MUTATED INTERLEUKIN-34 (IL-34) POLYPEPTIDES AND USES THEREOF IN THERAPY

Abstract

Interleukin-34 is a cytokine that is involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. The involvement of IL-34 has been shown in areas as diverse as neuronal protection, autoimmune diseases, infection, cancer, and transplantation. Recent work has also demonstrated a new and possible therapeutic role for IL-34 as a Foxp3.sup.+ Treg-secreted cytokine mediator of transplant tolerance. New mutated IL-34 polypeptides have been generated, which can be used as agonists or antagonists.

Claims

1-17. (canceled)

18. A mutated interleukin-34 (IL-34) polypeptide comprising an amino acid sequence selected from the group consisting of: a) the amino acid sequence ranging from the asparagine residue at position 21 of SEQ ID NO: 1 to the proline residue at position 242 of SEQ ID NO: 1, and comprising at least one mutation selected from the group consisting of: a substitution of the serine residue at position 100 by a phenylalanine residue (S100F), a substitution of the threonine residue at position 36 by a tyrosine residue (T36Y), a substitution of the glutamine residue at position 131 by a phenylalanine residue (Q131F), a substitution of the threonine residue at position 36 by a tryptophan residue (T36W), a substitution of the threonine residue at position 36 by a phenylalanine residue (T36F), and a substitution of the histidine residue at position 56 and of the glycine residue at position 112 by a cysteine residue (H56C and G112C), b) an amino acid sequence having at least 80% of identity with a sequence defined in a), provided that said mutated IL-34 polypeptide is an agonist of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138, and c) a fragment of an amino acid sequence defined in a) or b), provided that said mutated IL-34 polypeptide is an agonist of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

19. The mutated IL-34 polypeptide according to claim 18, said polypeptide comprising an amino acid sequence selected from the group consisting of: d) the amino acid sequence ranging from the asparagine residue at position 21 of SEQ ID NO: 1 to the proline residue at position 242 of SEQ ID NO: 1, wherein the serine residue at position 100 is substituted by a phenylalanine residue (S100F), and further comprising at least one mutation selected from the group consisting of: a substitution of the threonine residue at position 36 by a tyrosine residue (T36Y), a substitution of the glutamine residue at position 131 by a phenylalanine residue (Q131F), a substitution of the threonine residue at position 36 by a tryptophan residue (T36W), a substitution of the threonine residue at position 36 by a phenylalanine residue (T36F), and a substitution of the histidine residue at position 56 and of the glycine residue at position 112 by a cysteine residue (H56C and G112C), e) an amino acid sequence having at least 80% of identity with a sequence defined in a), provided that said mutated IL-34 polypeptide is an agonist of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138, and f) a fragment of an amino acid sequence defined in a) or b), provided that said mutated IL-34 polypeptide is an agonist of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

20. A fusion protein consisting of the mutated IL-34 polypeptide according to claim 18 fused to a heterologous polypeptide.

21. The fusion protein according to claim 20, wherein the heterologous polypeptide is an Fc region.

22. An isolated, synthetic or recombinant nucleic acid encoding for the mutated IL-34 polypeptide according to claim 18 or encoding for a fusion protein consisting of said mutated IL-34 polypeptide fused to a heterologous polypeptide.

23. A vector comprising the nucleic acid according to claim 22.

24. A host cell comprising the nucleic acid according to claim 22 and/or a vector comprising said nucleic acid.

25. A pharmaceutical composition comprising the mutated IL-34 polypeptide according to claim 18, a fusion protein consisting of said mutated IL-34 polypeptide fused to a heterologous polypeptide, a nucleic acid encoding for said mutated IL-34 polypeptide or for said fusion protein, or a vector comprising said nucleic acid, with at least one pharmaceutically acceptable excipient, and optionally at least one sustained-release matrix.

26. A mutated IL-34 polypeptide comprising an amino acid sequence selected from the group consisting of: g) the amino acid sequence ranging from the asparagine residue at position 21 of SEQ ID NO: 1 to the proline residue at position 242 of SEQ ID NO: 1, and comprising at least one mutation selected from the group consisting of: a substitution of the serine residue at position 100 by an aspartic acid residue (S100D), and a substitution of the glutamine residue at position 131 by an arginine residue (Q131R), h) an amino acid sequence having at least 80% of identity with a sequence defined in a), provided that said mutated IL-34 polypeptide is an antagonist of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138, and i) a fragment of an amino acid sequence defined in a) or b), provided that said mutated IL-34 polypeptide is an antagonist of at least one receptor selected from the group consisting of CSF-1R, PTP-ζ and CD138.

27. A fusion protein consisting of the mutated IL-34 polypeptide according to claim 26 fused to a heterologous polypeptide.

28. The fusion protein according to claim 27, wherein the heterologous polypeptide is an Fc region.

29. An isolated, synthetic or recombinant nucleic acid encoding for the mutated IL-34 polypeptide according to claim 26 or encoding for a fusion protein consisting of said mutated IL-34 polypeptide fused to a heterologous polypeptide.

30. A vector comprising the nucleic acid according to claim 29.

31. A host cell comprising the nucleic acid according to claim 29 and/or a vector comprising said nucleic acid.

32. A pharmaceutical composition comprising the mutated IL-34 polypeptide according to claim 26, a fusion protein consisting of said mutated IL-34 polypeptide fused to a heterologous polypeptide, a nucleic acid encoding for said mutated IL-34 polypeptide or for said fusion protein, or a vector comprising said nucleic acid, with at least one pharmaceutically acceptable excipient, and optionally at least one sustained-release matrix.

33. A method of inducing immune tolerance in a subject in need thereof, comprising administering to said subject the mutated IL-34 polypeptide according to claim 18, a fusion protein consisting of said mutated IL-34 polypeptide fused to a heterologous polypeptide, a nucleic acid encoding for said mutated IL-34 polypeptide or for said fusion protein, or a vector comprising said nucleic acid.

34. A method of preventing or reducing transplant rejection in a subject in need thereof, comprising administering to said subject the mutated IL-34 polypeptide according to claim 18, a fusion protein consisting of said mutated IL-34 polypeptide fused to a heterologous polypeptide, a nucleic acid encoding for said mutated IL-34 polypeptide or for said fusion protein, or a vector comprising said nucleic acid.

35. A method of preventing or treating neurodegenerative diseases, autoimmune diseases, unwanted immune response against therapeutic proteins or allergies in a subject in need thereof, comprising administering to said subject the mutated IL-34 polypeptide according to claim 18, a fusion protein consisting of said mutated IL-34 polypeptide fused to a heterologous polypeptide, a nucleic acid encoding for said mutated IL-34 polypeptide or for said fusion protein, or a vector comprising said nucleic acid.

36. An in vitro/ex vivo method of obtaining a population of immunosuppressive macrophages, comprising culturing a population of monocytes in a medium comprising the mutated IL-34 polypeptide according to claim 18.

37. A method of treating cancer or a bone disease involving abnormal proliferation in a subject in need thereof, comprising administering to said subject the mutated IL-34 polypeptide according to claim 26, a fusion protein consisting of said mutated IL-34 polypeptide fused to a heterologous polypeptide, a nucleic acid encoding for said mutated IL-34 polypeptide or for said fusion protein, or a vector comprising said nucleic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0417] FIG. 1: The free energy of mutation. Predicted effects of the single mutation of residues H56, G112, P59, L109, 160 and V108 to cysteine at pH 7.5.

[0418] FIG. 2: The free energy of mutation. Predicted effect of the mutation of residue P59 in all amino acids at pH 7.5.

[0419] FIG. 3: The free energy of mutation. Predicted effect of the mutation of several residues at the IL-34/CSF-1R interface in all amino acids at pH 7.5 (FIG. 3A, T36; FIG. 3B, FIG. 3F40; FIG. 3C, K44; FIG. 3D, S100; FIG. 3E, E103; FIG. 3F, T124; FIG. 3G, L125; FIG. 3H, L127; FIG. 3I, N128; FIG. 3J, Q131; FIG. 3K, S147; FIG. 3L, N150; FIG. 3M, L186 and FIG. 3N, N187).

[0420] FIG. 4: Interaction and kinetics of IL-34 muteins with CF1R. SPR experiments were performed on a Biacore T200 (GE Healthcare) for different IL-34 mutants (FIG. 4A, wt IL-34; FIG. 4B, S100F; FIG. 4C, T36Y; FIG. 4D, Q131F; FIG. 4E, T36F; FIG. 4F, T36W, FIG. 4G, Q131R; FIG. 4H, S100D; FIG. 4I, P59K; FIG. 4J, N150; FIG. 4K, G112C/H56C and FIG. 4L, T124F).

[0421] FIG. 5: Binding parameters of the different IL-34 mutant proteins. The affinity (KD), kinetics parameters (ka and kd) and the resonance maximum (Rmax) of IL-34 over CSF-1R were determined by using series of proteins dilutions in a “Single Cycle Kinetics” (SCK) model.

[0422] FIG. 6: Different effects of the IL-34 mutants on the viability of cultured monocytes after sorting from human total PBMCs. FIG. 6A, Percentage of live cells after 3 days of culture with the wild-type IL-34 (IL-34 WT), with each IL-34 mutant or medium control.

[0423] FIG. 6B, Absolute number of live cells represented as a percentage of live cells related to the percentage of live cells obtained in the condition with WT IL-34 (set as 100% and represented by the dotted line) after 3 days of culture with the WT IL-34, with each IL-34 mutant or medium control. FIG. 6C, Percentage of CD14.sup.+ cells in the monocyte population in a dose-response curve of IL-34 WT, IL-34 mutants or medium control (no cytokine as a negative control), from 1.5 to 200 ng/ml.

[0424] FIG. 7: Differential phosphorylation of Akt and ERK1/2 in response to WT IL-34 or IL-34 mutants in CD14.sup.+ monocytes sorted from human total PBMCs. FIG. 7A-B, Flow cytometry analysis of the phosphorylation of Akt (FIG. 7A) and ERK1/2 (FIG. 7B) at 1, 3 and 5 minutes following contact with IL-34 WT, S100F, T36W, T36Y, T36F, Q131F, Q131R, N150E or IL-34-Fc IL-34 mutants (no cytokine as a negative control). Results are shown as the mean fluorescence intensity of pAkt-AF647 and pERK1/2-AF647 compared to T0.

[0425] FIG. 8: Prediction of the free energy of mutation (ΔΔGmut) of some residues of IL-34 located in the interface with CSF-1R. The ΔΔGmut at pH 7.4 was calculated for the substitutions of residues Thr36 (T36) of IL-34 by Tyr (Y) and the substitutions of residues Ser100 (S100) and Gln131 (Q131) of IL-34 by Phe (F). The input data was the atomic coordinates of IL-34/CSF-1R complex (pdb code 4DKD). The effects of double (T36Y/S100F, T36Y/Q131F and S100F/Q131F) or triple (T36Y/S100F/Q131F) mutations have also been predicted.

[0426] FIG. 9: Interaction, kinetics and binding parameter of the WT IL-34 and mutant S100F with CD138. FIG. 9A-B, SPR experiments were performed on a Biacore T200 (GE Healthcare) for the WT IL-34 (FIG. 9A) and mutant S100F (FIG. 9B). FIG. 9C, The affinity parameter (KD) of the WT IL-34 and mutant S100F over CD138 were determined by using series of proteins dilutions in a “Single Cycle Kinetics” (SCK) model.

[0427] FIG. 10: Interaction, kinetics and binding parameter of the WT IL-34 and mutant 5100F with PTP-ζ. FIG. 10A-B, SPR experiments were performed on a Biacore T200 (GE Healthcare) for the WT IL-34 (FIG. 10A) and mutant S100F (FIG. 10B). FIG. 10C, The affinity parameter (KD) of the WT IL-34 and mutant S100F over CD138 were determined by using series of proteins dilutions in a “Single Cycle Kinetics” (SCK) model.

EXAMPLES

Example 1

Method

[0428] The prediction of the effect of several IL-34's residues mutations to cysteine on the stability of a dimeric form of IL-34 was performed by calculating the mutation energy (difference between the binding free energies of the mutant and the wild type) using the method developed by Spassov and Yan (Spassov and Yan, Proteins 2013; 81:704-714) accessible in the protocol “Calculate Mutation Energy (Binding)” implemented under Discovery Studio (DS) (Dassault Systemes BIOVIA software, San Diego, Calif.).

[0429] Briefly, the X-ray crystal structure of IL-34 dimeric form (Protein Data Bank code 4DKC) was used. The structure was first prepared by adding hydrogen atoms, removing the water molecules and inserting the missing atoms or loop regions using the Prepare Protein tool and CHARMm Polar H forcefield within DS2017. The mutation energy was then calculated for residues at the interface of the IL-34 dimer and at the IL-34/CSF-1R interface. The effect of the mutation was defined as stabilizing if mutation energy was less than −0.5 kcal/mol, as neutral if mutation energy is between −0.5 to 0.5 kcal/mol and as destabilizing if mutation energy is greater than 0.5 kcal/mol.

Results

Stabilization of the Dimer of IL-34

[0430] The mutation energy was calculated for the following mutations: H56C, G112C, P59C, L109C, I60C and V108C.

[0431] Mutations H56C, G112C and V108C are predicted to be neutral, whereas mutations P59C, L109C and I60C are predicted to be destabilizing (FIG. 1). The double mutation H56C/G112C is not expected to have a detrimental effect on the stability of the IL-34 dimer but could lead to the formation of disulfide bridges stabilizing the dimer (FIG. 1).

Destabilization of the Dimer of IL-34

[0432] The mutation energy was calculated for P59. Mutations P59K and P59R should prevent the formation of the IL-34 dimer (FIG. 2).

Stabilization of IL-34/CSF-1R Interface

[0433] The following mutations should potentially stabilize the complex IL-34/CSF-1R (FIG. 3): [0434] T36Y, T36F or T36W [0435] S100D or S100F [0436] T124F or T124W [0437] N128Y or N128F [0438] Q131R, Q131F [0439] S147E [0440] N150E [0441] L186R

Example 2

[0442] PBMCs from healthy volunteer (HV) blood are isolated by Ficoll gradient (CMSMSL01-01; Eurobio), and T, B and NK cells are depleted thanks to anti-CD3 (clone OKT3), anti-CD19 (clone HIB19; BD Biosciences) and anti-CD56 (clone MY31; BD Biosciences) antibodies using magnetic beads. Monocytes are then sorted according to forward scatter (FSC), side scatter (SSC) morphologic parameters and positive staining of CD14 (clone M5E2; BD Biosciences). Fresh sorted monocytes are washed and seeded at 1×10.sup.6/ml in complete medium (RPMI 1640, 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% FCS supplemented with an amount of the polypeptides (P1), (P2) and (P3) of the present invention. On day 2, cells are stimulated or not with 100 ng/mL of LPS (L4391; Sigma-Aldrich) for 24h. On day 3, cells are harvested and stained with anti-CD14 (clone M5E2; BD Biosciences), anti-CD16 (clone 3G8; BD Biosciences), anti-CD163 (clone GHI/61; BD Biosciences), anti-CD206 (clone 19.2; BD Biosciences), anti-CD209a (clone DCN46; BD Biosciences), anti-CD169 (clone 7-239; BD Biosciences), anti-CD80 (clone L307.4; BD Biosciences), anti-CD86 (clone 2331; BD Biosciences), anti-CD40 (clone 5C3; BD Biosciences) and anti-HLA-DR (clone G46-6; BD Biosciences). Fluorescence is measured with a FACSCanto II flow cytometer (BD Biosciences) and FlowJo software is used to analyze data. Cells are first gated by their morphology; dead cells are excluded by selecting DAPI-viable cells to analyze the expression of the different markers among the CD14.sup.+CD16.sup.− and CD14.sup.+CD16.sup.+ monocytes.

Example 3

[0443] For cell signaling experiments, cell lines are serum starved (0.1% FBS) for 48 hours and treated with the polypeptides (P1), (P2) and (P3) of the present invention (10 ng/ml) for 20 minutes at which point the samples are harvested. Samples are then analyzed by western blot using Tris/Glycine buffer and transferred onto a hybond-P membrane (Amersham, GE, Fairfield, Conn., USA). All protein samples are quantified by using a BCA assay to ensure similar protein quantities in all lanes of the western gel (Thermo Scientific Inc.). Antibodies used in western blot experiments are CSF-1R (sc692, 1:1000, Santa Cruz, Santa Cruz, Calif., USA), phospho-tyrosine (sc-508, 1:1000, Santa Cruz), phospho-ERK (sc-7383, 1:1000, Santa Cruz), total ERK (sc-94, 1:1000, Santa Cruz), and β-actin (A2228, 1:10,000, Sigma-Aldrich). All antibodies are incubated with the blot overnight at 4° C. in 5% BSA TTBS. The secondary antibodies mouse IgG-HPR (sc-2061, 1:10,000, Santa Cruz,) or rabbit IgG-HPR (sc-2030, 1:10,000, Santa Cruz,) are incubated for 1 hour at room temperature in 5% milk TTBS. The signal is detected using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific Inc., Waltham, Mass., USA).

Example 4

Material and Methods

Reagents and Material

[0444] Series S CMS sensor chips and HBS-P buffer (10 mM HEPES buffer with 2.7 mM KCl, 150 mM NaCl, and 0.05% surfactant P20, pH 7.4), Amine Coupling Kit [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); 1.0M ethanolamine (pH 8.5)], immobilization buffer (sodium acetate pH 5.0), regeneration solutions (NaOH 10 mM, for analysis) were purchased from GE Healthcare Life Science (Uppsala, Sweden).

Surface Plasmon Resonance (SPR) Analysis

[0445] SPR experiments were performed on a Biacore T200 (GE Healthcare) at 25° C. HBS-P buffer was filtered through a 0.45 μm membrane filter and degassed prior to use. First, recombinant Human CSF-1 receptor from Sino Biological (CSF-1R, ref. K10161-H08H) was immobilized on the chip surface by amine coupling. Briefly, recombinant CSF-1 receptor was diluted to 20 μg/mL in 10 mM sodium acetate solution at pH 5.0. The diluted CSF-1R was soon covalently immobilized to a flow cell of CMS sensor chip via primary amine group, using standard Amine Coupling Kit. IL-34 muteins were then analyzed in a “Single Cycle kinetics” (SCK) models over the CSF-1R immobilized chip.

Interaction and kinetics of IL-34 muteins with CSF-1R

[0446] The affinity (KD), kinetics parameters (ka and kd) and the maximum of resonance (Rmax) of IL-34 over CSF-1R were determined by using series of proteins dilutions in a “Single Cycle Kinetics” (SCK) model. IL-34 muteins as the analytes were diluted in HBS-P buffer with concentrations ranging from 25 nM to 400 nM, which flowed over the immobilized CSF-1R and the obtained response units (RUs) were recorded. The flow rate was at 30 μL/min with 120s for binding and 600s for dissociation. Then, the sensor chip surface was regenerated with 10 mM NaOH for 30s. The dissociation equilibrium constant, KD, kinetics parameters, kd and ka and Rmax were determined by direct curve fitting of the sensorgrams to a Langmuir 1:1 model of interaction.

Results

[0447] The biacore analysis shows that several mutants of IL-34 have a higher affinity to CSF-1R than the WT IL-34, in particular mutants S100F, T36Y, Q131F, T36F, T36W, Q131R and S100D, suggesting that these mutants will be more efficient than the WT IL-34 (FIG. 4 A-H and FIG. 5). In contrast, the P59K, N150E, G112/H56C and T124F mutants of IL-34 have a decreased or similar affinity to CSF-1R as compared to the WT IL-34 (FIG. 4 I-L and FIG. 5).

Example 5

Material and Methods

[0448] Buffy coat was obtained from the Etablissement Francais du Sang (EFS, Nantes, France) from anonymous healthy individuals. PBMCs were obtained by Ficoll gradient, then monocytes were isolated by magnetic-bead separation (Classical Monocyte Isolation Kit, Miltenyi Biotec). CD14.sup.+ monocytes were cultured in flat-bottom 96-well plates at 10.sup.6 cells/ml in complete medium (RPMI 1640, 10% FBS, 1% penicillin-streptomycin, 1% glutamine, 1% AANE, 1% Hepes, 1% sodium pyruvate), with 100 ng/ml final concentration of WT IL-34 or mutants: 5100F, 5100D, T36W, T36Y, T36F, Q131F, Q131R, N150E, C112/C56, P59K, T124F and IL-34 Fc (FIG. 6A and FIG. 6B) and ranging from 1.5 to 200 ng/mL (FIG. 6C). Medium with no added cytokines (No cytokine) was used as negative control. At day 3, cells were harvested by flushing in PBS 2% FBS 2 mM EDTA, and further used for viability staining and phenotypic analysis by flow cytometry. Absolute number of cells were analysed using counting beads (123count eBeads™ Counting Beads, ThermoFisher Scientific).

Results

[0449] Mutants T36F, S100F, Q131F, T36W, T36Y, N150E and IL-34-Fc are as efficient as WT IL34 in inducing survival of monocytes/macrophages (FIG. 6A). S100F, T36Y, Q131F and IL-34-Fc were particularly interesting since they show a stronger capacity to maintain survival of the cells (FIG. 6B).

[0450] Interestingly, Q131R and 5100D that have a better binding affinity to CSF-1R than WT IL34 inhibit macrophage survival (FIGS. 6A and B) and thus act as antagonists. Futhermore, analysis of CD14 expression by monocytes in presence of decreasing concentration of the IL-34 mutants showed that S100F, Q131F and T36Y mutants were more efficient at lower concentration at differentiating CD14.sup.+ monocytes than WT IL-34 at the same concentration (FIG. 6C).

Example 6

Material and Methods

[0451] For the analysis of Akt and ERK1/2 phosphorylation (pAkt and pERK1/2 respectively), freshly sorted CD14.sup.+ monocytes were cultured in FBS-free medium (RPMI 1640, 1% penicillin-streptomycin, 1% glutamine, 1% AANE, 1% Hepes, 1% sodium pyruvate) in low attachment 96-well plates, with the WT IL-34 or the different S100F, T36W, T36Y, T36F, Q131F, Q131R, N150E and IL-34-Fc mutants at a 100 ng/ml concentration, for 1, 3 and 5 minutes. Analysis was performed by flow cytometry, using the phospho-Akt (Ser473) and phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) primary antibodies (reference #4060 and #4370, Cell Signalling), and goat anti-rabbit IgG(H+L)-AF647 (ref A21245, Life Technologies) secondary antibody, following the BD Biosciences Phosflow protocol (using the BD Cytofix Fixation buffer and BD Phosflow Perm Buffer III, BD Biosciences).

Results

[0452] Flow cytometry analyses of pAkt and pERK1/2 were then performed to evaluate functionally the signalization induced by the binding of each mutant of IL-34 to CSF-1R. Flow cytometry analyses of pAkt show an increased phosphorylation for T36F, IL-34-Fc, S100F, Q131F, T36W and T36Y mutants compared to WT IL-34 after 3 minutes and for most of them (T36F, IL-34-Fc, Q131F, T36W and T36Y) also after 5 minutes (FIG. 7A). In contrast, the N150E mutant is less efficient than WT IL-34 in inducing phosphorylation of Akt after 3 minutes and as efficient as WT IL-34 after 5 minutes. Finally, the Q131R mutant of IL-34 does not induce any phosphorylation of Akt after 3 or 5 minutes. Flow cytometry analyses of pERK1/2 show an increased phosphorylation for IL-34-Fc, S100F, Q131F and T36Y mutants compared to wt IL-34 after 3 minutes and for Q131F and T36Y also after 5 minutes (FIG. 7B). Furthermore, the T36W and T36F mutants show the same kinetic as WT IL-34. In contrast, the N150E mutant is again less efficient than WT IL-34 after 3 and 5 minutes. Moreover, as already observed for pAkt, the Q131R mutant does not induce any phosphorylation of ERK1/2 (FIG. 7B).

Example 7

Material and Methods

[0453] The ΔΔGmut at pH 7.4 was calculated for the substitutions of residues Thr36 (T36) of IL-34 by Tyr (Y) and the substitutions of residues Ser100 (S100) and Gln131 of IL-34 by Phe (F), using the method of Spassov and Yan (Spassov and Yann, 2013) implemented under Discovery Studio (Dassault Systemes BIO VIA Release 2017, San Diego) in the protocol “Calculate Mutation Energy (Binding)”. The input data was the atomic coordinates of IL-34/CSF-1R complex (pdb code 4DKD).

Results

[0454] In concordance with the results obtained in the previous examples, mutations T36Y, S100F and Q131F were stabilizing in silico (ΔΔGmut<−0.5) (FIG. 8). It was thus hypothesized that combining two or three of those mutations could be even more stabilizing than one mutation alone. Interestingly, the effect of double mutations was predicted to be as or even slightly more stabilizing than simple ones. The combination of the triple mutations was predicted to be the more stabilizing.

Example 8

Material and Methods

Reagents and Material

[0455] Series S CMS sensor chips and HBS-P buffer (10 mM HEPES buffer with 2.7 mM KCl, 150 mM NaCl, and 0.05% surfactant P20, pH 7.4), Amine Coupling Kit [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); 1.0M ethanolamine (pH 8.5)], immobilization buffer (sodium acetate pH 5.0), regeneration solutions (NaOH 10 mM, for analysis) were purchased from GE Healthcare Life Science (Uppsala, Sweden).

Surface Plasmon Resonance (SPR) Analysis

[0456] SPR experiments were performed on a Biacore T200 (GE Healthcare) at 25° C. HBS-P buffer was filtered through a 0.45 μm membrane filter and degassed prior to use. First, recombinant Human CD138 from Sino Biological (SDC1, Syndecan-1, ref 11429-H08H) and PTPz from Aviva System Biologic (ref OPCA02772) was immobilized on the chip surface by amine coupling. Briefly, recombinant CD138 or PTP-ζ was diluted to 20 μg/mL in 10 mM sodium acetate solution at pH 5.0. The diluted CD138 or PTP-ζ was soon covalently immobilized to a flow cell of CMS sensor chip via primary amine group, using standard Amine Coupling Kit. IL-34 muteins were then analyzed in a “Single Cycle kinetics” (SCK) models over the CD138 or PTP-ζ immobilized chip.

[0457] Interaction and kinetics of IL-34 muteins with CD138 or PTP-ζ

[0458] The maximum of resonance (Rmax) of IL-34 over CD138 or PTP-ζ were determined by using series of proteins dilutions in a “Single Cycle Kinetics” (SCK) model. IL-34 muteins as the analytes were diluted in HBS-P buffer with concentrations ranging from 25 nM to 400 nM, which flowed over the immobilized CD138 or PTP-ζ and the obtained response units (RUs) were recorded. The flow rate was at 30 μL/min with 120s for binding and 600s for dissociation. Then, the sensor chip surface was regenerated with 10 mM NaOH for 30s. The Rmax were determined by direct curve fitting of the sensorgrams to a Langmuir 1:1 model of interaction.

Results

[0459] The biacore analysis shows that the S100F mutant of IL-34 has a higher affinity to CD (FIG. 9A-C) and PTP-ζ (FIG. 10A-C) than the WT IL-34, suggesting that this mutant will be more efficient than the WT IL-34.

REFERENCES

[0460] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.