x 10-7 D21G - + not binding R44E x 10-7 I46A not measured E137Q binds E137R N143D x 10-7 K139E x 10-7 T140G not measured W142G x 10-7 N143D x 10-7 N143E x 10-7 N143R x 10-7 K144E not measured M147A not measured

19 Does an IL-7 mutant that is deficient in signaling through the common gamma chain reduce IL-7 wild type signaling? 0,6 +0ng/ml N143D 0,5 +1ng/ml N143D 0,4 +10ng/ml N143D 0,3 +50ng/ml N143D +100ng/ml N143D 0,2 0,1 0 0ng/ml IL-7 C-His 1ng/ml IL-7 C-His 10ng/ml IL-7 C-His Although the IL-7 mutants D21G, K139E, N143R, N143D are deficient in supporting survival and proliferation of the IL-7 dependent cell line Tib-293, they do not block signaling by IL-7 wt.

20 Conclusions of IL-7 expression and purification, and mutagenesis to obtain a competitive inhibitor for IL-7 signaling IL-7 secreted by Pichia pastoris is hypergycosylated, enzymatic deglycosylation interferes with biological activity testing. Purification (denaturating CEX or IMAC) of E. coli expressed Il-7 prior to refolding increases refolding yield, and reduces endotoxin concentration. All tested IL-7 mutants could be expressed, and refolded from E. coli inclusion bodies, although the yield of monomeric protein was in some cases reduced compared to the IL-7 C-His wt control. IL-7 mutants at the positions K139 (K139E) and N143 (N143D, N143R, N143K) do not induce IL-7 mediated signal transduction, but do bind to the IL-7 R alpha subunit. The IL-7 mutant D21G does not induce IL-7 mediated signal transduction, but does not bind to the IL-7 R alpha subunit. The IL-7 mutants D21G, K139E and N143D do not compete with IL-7 activity in cell culture, possibly because of a buffering effect as was described for the soluble IL-7 R alpha H6 form (Lundstroem et al., 2013).

21 Medivir has a Research and Development focus on cancers of high unmet medical need, and is located in Huddinge (Stockholm area) Medivir has expertise in the design and synthesis of small molecule protease and polymerase inhibitors The Protein Sciences team provides proteins for structure-activity relationship based drug discovery Purification Expression of USP14 and for the Development purification of Small of USP14 Molecule for Compunds the development for Cancer Therapy of small molecule inhibitors for 12th P4EU meeting Prague, cancer therapy ralf.paul@medivir.com Improving life for cancer patients through transformative drugs Expression in prokaryotic and eukaryotic hosts, purification of monodisperse proteins for NMR and crystallography Binding and activity assays by e.g. Biacore, ITC, and microscale thermophoresis

22 Thank you! Ezio Bonifacio Christian Stumpp (CRTD) Denise Kühn (CRTD) Denise Walther (CRTD) Jörg Heinrich (B-Cube Dresden) Magdalena Zaremba-Czogalla (CRTD) M. Teresa Pisabarro (Biotec Dresden) Maria Schreiber (CRTD) Sabine Knappe Sergey Samsonov (Biotec Dresden) Dean Derbyshire Esmeralda Woestenenk Ewa Ordzywol Gun Sternberg Ian Henderson Jimmy LIndberg Kerstin Böhm Mark Albertella Mikaela Rapp Richard Bethell Sofia Unnerstal Tatiana Agback and for your attention!

23 A model for the interaction of IL-7 with the common gamma chain receptor subunit as a starting point for a competitive inhibitor Mc Elroy et al., Structure 17, 54 65, 2009

24 Effect of endotoxin on the proliferation of the IL-7 dependent cell line Tib-293 Endotoxin (U/µg IL-7) h IL-7 wt h IL-7wt CEX) m N-His IL h IL-7 C-His Endotoxin concentrations in the His-tagged IL-7 normalized to the amount tested in the activity assays are 3 to 5 orders of magnitude lower than an endotoxin concentration that had no apparent effect on survival and proliferation of the Tib-293 cell line.

25 2 nd round of mutagenesis based upon the energy contribution calculation, and on the D-helix glycine screen A helix M DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAA G G G QDA E aa mini helix B helix B helix C helix RKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQ D helix KKLNDLCFLKRLLQEIKTCWNKILMGTKEH G GGGGG GGG E Q E DE A Q K E K R aa GENLYFQGGHHHHHH

26 pstat5 (MFI) Activity tests of 2 nd round Il-7 C His mutants on human CD4+ pstat5 phosphorylation Manuscript in preparation