NOVEL G-CSF MIMICS AND THEIR APPLICATIONS
20230227520 · 2023-07-20
Assignee
- Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. (München, DE)
- Eberhard Karls Universität Tübingen (Tübingen, DE)
Inventors
- Mohammad ELGAMACY (Tübingen, DE)
- Birte HERNANDEZ ALVAREZ (Dettenhausen, DE)
- Yulia SKOKOWA (Kusterdingen, DE)
Cpc classification
A61K45/06
HUMAN NECESSITIES
C07K14/535
CHEMISTRY; METALLURGY
A61P7/00
HUMAN NECESSITIES
International classification
C07K14/535
CHEMISTRY; METALLURGY
C12N5/00
CHEMISTRY; METALLURGY
A61P7/00
HUMAN NECESSITIES
Abstract
The present invention relates to a protein having G-CSF-like activity comprising a) one or two polypeptide chains; b) a bundle of four α-helices; and c) two or three amino acid linkers that connect contiguous bundle-forming α-helices that are located on the same polypeptide chain, wherein each amino acid linker has a length between 2 and 20 amino acids. The invention also provides for a polynucleotide and a vector encoding the protein of the invention, host cells comprising said polynucleotide, a method for producing the protein of the invention and a pharmaceutical composition comprising the protein of the invention. The invention further relates to uses of the proteins of the invention as a research reagent and the use of the protein and/or pharmaceutical composition comprising the same as a medicament, e.g., for use in increasing stem cell production, for use in inducing hematopoiesis and/or for use in mobilizing hematopoietic stem cells.
Claims
1. A protein comprising: a) one or two polypeptide chains; b) a bundle of four α-helices; and c) two or three amino acid linkers that connect contiguous bundle-forming α-helices that are located on the same polypeptide chain, wherein each amino acid linker has a length between 2 and 15 amino acids; wherein the protein comprises one or more G-CSF receptor (G-CSF-R) binding sites; and wherein the protein has a melting temperature (T.sub.m) of at least 74° C.
2. The protein according to claim 1, wherein each G-CSF receptor binding site individually comprises six to eight amino acid residues having a similar structure and a similar spatial orientation towards each other as the amino acid residues Lysine 16, Glutamate 19, Glutamine 20, Arginine 22, Lysine 23, Aspartate 27, Aspartate 109, and Aspartate 112 of human G-CSF.
3. The protein according to claim 1, wherein the protein a) binds to G-CSF-R with an affinity of less than 10 μM; and/or b) has G-CSF-like activity, in particular wherein the G-CSF-like activity comprises at least one, preferably at least two, more preferably at least three, most preferably all of the following activities: (i) induction of granulocytic differentiation of HSPCs; (ii) induction of the formation of myeloid colony-forming units from HSPCs; (iii) induction of the proliferation of NFS-60 cells; and/or (iv) activation of the downstream signaling pathways MAPK/ERK and/or JAK/STAT; and/or c) induces the proliferation of NFS-60 cells, in particular wherein the protein induces the proliferation of NFS-60 at a half maximal effective concentration (EC50) of less than 100 μg/mL; and/or d) induces the proliferation and/or differentiation of cells comprising one or more G-CSF receptor on the cell surface, in particular wherein the cell is a hematopoietic stem cell or a cell deriving thereof, more preferably wherein the cell is a common myeloid progenitor or a cell deriving thereof, even more preferably wherein the cell is a myeloblast or a cell deriving thereof.
4-8. (canceled)
9. The protein according to claim 1, wherein the calculated contact order number of said protein is lower than the calculated contact order number of human G-CSF (SEQ ID NO:1); and/or wherein the protein has a molecular mass between 13 and 18 kDa; and/or wherein the protein comprises no disulfide bonds; and/or wherein the protein comprises no disulfide bonds and/or wherein the protein is not glycosylated.
10-12. (canceled)
13. The protein according to claim 1, wherein the α-helices that form the bundle of four α-helices are located on a single polypeptide chain, in particular wherein the single polypeptide chain comprises a four-helix bundle arrangement, in particular wherein the four-helix bundle arrangement has an up-down-up-down topology.
14-15. (canceled)
16. The protein according to claim 13, wherein the single polypeptide chain comprises an amino acid sequence having at least 60%, 70%, 80%, 90% amino acid sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO: 4, SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22 and SEQ ID NO:25; in particular wherein the single polypeptide chain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO: 4, SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22 and SEQ ID NO:25.
17. (canceled)
18. The protein according to claim 1, wherein the α-helices that form the bundle of four α-helices are located on two separate polypeptide chains, in particular wherein each of the two polypeptide chains contributes two α-helices to the bundle of four α-helices and/or wherein each of the two polypeptide chains comprises a helical-hairpin motif; and/or wherein the two polypeptide chains form a dimer.
19-21. (canceled)
22. The protein according to claim 18, wherein both polypeptide chains comprise an amino acid sequence having at least 60%, 70%, 80%, 90% amino acid sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:32 and SEQ ID NO:33; in particular wherein both polypeptide chains comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:32 and SEQ ID NO:33.
23. (canceled)
24. The protein according to claim 1, wherein the spatial orientation and molecular interaction features of at least two, at least three, at least four, at least five, at least six, at least seven of the amino acid residues Lysine 16, Glutamate 19, Glutamine 20, Arginine 22, Lysine 23, Aspartate 27, Asparagine 109, and Aspartate 112 of human G-CSF (SEQ ID NO:1) are preserved.
25. The protein according to claim 1, wherein the protein comprises or consists of an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:5, wherein the protein comprises one or more G-CSF receptor (G-CSF-R) binding sites; and wherein the protein has a melting temperature (T.sub.m) of at least 75° C.
26-35. (canceled)
36. The protein according to claim 1, wherein the protein comprises or consists of an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:6, wherein the protein comprises one or more G-CSF receptor (G-CSF-R) binding sites; and wherein the protein has a melting temperature (T.sub.m) of at least 74° C., in particular wherein the protein binds to G-CSF-R with an affinity of less than 10 μM.
37-46. (canceled)
47. The protein according to claim 1, wherein the protein comprises or consists of an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:14, wherein the protein comprises one or more G-CSF receptor (G-CSF-R) binding sites; and wherein the protein has a melting temperature (T.sub.m) of at least 75° C., in particular wherein the protein binds to G-CSF-R with an affinity of less than 10 μM.
48-57. (canceled)
58. The protein according to claim 1, wherein the protein comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:19, wherein the protein comprises one or more G-CSF receptor (G-CSF-R) binding sites; and wherein the protein has a melting temperature (T.sub.m) of at least 75° C., in particular wherein the protein comprises two polypeptide chains, preferably wherein the two polypeptide chains of the protein comprise identical amino acid sequences, in particular wherein the protein binds to G-CSF-R with an affinity of less than 10 μM.
59-68. (canceled)
69. The protein according to claim 1, wherein the protein comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:32, wherein the protein comprises one or more G-CSF receptor (G-CSF-R) binding sites; and wherein the protein has a melting temperature (T.sub.m) of at least 75° C., in particular wherein the protein comprises two polypeptide chains, preferably wherein the two polypeptide chains of the protein comprise identical amino acid sequences, in particular wherein the protein binds to G-CSF-R with an affinity of less than 10 μM.
70-79. (canceled)
80. A fusion protein comprising a first protein domain and a second protein domain, wherein the first protein domain and/or the second protein domain comprises a protein according to claim 1.
81. The fusion protein according to claim 80, wherein the first protein domain and the second protein domain are linked by a peptide linker, in particular wherein the peptide linker is a glycine-serine linker and/or wherein the linker has a length of 5 to 50 amino acid residues and/or wherein the first protein domain and the second protein domain comprise identical amino acid sequences.
82-98. (canceled)
99. A method of treating neutropenia in a subject, the method comprising administering an effective amount of the protein according to claim 1 to the subject.
100. (canceled)
101. A method of mobilizing stem cells in a subject, the method comprising administering an effective amount of the protein according to claim 1 to the subject.
102-104. (canceled)
105. A method for proliferating and/or differentiating cells in a cell culture, the method comprising the steps of: a) providing a plurality of cells in a cell culture; b) contacting said cells with the protein according to claim 1.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
[0519] Aspects of the present invention are additionally described by way of the following illustrative non-limiting examples that provide a better understanding of embodiments of the present invention and of its many advantages. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques used in the present invention to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should appreciate, in light of the present disclosure that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1: In Silico Design of Protein Variants
[0520] The first stage of the inventors approach was to convert the two bundle-spanning loops between α-helices A and B and α-helices C and D into two short de novo designed loops, which obligates the redesign of an up-up-down-down four-helix-bundle into an up-down-up-down four-helix-bundle. This is expected to bring the contact order of an idealized bundle to a theoretical minimum, and also decreases the domain sequence length by almost a third of the wild-type sequence length. This was followed by three more stages of redesign to improve the core packing, optimization of the loop landing sites to the best scoring new loop compositions, and redesign all of the newly surface exposed residues after removing the loops. This was done while maintaining site II conformationally and compositionally fixed.
[0521] Geometric Search Algorithm
[0522] At the first stage the inventors aimed at systematically searching the PDB for finding accommodating structural scaffolds to host the essential site II residues, namely: K16, E19, Q20, R22, K23, D27, D109, and D112 (
[0523] Loop Design
[0524] Novel loops were constructed through the automatic modeling of three- or four-residue long loops was performed covering the all sequence combinations of the involved residue types, which comprised: G, D, P, S, L, N, T, E, K for three-residue-loops, and G, D, P, S, L, N, T, K for four-residue-loops. A novel loop energetics evaluation routine was devised to perform adaptively directed generalized-ensemble sampling, based on a theoretical framework demonstrating the approximate equivalence of serial tempering to systematic umbrella sampling. The conformational homogeneity, quantified through a measure of local mean square structural deviations, of the resulting simulation trajectories was used to rank the candidate loop sequences for stability.
[0525] Sequence Optimization for Stability Enhancement
[0526] Sequence and conformer sampling were performed to the designs upon retrofitting the selected scaffolds with disembodied residues, using the RosettaScripts framework. In addition to an RMSD constraint on the binding epitope, a previously described core packing protocol was used. That comprised steps of interleaved Monte Carlo sequence and side chain and backbone conformer sampling iterations. The sequence sampling was directed to most core residues and to solvent-exposed hydrophobic residues. The scoring functions used were the talaris2013 energy function and the packstat packing score. While the energy function was used to bias the sampling towards lower energy decoys, the top decoys were forwarded for further evaluation based on the packing quality, where the latter was further judged by the ruggedness of the radial distribution function g(r) as given by the definite integral ∫.sub.0.sup.4|dg(r)/dr|dr.
[0527] In Silico Affinity Maturation
[0528] Mutations were systematically sampling for residues around the binding epitope of the artificial GCSF to lower the potential energy of the modelled receptor-design complexes. The modeled complexes were based on the native GCSF-GCSFR complex (PDB:2D9Q), where the design models were aligned by their binding pharmacophore to the native ligand and further annealed in implicit solvent to refine their docked posses. For a more accurate evaluation for the binding free energy of the complexes, potential of mean force (PMF) [37] simulations were used to estimate the binding free energy (to the of the GCSF receptor CRH domains) generated decoys.
[0529] As a result, eight protein designs, namely Boskar_1 (SEQ ID NO:2), Boskar_2 (SEQ ID NO:3), Boskar_3 (SEQ ID NO:4), Boskar_4 (SEQ ID NO:5), Moevan (SEQ ID NO:6), Sohair (SEQ ID NO:14), Disohair_1 (SEQ ID NO:18) and Disohair_2 (SEQ ID NO:19) have been obtained with the strategy described above.
Example 2: Expression and Purification
[0530] The synthetic genes encoding the protein variants designed in Example 1 were ordered and cloned in-frame with an N-terminal hexa-His-tag and a thrombin cleavage site into the NdeI and XhoI sites of the pET28a(+) expression vector harboring a kanamycin resistance gene as a selection marker. The plasmids were transformed by heat-shock in chemically competent E. coli BL21(DE3) cells. For protein expression, the cells were grown in LB medium and expression was induced with IPTG at OD.sub.600 of 0.5-1 followed by incubation overnight at 25° C. For expression of isotopically labeled protein, a pre-culture in LB medium was grown, cells were collected, washed twice in PBS buffer, and resuspended in M9 minimal medium (240 mM Na.sub.2HPO.sub.4, 110 mM KH.sub.2PO.sub.4, 43 mM NaCl), supplemented with 10 μM FeSO.sub.4, 0.4 μM H.sub.3BO.sub.3, 10 nM CuSO.sub.4, 10 nM ZnSO.sub.4, 80 nM MnCl.sub.2, 30 nM CoCl.sub.2, and 38 μM kanamycin sulfate to an OD.sub.600 of 0.5-1. After 40 minutes of incubation at 25° C., 2.0 gram .sup.15N-labelled ammonium chloride (Sigma-Aldrich cat. nr. 299251) and 6.25 gram .sup.13C D-glucose (Cambridge Isotope Laboratories, Inc. cat. nr. CLM-1396) were added to a 2.5 L culture. Following another 40 minutes of incubation, IPTG was added to 1 mM final concentration to induce overnight expression. Cells were collected by centrifugation at 5,000 g for 15 minutes, lysed using a Branson Sonifier S-250 (Fisher Scientific) in hypotonic 50 mM Tris-HCl buffer supplemented with cOmplete protease cocktail (Sigma-Aldrich cat. nr. 4693159001) and 3 mg of lyophilized DNase I (5200 U/mg; Applichem cat. nr. A3778). The insoluble fraction was pelleted by centrifugation at 25,000 g for 50 minutes, and the soluble fraction was filtered (0.45 μm filter pore size) and directly applied to a Ni-NTA column. For wild-type G-CSF, from the expressed protein was extracted from the insoluble fraction of lysed E. coli cells by stirring the pellet in 8 M guanidinium chloride solution for 2 hours at 4° C. The mixture was gradually diluted to 1 M guanidinium chloride in 4 steps over 4 hours, and loaded directly onto a Ni-NTA column. A 5 mL HisTrapFF immobilized nickel column (GE Healthcare Life Sciences cat. nr. 17-5255-01) was used for this purpose, washed consecutively with 30 mL 150 mM NaCl, 30 mM Tris buffer (pH 8.5) containing 0, 30 and 60 mM imidazole. Bound protein was eluted with a linear gradient from 60-500 mM imidazole and fractions were collected. The eluate was concentrated using 3 kDa MWCO centrifugal filters (Merck Millipore cat. nr. UFC901024) and loaded onto a Superdex 75 gel filtration column (GE Healthcare Life Sciences cat. nr. 17517401) equilibrated with gel filtration buffer, which was always Phosphate Buffered Saline (PBS) pH 7.4 which is favorable for NMR, CD, and cell culture. An ÄktaFPLC system (GE Healthcare Life Sciences) was used for all chromatography runs.
[0531] The inventors expressed all newly designed proteins in E. coli, where all protein variants were efficiently expressed as soluble protein. After the IMAC and preparative size exclusion chromatography, the non-optimized final purification yield of the designs was at least 15 mg per liter culture.
[0532] In comparison, filgrastim (recombinant human G-CSF) is only insolubly expressed in E. coli and has to be refolded from inclusion bodies prior to purification. The optimized production yield in the pharmacopoeia-mandated expression host, E. coli, was 3.2 mg/Liter culture [11].
Example 3: Biophysical Analyses
[0533] Thermal unfolding was measured by CD spectroscopy monitoring loss of secondary structure. The temperature was monitored and regulated by a Peltier element which was connected to the CD spectroscopy unit. The temperature was measured in the cuvette jacket that is made of copper. Samples (0.5 mL) of concentrations between 0.3 and 6 mg/mL were loaded into 2 mm path length cuvettes. Spectral scans of mean residual ellipticity were done at a resolution of 0.1 nm, across the range of 240-195 nm. Melting curves tracked the mean residual ellipticity at a wavelength of 222 nm across a temperature range of 20 to 100° C. Melting temperature was extracted as the value of T.sub.m (where
), where an inflection is observed.
[0534] Circular dichroism spectra of diSohair_2 (SEQ ID NO:19), Moevan (SEQ ID NO:6) and Boskar_4 (SEQ ID NO:5) showed strong alpha-helical content, with characteristic minima of almost double intensities compared to that of G-CSF at the same concentration. Strong NMR signal dispersion also indicated well folded proteins for Moevan (SEQ ID NO:6), Sohair (SEQ ID NO:14), and Boskar_4 (SEQ ID NO:5). Thermal melting measured by circular dichroism of the most active design Boskar_4 (SEQ ID NO:5), and diSohair_2 (SEQ ID NO:19), showed thermal stability up to 100° C. accompanied by only a slight decrease in helicity, which was fully reversible upon cooling (
Example 4: Protease Sensitivity Assay
[0535] Previous studies have established the negative feedback loop of granulopoiesis, where GCSF-induced neutrophils in-turn release neutrophil elastase (NE) that strongly antagonises GCSF through its GCSF-directed protease activity. NE concentration in serum was shown to be directly correlated to neutrophil count, and is demonstrated to be the major degrading protease of GCSF [18, 19]. The inventors have thus compared three of their protein designs against filgrastim USP standard to assess their NE degradation sensitivity.
[0536] Purified human neutrophil elastase was obtained from Enzo Life Science (cat. nr.: BML-SE284-0100). The elastase was reconstituted in PBS buffer (pH 7.4) to a stock concentration of 20 IU/mL. Digestion reactions were conducted in PBS buffer with final concentrations of 300 μg/mL of the protein of interest and 1 U/mL of neutrophil elastase. The reaction mixture was incubated at 37° C. and digestion samples were withdrawn, immediately mixed with SDS sample buffer (450 mM Tris HCl, 12% Glycerol, and 10% SDS) and flash-frozen in liquid nitrogen bath to stop the reaction, after 5, 15 and 30 minutes from the reaction start. Frozen samples were then heated at 85° C. for 10 minutes before loading on Novex™ 16% Tricine Protein Gels (ThermoFisher Scientific; cat. nr. EC6695BOX). The SDS-PAGE gels were incubated overnight in fixing solution (30% ethanol, 10% acetic acid), and then stained using colloidal coomassie dye.
[0537] The results show that Moevan (SEQ ID NO:6) and human G-CSF are very susceptible to NE proteolysis, while Boskar_4 (SEQ ID NO:5) and Disohair_2 (SEQ ID NO:19) are much more resistant to NE (
Example 5: In-Cell Activity Testing
[0538] For testing the functionality of the newly designed protein variants in cells, the inventors analyzed the proliferation of NFS-60 cells. The growth and maintenance of viability of this murine myeloblastic cell line is dependent on IL-3. NFS-60 cells are also highly responsive to IL-3, GM-CSF, G-CSF, and erythropoietin and therefore commonly used to assay human and murine G-CSF activity.
[0539] NFS-60 cells were cultured in GM-CSF-containing RPMI 1640 medium ready-to-use, supplemented with L-glutamine, 10% KMG-5 and 10% FBS (cls, cell line services). Before each assay, cells were pelleted and washed three times with cold non-supplemented RPMI 1640 medium. After the last washing step, cells were diluted at a density of 6×10.sup.5 cells/mL in RPMI 1640 containing glutamine and 10% FBS. In order to analyze cell proliferation, NFS-60 cells were grown in the presence of varying concentrations of G-CSF wild-type and designed variants. For this, fivefold dilution series were prepared from stock solutions of wild type G-CSF (40 ng/mL) and newly designed protein variants (40 μg/mL) in RPMI 1640 medium supplemented with glutamine and 10% FBS. 75 μL of each dilution were mixed with the same volume of washed cells in a 96 well plate yielding a final cell density of 3×10.sup.5 cells/mL and G-CSF concentrations varying from 0.00001-20 ng/mL for wild type and 0.01-20,000 ng/μL for the designs. Each 96 well plate contained triplicates of each dilution and the according blanks, including wells containing cells seeded in RPMI 1640 medium supplemented with L-glutamine, 10% KMG-5 and 10% FBS (cls, cell line services) and wells containing medium solely. Following incubation for 48 h at 37° C. and 5% CO.sub.2, 30 μL of the redox dye resazurin (CellTiter-Blue® Cell Viability Assay, Promega) was added to the wells and incubation was continued for another hour. Cell viability was measured by monitoring the fluorescence of each well at a H4 Synergy Plate Reader (BioTek) using the following settings: excitation=560/9.0, Emission=590/9.0, read speed=normal, delay=100 msec, measurements/data Point=10. The data were analyzed and curves were plotted applying a four-parameter sigmoid fit using SigmaPlot (Systat Software).
[0540] Five different designs (Boskar_3 (SEQ ID NO:4), Boskar_4 (SEQ ID NO:5), Sohair (SEQ ID NO:14), Moevan (SEQ ID NO:6) and Disohair_2 (SEQ ID NO:19)) were analyzed in comparison to wild-type human G-CSF. In the assay, variant Boskar_4 (SEQ ID NO:5) had the highest activity of the five designs followed by Moevan (SEQ ID NO:6), Disohair_2 (SEQ ID NO:19), Boskar_3 (SEQ ID NO:4) and Sohair (SEQ ID NO:14) (
Example 6: Induction of In Vitro Granulocytic Differentiation of HSPCs
[0541] It was first evaluated whether G-CSF-like designs are capable to induce myeloid differentiation of human CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro. To study in vitro myelopoietic capacity of the designs, human CD34+ HSPCs were isolated from the bone marrow mononuclear cell fraction of two healthy donors by magnetic bead separation using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech #130-046-703, Germany). CD34+ cells were cultured at a density of 2×10.sup.5 cells/mL in Stemline II Hematopoietic Stem Cell Expansion medium (Sigma Aldrich, #50192) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine and 20 ng/mL IL-3, 20 ng/mL IL-6, 20 ng/mL TPO, 50 ng/mL SCF and 50 ng/mL FLT-3L. For liquid culture granulocytic differentiation, expanded CD34+ cells (2×10.sup.5 cells/mL) were incubated for 7 days in RPMI 1640 GlutaMAX supplemented with 10% FBS, 1% penicillin/streptomycin, 5 ng/mL SCF, 5 ng/mL IL-3, 5 ng/mL GM-CSF and 10 ng/mL of rhG-CSF, or 10 μg/mL of each design, respectively. Medium was exchanged every second day. On day 7, medium was changed to RPMI 1640 GlutaMax supplemented with 10% FBS, 1% penicillin/streptomycin and 10 ng/mL rhG-CSF, or 10 μg/mL of each design, respectively. Medium was exchanged every second day until day 14. On day 14, cells were analyzed by flow cytometry using the following antibodies: mouse anti-human CD45 (Biolegend, #304036), mouse anti-human CD11b (BD, #557754), mouse anti-human CD15 (BD, #555402), and mouse anti-human CD16 (BD, #561248) on a FACSCanto II instrument. Of note, FACS analysis revealed differentiation of HSPCs isolated from two healthy donors in myeloid/granulocytic cells, co-expressing cell surface markers of granulocytes, such as CD15+CD11b+, CD16+CD11b+, CD15+CD16+ cells, in the presence of designs to the levels comparable to that of rhG-CSF. HSPCs of healthy donor 1 were stimulated with rhG-CSF, Boskar_3 (SEQ ID NO:4), or Boskar_4 (SEQ ID NO:5) (
[0542] It was also analyzed whether myeloid cells generated in the presence of the designs will have the typical cell morphology of mature neutrophils. Cell morphology was evaluated on cytospin preparations. For this, cells were isolated on day 14 of culture, 10×10.sup.4 cells per cytospin slide were centrifuged at 400 g for 5 min at room temperature using a Thermo Scientific Cytospin 4 Cytocentrifuge. Wright-Giemsa-stained cytospin slides were prepared using Hema-Tek slide stainer (Ames) and evaluated using a Nikon Inverted Microscope. As expected, a vast majority of cells cultured in the presence of rhG-CSF or designs revealed the typical and highly specific morphology of neutrophilic granulocytes with multilobed nuclei (
[0543] These data clearly demonstrate biological activity of designs towards granulocytic differentiation of human hematopoietic stem and progenitor cells.
Example 7: Induction of Formation of Myeloid Colony-Forming Units (CFUs) from HSPCs
[0544] It was further tested whether the designs induce the formation of myeloid colony-forming units (CFUs) from healthy donor HSPCs. This would be an additional proof of the biological activity of designs on the hematopoietic stem cells. For this, CD34+ HSPCs at a concentration of 10.000 cells/mL medium were plated in 35 mm cell culture dishes in 1 mL Methocult H4230 medium (Stemcell Technologies) supplemented with 2% FBS, 10 μg/mL of 100× Antibiotic-Antimycotic Solution (Sigma) and 50 ng/mL of rhG-CSF, or 1 μg/mL of Boskar_3, Boskar_4, DiSohair_2 or Moevan, respectively. Cells were cultured at 37° C., 5% CO2. Colonies were counted on day 14.
[0545] Indeed, myeloid CFUs were observed in the HSPC cultures in the presence of the designed proteins. Although the number of CFU colonies induced by Boskar_3 (SEQ ID NO:4), Boskar_4 (SEQ ID NO:5), Moevan (SEQ ID NO:6) and DiSohair_2 (SEQ ID NO:19) was much lower than the number stimulated by rhG-CSF, the typical myeloid cell morphology of CFUs was visible in all groups (
Example 8: Activation of G-CSF Receptor Downstream Intracellular Signaling Pathways in Human Hematopoietic Stem Cells
[0546] Binding of rhG-CSF to G-CSFR activates a cascade of intracellular signaling pathways, including phosphorylation of downstream proteins, such as STAT3, STAT5, or MAPK, which ultimately induces granulocytic differentiation of HSPCs. Therefore, it was investigated whether the designs are capable of inducing phosphorylation of these proteins in CD34+ HSPCs. For this, CD34+ cells were cultured in Stemline® II Hematopoietic Stemcell Expansion Medium (Sigma-Aldrich; #50192) supplemented with 10% FBS (Sigma-Aldrich; #F7524; batch-no. BCBW7154), 1% L-Glutamine (Biochrom; #K0283), 1% Pen/Strep (Biochrom; #A2213) and a premixed Cytokine Cocktail containing rh-IL3 (PeproTech; #200-03), rh-IL6 (Novus Biologicals; #NBP2-34901), rh-TPO, rh-SCF (both R&D Systems; TPO #288-TP200; SCF #255-SC-200) and rh-Flt-3L (BioLegend; #550606). The final concentration of IL-3, IL-6 and TPO was 20 ng/ml, and for SCF and Flt-3L 50 ng/ml. On day 6 of culture, serum- and cytokine-starved (3 h) CD34+ HSPCs were treated with rhG-CSF, Moevan (SEQ ID NO:6) or DiSohair_2 (SEQ ID NO:19) (10 μg/mL for Moevan and Di-Sohair_2), respectively, for 10, 15 or 30 min, fixed in 4% PFA (Merck; #P6148) for 15 min at room temperature, and permeabilized for 30 min by slowly adding ice-cold methanol (C. Roth; #7342.1) to a final concentration of 90%. Cells were left overnight in methanol at −20° C. and stained on the next day with specific antibodies recognizing phosphorylated signaling effectors (phospho-Stat3 (Tyr705) (D3A7) XP rabbit mAb (Cell Signaling; #9145); phospho-Stat5 (Tyr694) (C1105) rabbit mAb (Cell Signaling; #9359), and phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) mouse mAb (Cell Signaling; #9106) or respective isotype control antibody (anti-mouse IgG (H+L), F(ab′)2 fragment (Alexa Fluor® 488 Conjugate) (Cell Signaling; #4408; goat anti-rabbit IgG H+L (Alexa Fluor® 488) (abcam; #ab150077) by incubation for 20 minutes on ice in PBS/2% BSA. After that, cells were washed twice in ice-cold PBS/2% BSA and analyzed by FACS. To determine the background-corrected fluorescent signal from the corresponding phosphorylated proteins, the fluorescent signal of the appropriate isotype control estimated at each time point of stimulation was subtracted from the specific phospho-protein signal.
[0547] Indeed, time-dependent tyrosine phosphorylation of p44/42 MAPK (Erk1/2) in HSPCs treated with Moevan (SEQ ID NO:6) or DiSohair_2 (SEQ ID NO:19), respectively, was observed to a similar degree as in rhG-CSF treated cells (
Example 9: In-Cell Activity Testing with Further Designs
[0548] NFS-60 cells were cultured in IL-3-containing RPMI 1640 medium, supplemented with L-glutamine, 10% KMG-5 and 10% FBS (CLS, cell line services). Before each assay, cells were pelleted and washed three times with cold non-supplemented RPMI 1640 medium. After the last washing step, cells were diluted at a density of 6×10.sup.5 cells/ml in RPMI 1640 containing glutamine and 10% FBS. In order to analyze cell proliferation, NFS-60 cells were grown in the presence of varying concentrations of G-CSF wild-type and designed variants. For this, five-fold dilution series were prepared from stock solutions of the designs (Moevan t2=60.2 μg/ml, Boskar4 t2=2 μg/ml, bika1=26.8 μg/ml, bika2=1.07 μg/ml, Sohair2_15 rl=26.8 μg/mL, Boskar4_15 rl=26.8 μg/ml, Boskar4_st2=26 μg/mL, Moevan_st2=26 μg/mL) in RPMI 1640 medium supplemented with glutamine and 10% FBS. 75 μl of each dilution were mixed with the same volume of washed cells in a 96-well plate yielding a final cell density of 3×10.sup.5 cells/ml and designed protein concentrations varying from 0.0001-60,000 ng/mL. Each 96-well plate contained triplicates of each dilution and the according blanks, including wells containing cells seeded in RPMI 1640 medium supplemented with L-glutamine, 10% KMG-5 and 10% FBS (cls, cell line services) and wells containing medium only. For endpoint analysis, following incubation for 48 h at 37° C. and 5% CO.sub.2, 30 μl of the redox dye resazurin (CellTiter-Blue® Cell Viability Assay, Promega) was added to the wells, and incubation was continued for another hour. Cell viability was measured by monitoring the fluorescence of each well at a H4 Synergy Plate Reader (BioTek) using the following settings: excitation=560 nm±9 nm, Emission=590 nm±9 nm, read speed=normal, delay=100 ms, measurements per data point=10. The data were analysed and curves were plotted applying a four-parameter sigmoid fit using SigmaPlot (Systat Software).
[0549] The inventors surprisingly found that dimerization of protein designs results in more active variants. For example, it has been demonstrated that the variant boskar4_t2, comprising two boskar_4 variants connected via a 24 amino acid GS-rich linker, induced the proliferation of NFS-60 cells with an EC.sub.50 of 4.2 ng/mL. More importantly, the dimeric variant boskar_4_st2, comprising a 6 amino acid GS-linker induced the proliferation of NFS-60 cells even with an EC50 of 0.202 ng/mL (Table 7). In comparison, the parent variant boskar_4 induced the proliferation of NFS-60 cells with an EC.sub.50 of 27 ng/mL (Table 5). Variant boskar4_15 rl, comprising a 15 amino linker between helices 2 and 3 induced the proliferation of NFS-60 cells with an EC.sub.50 of 48.5 ng/mL (Table 7).
[0550] Similarly to boskar_4, dimerization of Moevan also resulted in higher activity. The designs moevan_t2 (24 amino acid GS-linker) and moevan_st2 (6 amino acid GS-linker) induced proliferation of NFS-60 cells with EC.sub.50 values of 47.1 ng/mL and 8.89 ng/mL, respectively (Table 5). The parent variant Moevan induced proliferation of NFS-60 cells with an EC.sub.50 of 356 ng/mL (Table 7).
[0551] Variant disohair2_15 rl comprises two disohair2 designs connected via a 15 amino acid GS-linker. The activity of this variant was increased compared to the variant Disohair_2 (228 ng/mL compared to 396 ng/mL, Tables 5 and 7).
[0552] The two designs bika1 and bika2 have been demonstrated to induce the proliferation of NFS-60 cells with an EC.sub.50 of 63 ng/mL and 98 ng/mL respectively (Table 7).
Example 10: Analysis of the Binding Epitope in Boskar_4
[0553] To evaluate the structural precision of the design process, the inventors determined the structure of Boskar4. The structure was determined using the CoMAND method (Conformational Mapping by Analytical NOESY Decomposition), a protocol that provides unbiased structure determination driven by a residue-wise R-factor tracking the match between experimental and back-calculated NOESY spectra. In the CoMAND protocol, a 3D-CNH-NOESY spectrum is divided into 1D sub-spectra, each representing contacts to a single backbone amide proton, thus representing the structural environment at and around the respective residue. Spectral decomposition is then performed, which yields the local backbone dihedral angles for all residues where strips are available. In a subsequent stage, the R-factor is used as a selection criterion for frame-picking from equilibrium MD trajectories, yielding the final structure ensemble.
[0554] The CNH-NOESY spectra of Boskar4 provided 98 strips, after excluding strips containing overlapped intensities. CoMAND factorization calculations were performed on these strips, yielding backbone dihedrals, that were both consistent with the values predicted from chemical shift profiles by TALOS-N, as well as the lowest energy Rosetta ab initio folding decoy. Refinement was done by running 1 μs of explicit solvent NPT sampling followed by the frame picking step, where the global average R-factor minimization converged after the picking of 12 frames. This final ensemble yielded an average R-factor of 0.36±0.11 over 89 spectra (Table 8). The ensemble deviated by an average of 1.34 Å from the average structure, and 2.59 Å from the design model (
[0555] For Moevan, the CNH-NOESY spectra provided sub-spectra 205 for 102 amide protons, with those missing mainly due to unassigned resonances spanning two ranges (residues 1-8 and 65-67) where the latter stretch was a disordered loop in the template structure. The inventors applied CoMAND factorization calculations to these sub-spectra, yielding backbone dihedrals both consistent with the values predicted from chemical shift profiles by TALOS-N and having the lowest energy Rosetta ab initio folding decoy. Due to its high conformational heterogeneity, the refinement simulations for Moevan were carried out under a set of unambiguous distance restraints. During the frame-picking stage, R-factor minimization converged at 17 frames, three of which were rejected on the basis of distance restraint violations, leaving 14 frames constituting the final ensemble. The ensemble deviated by an average of 1.8 Å from the average structure, and 2.5 Å from the design model (
[0556] For Sohair, the inventors extracted 146 CNH-NOESY sub-spectra out of a total length of 154 residues (excluding the purification tag). Due to the significant pseudo-symmetry in the sequence and chemical environment, 29 of these had overlapped intensities. Performing CoMAND factorization on the non-overlapped strips, the inventors obtained backbone dihedrals consistent with TALOS-N predictions, which are in turn in line with the dihedral values of the lowest energy Rosetta ab initio folding decoy. The final, refined ensemble compiled by R-factor minimization yielded 19 frames, with an RMSD of 1.8 Å from the average structure. Although the final ensemble has an average RMSD of 2.9 Å to the design model (
[0557] Methods:
[0558] All spectra were recorded at 310 K on Bruker AVIII-600 and AVIII-800 spectrometers. Backbone sequential and aliphatic side chain assignments were completed using standard triple resonance experiments, while aromatic assignments were made by linking aromatic spin systems to the respective CβH2 protons in a 2D-NOESY spectrum. Structures were calculated using the CoMAND method, which exploits the high accuracy that can be obtained in back-calculating NOESY spectra with indirect 13504 C dimensions. The CoMAND method involves spectral decomposition of one-dimensional sub-spectra extracted from a 3D-CNH-NOESY spectrum. These sub-spectra are chosen from a search area centered on assigned 15506 N-HSQC positions and thus contain only cross-peaks to a specific amide proton. Residues with overlapping search areas were examined separately. In most cases strips with acceptable separation of signals could be obtained. Where this was not possible, the residues were flagged as overlapped and a joint strip constructed by summing those at the estimated maxima of the respective components. These 1D strips were decomposed against a library of spectra back-calculated by systematic sampling over a local dihedral angle space, yielding estimates of backbone and side chain dihedral angles for each residue. In this work however, the inventors have excluded heavily overlapped strips since there were only few overlaps. Later stages of the protocol involve conformer selection aimed at minimizing a quantitative R-factor expressing the match between the experimental strips and back-calculated spectra, or a fold-factor designed to isolate the contribution to the R-factor from long-range NOESY contacts.
[0559] For initial model building, unrestrained Rosetta ab initio folding simulations were performed and generated 10,222 decoys. The corresponding CNH-NOESY spectra of these decoys were back-calculated to evaluate the structure-averaged fold-factors. The decoy with the lowest fold-factor was used to seed five independent unrestrained molecular dynamics simulations. These refinement simulations were carried out using the CHARMM36 force field in explicit solvent using the polarizable TIP3P water model. Trajectories of a total length of approximately 1 μs were run, with frames collected every 100 ps. An initial refined ensemble was compiled through a global greedy minimization of the R-factor as previously described, which converged on a total of 12 frames.
Example 11: Binding of the Protein Designs to G-CSF-R
[0560] To characterize the kinetics and affinity of interactions between the designs and the G-CSF receptor, the inventors performed surface plasmon resonance (SPR)-based measurements for Boskar3 and Boskar4 in comparison to rhG-CSF. Analysis of the kinetics across the injection dilution series, assuming 1:1 binding, resulted in dissociation constants (Kd) of 14 nM and 5.1 nM for Boskar3 and Boskar4, respectively. In comparison, the Kd determined for rhG-CSF was 335 μM (Table 9). Previous studies have reported Kd values for the G-CSF:G-CSFR interaction between 200 μM using SPR [38] and 1.4 nM using ITC [39]. To obtain a more detailed picture on the nature of the binding, the inventors fitted the highest concentration sensorgram curves using higher order kinetics models. These fitting attempts showed the second-order reaction model to better fit the data than a first-order model despite the same number of parameters in each model. This indicates that the binding reaction depends on two analyte molecules, yielding Kd values of 4.4 μM, 6.1 μM, 86 nM, for Boskar3, Boskar4, and rhG-CSF, respectively. While this higher-order interaction model better explains the data than a 1:1 binding model, a clear deviation remained for rhG-CSF sensorgrams. While this may point to different interaction modes between the two designs and rhG-CSF with the G-CSFR, it demonstrates that the binding form of the designs is plausibly dimeric. Size-exclusion chromatography of the designs indeed show that the designs partition between monomeric and dimeric forms (
[0561] To characterize the kinetics and affinity of interactions between the designs and the G-CSF receptor, the inventors performed surface plasmon resonance-based measurements for Moevan and diSohair2 in comparison to rhG-CSF (Table 10). Analysis of the kinetics across the injection dilution series, assuming 1:1 binding, resulted in dissociation constants (Kd) of 4.5 μM, 21.0 nM, and 1.1 nM for diSohair2 (
[0562] The Moevan control and diSohair control refers to the unmutated scaffold protein sequences of both diSohair2 (PDB: 5J73) and Moevan (PDB: 2QUP), lacking the G-CSF-R binding epitope.
[0563] Methods:
[0564] Single-cycle kinetics experiments were performed on a Biacore X100 system (GE Healthcare Life Sciences). G-CSF Receptor (G-CSFR) (R&D systems 381-GR-050/CF) was diluted to 50 μg/ml in 10 mM acetate buffer pH 5.0 and immobilized on the surface of a CM5 sensor chip (GE Healthcare 29149604) using standard amine coupling chemistry. The designs and rhG-CSF (USP RS Filgrastim, Sigma-Aldrich 1270435) were diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% v/v Tween-20). Analyses were conducted at 25° C. at a flow rate of 30 μl/min. Five sequential 10-fold increasing concentrations of the sample solution (for the designs from 0.5 nM to 50 μM, and for rhG-CSF from 0.05 to 500 nM) were injected over the functionalized sensor chip surface for 180 s, followed by a 180 s dissociation with running buffer. At the end of each run, the sensor surface was regenerated with a 240 s injection of 10 mM glycine-HCl pH 2.0. Each experiment was performed two times for rhG-CSF, Boskar3, Boskar4, diSohair2, Moevan, and Moevan_t2. Association rate (ka), dissociation rate (kd), and equilibrium dissociation (Kd) constants were initially obtained by global fitting of the experimental reference-subtracted data to a 1:1 interaction model using the Biacore X100 evaluation software (v.2.0.1). To evaluate if a kinetics model that depends on double the analyte stoichiometry improves the goodness of fit to the data, the following rate integral was used:
[0565] where R(t) is the normalized response at time tin normalized response units (and time t is in seconds), and Rmax is the maximum normalized response (i.e. R(180 s)), at analyte concentration C, given association and dissociation intervals of 180 s each. The goodness of fit was evaluated by the χ2 as:
[0566] where Rfit is the R(t) function with minimum sum of square deviation from the observed sensorgram curve Robs, optimizing ka and kd, individually, within the bounds [10, 1×10.sup.6] and [1×10.sup.−5, 0.1], respectively. The optimization was performed using the Nelder-Mead method at a tolerance of 1×10.sup.−8 and a maximum number of iterations of 1×10.sup.4. The coefficient of determination R.sup.2 was calculated as:
[0567] where <⋅> is the vector average.
Example 12: Activation of G-CSFR Signaling by Boskar3 and Boskar4
[0568] To evaluate the dependency of the response to the designed proteins on G-CSFR expression, the inventors knocked out G-CSFR in NFS-60 cells using CRISPR/Cas9-mediated mutagenesis. For this, the inventors synthesized guide RNA (gRNA) specifically targeting exon 4 of CSF3R (cut site: chr4 [+126,029,810: −126,029,810]) to introduce stop-codon or frameshift mutations in the extracellular part of all G-CSFR isoforms. The inventors generated pure G-CSFR KO NFS-60 cell clones that have one nucleotide deletion on each allele, as assessed by Sanger sequencing and tracking of indels by decomposition (TIDE) analysis. In contrast to wild type cells, G-CSFR KO NFS-60 cells did not respond to treatment with rhG-CSF, Boskar3 or Boskar4 (
[0569] Methods:
[0570] A specific guide RNA (sgRNA) for knock-out of the CSF3R gene (cut site: chr4 [+126.029.810: −126.029.810], NM_007782.3 and NM_001252651.1, exon 4, 112 by after ATG; NP_031808.2 and NP_001239580.1 p.L38) was designed using CCTop at (http://crispr.cos.uni-heidelberg.de) [54]. Electroporation of NFS-60 cells was carried out using the Amaxa nucleofection system (SF cell line 4D-Nucleofector kit, #V4XC-2012) according to the manufacturer's instructions. Briefly, 1×106 cells were electroporated with assembled sgRNA (8 μg) and HiFi Cas9 nuclease protein (15 μg) (Integrated DNA Technologies). Clonal isolation of single-cell derived NFS-60 cells was performed by limiting dilution followed by an expansion period of 3 weeks. Genomic DNA of each single-cell derived NFS-60 clones was isolated using QuickExtract DNA extraction solution (Lucigen #QE09050). PCR was carried out with mouse CSF3R-specific primers (forward: 5′-GGCATTCACACCATGGGGCACA-3′, reverse: 5′-GCCTGCGTGAAGCTCAGCTTGA-3′) and the GoTaq Hot Start Polymerase Kit (Promega, #M5006) using 2 μl of gDNA template for each PCR reaction. In vitro cleavage assay was done by adding 1 μM Cas9 RNP assembled by the same sgRNA used for the knock-out experiment to 3 μL of each PCR product. The PCR reactions were incubated at 37° C. for 60 min and run on a 1% agarose gel. The PCR products that showed no cleavage were purified by ExoSAP (ratio 3:1), which is a master mix of one-part Exonuclease I 20 U/μl (Thermo Fisher Scientific, #EN0581) and two parts of FastAP thermosensitive alkaline phosphatase 1 U/μl (Thermo Fisher Scientific, #EF0651). Sanger sequencing of purified PCR products was performed by Microsynth and analysed using the TIDE (Tracking of Indels by Decomposition) webtool.
Example 13: Activation of G-CSF Receptor Downstream Intracellular Signaling Pathways in Human Hematopoietic Stem Cells
[0571] Binding of G-CSF to G-CSFR rapidly activates a cascade of intracellular events, including phosphorylation of downstream effectors, e.g. Akt, STAT3, STAT5 or MAPK, that ultimately induce granulocytic differentiation. To test whether our designed proteins directly induce G-CSFR signaling, the inventors measured these immediate phosphorylation targets of G-CSFR signaling in CD34+ HSPCs. Indeed, the inventors found that Akt, STAT3, STAT5 and p44/42 MAPK (Erk1/2) were tyrosine phosphorylated in HSPCs treated with Boskar3 or Boskar4 to a similar degree as in rhG-CSF-treated cells (
[0572] Methods:
[0573] CD34+ 703 cells were cultured in Stemline II Hematopoietic Stemcell Expansion Medium (Sigma-Aldrich; #50192) supplemented with 10% FBS (Sigma-Aldrich; #F7524), 1% L-glutamine (Biochrom; #K0283), 1% penicillin/streptomycin (Biochrom; #A2213) and a premixed cytokine cocktail containing IL-3 (PeproTech; #200-03), IL-6 (Novus Biologicals; #NBP2-34901), TPO (R&D Systems; #288-TP200), rhSCF (R&D Systems; #255-SC-200) and Flt-3L (BioLegend; #550606). Final concentrations were 20 ng/ml for IL-3, IL-6 and TPO, and 50 ng/ml for SCF and Flt-3L. On day 6 of culture, serum- and cytokine-starved (4 h) CD34+ HSPCs were treated with 20 ng/ml of rhG-CSF, 10 μg/ml of Boskar3 or 10 μg/ml of Boskar4 for 30 or 60 min, fixed in 4% PFA (Merck; #P6148) for 15 min at room temperature, and permeabilised by slowly adding ice-cold methanol (C. Roth; #7342.1) to a final concentration of 90% and incubating for 30 min. Cells were left overnight in methanol at −20° C. and stained on the next day by incubation for 20 min on ice in PBS/2% BSA with specific antibodies recognizing the phosphorylated signaling effectors, phospho-Stat3 (Tyr705) (D3A7) XP rabbit mAb (Cell Signaling; #9145); phospho-Stat5 (Tyr694) (C1105) rabbit mAb (Cell Signaling; #9359); phospho AKT (Thr308) (244F9) rabbit mAb (Cell Signaling; #4056S), and phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) mouse mAb (Cell Signaling; #9106), or the respective Alexa Fluor 488-conjugated isotype control antibody, anti-mouse IgG (H+L) F(ab′)2 fragment (Cell Signaling; #4408) or goat anti-rabbit IgG H+L (Abcam; #ab150077). Thereafter, cells were washed twice in ice-cold PBS/2% BSA and analyzed by FACS. The background-corrected fluorescence signal was distinguished from the corresponding phosphorylated proteins by subtracting the fluorescence signal of the appropriate isotype control, estimated at each time point of stimulation, from the specific phospho-protein signal.
Example 14: Neutrophils Generated from Design-Treated HSPCs are Functional
[0574] To test whether the neutrophils differentiated by our designs can execute neutrophil-specific functions such as production of reactive oxygen species (ROS) and phagocytosis, the inventors evaluated in vitro activation of neutrophils generated from Boskar3- and Boskar4-treated HSPCs in liquid culture for 14 days. For that, cells were seeded at a density of 1×10.sup.5 cells/mL with or without 10 nM tMLP (Sigma, #F3506) and incubated for 30 min at 37° C. and 5% CO.sub.2. The level of hydrogen peroxide (H.sub.2O.sub.2), a reactive oxygen species (ROS), was measured with the ROS-Glo H.sub.2O.sub.2 Assay kit (Promega, #G8820) according to the manufacturer's protocol. The inventors first assessed H.sub.2O.sub.2 levels in N-Formylmethionyl-leucyl-phenylalanine (fMLP)-activated neutrophils and detected even higher ROS levels in Boskar-generated neutrophils compared to rhG-CSF-stimulated samples (
[0575] Methods:
[0576] Granulocytes from day 14 of liquid culture differentiation were cultured in RPMI 1640 medium supplemented with 0.5% BSA and pHrodo Green E. coli Bioparticles Conjugate (Essen Bio; #4616) according to the manufacturer's protocol (Essen Bio) at 37° C. and 5% CO2. Briefly, 1×10.sup.4 cells were seeded in 90 μl medium, and 10 μg of Bioparticles were added to a final volume of 100 μl. The cells were monitored for 8 h in an IncuCyte S3 Live-Cell Analysis System (Essen Bio) with a 10× objective. The analysis was conducted in IncuCyte S3 Software.
Example 15: The Designed Proteins Induce Myeloid Differentiation of HSPCs in Mice
[0577] The inventors next evaluated the effects of the designed proteins on the proliferation and myeloid differentiation of HSPCs in mice. The inventors treated C57BL/6 mice with rhG-CSF or G-CSF designs, Boskar3 and Boskar4 at a concentration of 300 μg/kg by intraperitoneal injection (i.p.) every second day for a total of three injections. Mice in the control group were treated with PBS using the same treatment scheme. Two days after the third injection, the number of CD11b+ myeloid cells and of Gr-1+ 311 neutrophilic granulocytes in the bone marrow of treated mice was evaluated. The inventors found that treatment of mice with rhG-CSF, Boskar3, or Boskar4 induces production of myeloid cells and neutrophils, as compared to the control PBS-treated group (
[0578] Methods:
[0579] C57BL/6 mice (The Jackson Laboratory) were maintained under pathogen-free conditions in the research animal facility of the University of Tübingen, according to German federal and state regulations (Regierungspräsidium Tübingen, K3/17). Mice were treated with intraperitoneal injections (i.p.) of rhG-CSF, Boskar3, or Boskar4 at a concentration of 300 μg/kg every second day for a total of three injections. Mice were sacrificed 2 days after the last injection. Mice in the control group were treated with PBS using the same schema. Bone marrow cells were isolated by flushing with a 22 G syringe, and filtered through a 0.45 μm cell strainer prior to counting and staining for flow cytometry analyses. For the analysis of Gr-1+ or CD11b+ myeloid cells, 0.5×10.sup.6 cells were transferred into FACS tubes and washed once with FACS buffer. Phycoerythrin (PE)-Cyanine7-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (clone RB6-8C5; eBioscience) or PE-conjugated anti-mouse CD11 b antibody (clone M1/70; BioLegend) was added to a final concentration of 1-5 μg/ml according to the manufacturer's instructions, and cells were incubated in the dark at 4° C. for 30 min. Thereafter, cells were washed twice with ice-cold FACS buffer. All centrifugation steps were conducted at 400×g, 4° C. for 5 min. Samples were measured on a LSR II cytometer and analyzed using BD FACSDiva software. For all FACS analyses, vital mononuclear cells were selected, and doublets were excluded based on scatter characteristics.
REFERENCES REFERRED TO HEREIN ABOVE
[0580] [1] Kinch, M. S., An overview of FDA-approved biologics medicines. Drug Discovery Today, 2015. 20(4): p. 393-398. [0581] [2] Kintzing, J. R., M. V. Filsinger Interrante, and J. R. Cochran, Emerging Strategies for Developing Next-Generation Protein Therapeutics for Cancer Treatment. Trends in Pharmacological Sciences, 2016. 37(12): p. 993-1008. [0582] [3] Zídek, Z., P. Anzenbacher, and E. Kmoničková, Current status and challenges of cytokine pharmacology. British Journal of Pharmacology, 2009. 157(3): p. 342-361. [0583] [4] Platanias, L. C., Mechanisms of type-I- and type-II-interferon-mediated signalling. Nature Reviews Immunology, 2005. 5: p. 375. [0584] [5] Dale, D. C., et al., Review: Granulocyte Colony-Stimulating Factor—Role and Relationships in Infectious Diseases. The Journal of Infectious Diseases, 1995. 172(4): p. 1061-1075. [0585] [6] Dale, D. C., et al., A systematic literature review of the efficacy, effectiveness, and safety of filgrastim. Supportive Care in Cancer, 2018. 26(1): p. 7-20. [0586] [7] Kuwabara, T., S. Kobayashi, and Y. Sugiyama, Pharmacokinetics and Pharmacodynamics of a Recombinant Human Granulocyte Colony-Stimulating Factor. Drug Metabolism Reviews, 1996. 28(4): p. 625-658. [0587] [8] Arvedson, T., J. O'Kelly, and B.-B. Yang, Design Rationale and Development Approach for Pegfilgrastim as a Long-Acting Granulocyte Colony-Stimulating Factor. Biodrugs, 2015. 29(3): p. 185-198. [0588] [9] Bishop, B., et al., Reengineering Granulocyte Colony-stimulating Factor for Enhanced Stability. Journal of Biological Chemistry, 2001. 276(36): p. 33465-33470. [0589] [10] Miyafusa, T., et al., Backbone Circularization Coupled with Optimization of Connecting Segment in Effectively Improving the Stability of Granulocyte-Colony Stimulating Factor. ACS Chemical Biology, 2017. 12(10): p. 2690-2696. [0590] [11] Vanz, A. L. S., et al., Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization. Microbial Cell Factories, 2008. 7(1): p. 13. [0591] [12] Zink, T., et al., Structure and Dynamics of the Human Granulocyte Colony-Stimulating Factor Determined by NMR Spectroscopy. Loop Mobility in a Four-Helix-Bundle Protein. Biochemistry, 1994. 33(28): p. 8453-8463. [0592] [13] Hill, C. D., et al., The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. Proc Natl Acad Sci USA, 1993. 90(11): p. 5167-5171. [0593] [14] Schneider, A., et al., The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis. J Clin Invest, 2005. 115(8): p. 2083-2098. [0594] [15] England, T. J., et al., Granulocyte-Colony Stimulating Factor (G-CSF) for stroke: an individual patient data meta-analysis. Sci Rep, 2016. 6: 36567. [0595] [16] Sanchez-Ramos, J., et al., Pilot study of granulocyte-colony stimulating factor for treatment of Alzheimer's disease. J Alzheimers Dis, 2012. 31(4): p. 843-855. [0596] [17] Altschul, S. F., et al., Basic local alignment search tool. J Mol Biol, 1990. 215(3): p. 403-410. [0597] [18] Carter, C. R. D., et al., The significance of carbohydrates on G-CSF: differential sensitivity of G-CSFs to human neutrophil elastase degradation. Journal of Leukocyte Biology, 2004. 75(3): p. 515-522. [0598] [19] μl Ouriaghli, F., et al., Neutrophil elastase enzymatically antagonizes the in vitro action of G-CSF: implications for the regulation of granulopoiesis. Blood, 2003. 101(5): p. 1752. [0599] [20] Plaxco, K. W., et al., Contact order, transition state placement and the refolding rates of single domain proteins. J Mol Biol, 1998, 277(4): p. 985-994. [0600] [21] Liles, W. C., Augmented mobilization and collection of CD34+ hematopoietic cells from normal human volunteers stimulated with granulocyte colony-stimulating factor by single administration of AMD3100, a CXCR-4 antagonist. Transfusion, 2005, 45: p. 295-300. [0601] [22] Flomemberg, N., et al., The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood, 2005, 106: p. 1867-1874. [0602] [23] Broxmeyer, H. E., et al., Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR-4 antagonist. J Exp Med, 2005, 201: p. 1307-1318. [0603] [24] Devine, S. M., et al., A pilot study evaluating the safety and efficacy of AMD3100 for the mobilization and transplantation of HLA-matched sibling donors hematopoietic stem cells in patients with advanced hematological malignancies. Blood, 2005, 106: p. 299-304. [0604] [25] Raso, S. W., et al., Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state. Protein Science, 2005, 14(9): p. 2246-2257. [0605] [26] Young, D. C., et al., Characterization of the receptor binding determinants of granulocyte colony stimulating factor. Protein Sci, 2997, 6(6): p. 1228-1236 [0606] [27] Layton, J. E., et al., Interaction of Granulocyte Colony-stimulating Factor (G-CSF) with its receptor: evidence that Glu.sup.19 of G-CSF interacts with Arg.sup.288 of the receptor. J Biol Chem, 1999, 274(25): p. 17445-17451. [0607] [28] Silva, D. A., et al., De novo design of potent and selective mimics of IL-2 and IL-15. Nature, 2019, 565, p. 186-191. [0608] [29] Jones, D. T., Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol, 1999, 292, p. 195-202. [0609] [30] Yang, Y., et al., SPIDER2: A Package to Predict Secondary Structure, Accessible Surface Area, and Main-Chain Torsional Angles by Deep Neural Networks. Methods Mol Biol, 2017, 1484, p. 55-63. [0610] [31] Wang, S., et al., DeepCNF-SS: Protein Secondary Structure Prediction Using Deep Convolutional Neural Fields. Sci Rep, 2016, 6, 18962. [0611] [32] Lupas, A., et al., Predicting coiled coils from protein sequences. Science, 1991, 252, p. 1162-1164. [0612] [33] Czekanska, E. M., Assessment of cell proliferation with resazurin-based fluorescent dye. Methods Mol Biol, 2011, 740, p. 27-32. [0613] [34] Kabsch, W., A discussion of the solution for the best rotation to relate two sets of vectors. Acta Cryst, 1978, 34, p. 827-828. [0614] [35] Skokowa, J., et al., Neutrophil elastase is severely down-regulated in severe congenital neutropenia independent of ELA2 or HAX1 mutations but dependent on LEF-1. Blood, 2009, 114, p. 3044-3051. [0615] [36] Velázquez-Campoy, A., et al., Isothermal Titration Calorimetry. Current Protocols in Cell Biology, 2004, 23, 17.8.1-17.8.24. [0616] [37] ElGamacy, M., et al., An Interface-Driven Design Strategy Yields a Novel, Corrugated Protein Architecture, ACS Synthetic Biology, 2018, 7 (9), 2226-2235. [0617] [38] Heinzelmann, P., et al., pH responsive granulocyte colony-stimulating factor variants with implications for treating Alzheimer's disease and other central nervous system disorders. Protein engineering, design & selection: PEDS, 2015. 28(10), 481-489. [0618] [39] Mine, S., et al., Thermodynamic Analysis of the Activation Mechanism of the GCSF Receptor Induced by Ligand Binding. Biochemistry, 2004. 43(9), 2458-2464. [0619] [40] Luo, P., et al., Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening. Protein Sci, 2002. 11(5), 1218-1226.
[0620] The application text refers to the following tables:
TABLE-US-00001 TABLE 1 Amino acid substitutions Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine; Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Tyr Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu Norleucine
TABLE-US-00002 TABLE 2 Sequence identities of the protein variants of the invention with human G-CSF Sequence identity with G- Highest local CSF over the sequence identity whole length Protein design with G-CSF of the protein Boskar_1 53 identical residues over 119 43% (SEQ ID NO: 2) residues of Boskar_1 (45% identity) Boskar_2 52/119 42% (SEQ ID NO: 3) (44%) Boskar_3 55/119 45% (SEQ ID NO: 4) (46%) Boskar_4 51/119 42% (SEQ ID NO: 5) (43%) Moevan 15/22(68%) 13% (SEQ ID NO: 6) Moevan_es1.1 14/22(64%) 12% (SEQ ID NO: 7) Moevan_es1.2 14/22(64%) 12% (SEQ ID NO: 8) Moevan_es1.3 14/22(64%) 12% (SEQ ID NO: 9) Moevan_ea1.1 12/22(55%) 10% (SEQ ID NO: 10) Moevan_ea1.2 12/22(55%) 10% (SEQ ID NO: 11) Moevan_ea1.3 12/22(55%) 10% (SEQ ID NO: 12) Moevan_ea1.4 12/22(55%) 10% (SEQ ID NO: 13) Moevan_ea2.5 11/18(61%) 13% (SEQ ID NO: 20) Moevan_ea2.6 11/18(61%) 12% (SEQ ID NO: 21) Moevan_ea2.7 11/19 12% (SEQ ID NO: 22) (58%) Sohair 11/23 7% (SEQ ID NO: 14) (48%) Sohair_esa1.1 No significant similarity found (SEQ ID NO: 15) Sohair_esa1.2 3/5(60%) 4% (SEQ ID NO: 16) Sohair_esa1.3 8/20(40%) 7% (SEQ ID NO: 17) Sohair_esa2.4 9/20(45%) 8% (SEQ ID NO: 23) Sohair_esa2.5 8/19(42%) 7% (SEQ ID NO: 24) Sohair_esa2.6 No significant similarity found (SEQ ID NO: 25) Disohair_1 11/23 15% (SEQ ID NO: 18) (48%) Disohair_2 11/23 15% (SEQ ID NO: 19) (48%)
TABLE-US-00003 TABLE 3 Amino acid residues involved in α-helices according to design models and G-CSF crystal structure (2D9Q). Protein Total design Helix 1 Helix 2 Helix 3 Helix 4 length G-CSF 11-37 74-90 101-122 143-171 174 Boskar_4 2-22 27-53 60-87 92-116 119 Moevan 3-33 36-63 71-93 99-117 118 Sohair 4-37 41-75 82-114 119-152 154 Disohair_2 4-37 41-75 4-37 41-75 76 Bika1 2-32 39-62 2-32 39-62 64
TABLE-US-00004 TABLE 4 Absolute contact orders of protein variants Protein design Absolute Contact Order G-CSF 18.60 Boskar_4 17.84 Moevan 9.42 Sohair 4.53 Disohair_2 4.53
TABLE-US-00005 TABLE 5 Amino acid sequences and EC50 for activating the proliferation of NFS-60 cells. The residues highlighted in grey are involved in the binding to the G-CSF receptor. NFS-60 EC50 Sequence (ng/mL) >boskar_1 (SEQ ID NO: 2) 2173 AALAAELAEIYKGLAEYQARLQSLEGISPELGPALDALRL VA
FA TTLAQAMEEKKTNLPQSFLL
AL
I
IQA
AAALREKLAATYTG TDRAAAAVEIAAQLEAFLEKAYEILRHLAAA >boskar_2 (SEQ ID NO: 3) 3225 AALAAELAEIMKGLQEYQARLKSLEGISPELGPALDALRL
MA
FA TTMAQMMEENPSDLPQSFLL
AL
I
IQA
AAALREKLAATYP NSQRAAAAVEIAAQLEAFLEKAYQILRHLAAA >boskar_3 (SEQ ID NO: 4) 768 AALAAVLAEIYKGLAEYQARLQSLEGISPELGPALDALRL
VA
FA TTIAQAMEENKGPLPQSFLL
AL
I
IQA
AAALREKLAATYPSS QRAAAAVEIAAQLEAFLEKAYEILRHLAAA >boskar_4 (SEQ ID NO: 5) 27 AALAAALAEIYKGLAEYQARLKSLEGISPELGPALDALRL
MA
FA TTMAQAMEEGLDSLPQSFLL
AL
I
IQA
AAALREKLAATYKG NDRAAAAVEIAAQLEAFLEKAYQILRHLAAA >moevan (SEQ ID NO: 6) 356 MEAAAAARDESAYL
LQ
M
IDA
AAALSETRTIEELDTFKL
VA
FV TTVVQLAEELEHRFGRNRRGRTEIYKIVKEVDRKLLDLTDAVLAKEKKGE DILNMVAEIKALLINIYK >disohair_1 (SEQ ID NO: 18) 2375 MTSDYIIEQIQRKQEEARL
VE
ME
LEEVKEASKRGVSSDQLLNLIL
L A
IITTLIQIIEESNEAIKELIKNQ >disohair_2 (SEQ ID NO: 19) 396 MTSDYIIEQIQRKQEEARL
VE
E
LEAVKEASKRGVSSDQLLNLIL
L A
IITTLIQIIEESNEAIKELIKNQ >sohair (SEQ ID NO: 14) 5053 MTSDYIIEQIQRKQEEARL
VE
ME
LEAVKEASKRGVSSDQLLNLIL
L A
IITTLIQIIEESNEAIKELIKNQKGPTSDYIIEQIQRDQEEARKKVEEAEER LERVKEASKRGVSSDQLLDLIRELAEIIEELIRIIRRSNEAIKELIKNQ >csf_2d9q|WILD_TYPE G-CSF 0.055 MSSLPQSFLL
CL
V
IQG
GAALQEKLCATYKLCHPEELVLLGHSL GIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLD TLQL
VA
FATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVL VASHLQSFLEVSYRVLRHLAQP
TABLE-US-00006 TABLE 6 Comparison of the protein designs with recombinant human G-CSF Chain length Yield Thermal (amino (per litre Contact- stability Protease Protein acids) culture) Solubility order (T.sub.m) resistance Moevan 118 >15 mg/L <5 mg/mL 9.42 74° C. − (SEQ ID NO: 6) DiSohair_2 76 >30 mg/L >30 mg/mL 4.53 >100° C. +++ (SEQ ID NO: 19) Boskar_4 119 >30 mg/L >15 mg/mL 17.84 >100° C. +++ (SEQ ID NO: 5) rhG-CSF 174 3.2 mg/L <4 mg/mL 18.60 57° C. − (SEQ ID NO: 1)
TABLE-US-00007 TABLE 7 Amino acid sequences and EC50 for activating the proliferation of NFS-60 cells The residues highlighted in grey are involved in the binding to the G-CSF receptor. NFS-60 EC.sub.50 Sequence (ng/mL) >boskar4_t2 (SEQ ID NO: 26) 4.2 AALAAALAEIYKGLAEYQARLKSLEGISPELGPALDALRL M
FA TTMAQAMEEGLDSLPQSFLL
AL
I
IQA
AAALREKLAATYKG NDRAAAAVEIAAQLEAFLEKAYQILRHLAAA GGGGSSGGGGSSGGGGSSGGGGSS AALAAALAEIYKGLAEYQARLKSLEGISPELGPALDALRL
MA
FA TTMAQAMEEGLDSLPQSFLL
AL
I
IQA
AAALREKLAATYKG NDRAAAAVEIAAQLEAFLEKAYQILRHLAAA >boskar4_st2 (SEQ ID NO: 27) 0.202 AALAAALAEIYKGLAEYQARLKSLEGISPELGPALDALRL
MA
FA TTMAQAMEEGLDSLPQSFLL
AL
I
IQA
AAALREKLAATYKG NDRAAAAVEIAAQLEAFLEKAYQILRHLAAA GGGGSS AALAAALAEIYKGLAEYQARLKSLEGISPELGPALDALRL
MA
FA TTMAQAMEEGLDSLPQSFLL
AL
I
IQA
AAALREKLAATYKG NDRAAAAVEIAAQLEAFLEKAYQILRHLAAA >boskar4_15rl (SEQ ID NO: 28) 48.5 AALAAALAEIYKGLAEYQARLKSLEGISPELGPALDALRL
MA
FA TTMAQAME GGGGSGGGGSGGGGS QSFLL
AL
I
IQA
AAALREKLAATYKGNDRAAAAVEIAAQLE AFLEKAYQILRHLAAA >moevan_t2 (SEQ ID NO: 29) 47.1 EAAAAARDESAYL
LQ
M
IDA
AAALSETRTIEELDTFKL
VA
FVT TVVQLAEELEHRFGRNRRGRTEIYKIVKEVDRKLLDLTDAVLAKEKKGED ILNMVAEIKALLINIYK GGGGSSGGGGSSGGGGSSGGGGSS EAAAAARDESAYL
LQ
M
IDA
AAALSETRTIEELDTFKL
VA
FVT TVVQLAEELEHRFGRNRRGRTEIYKIVKEVDRKLLDLTDAVLAKEKKGED ILNMVAEIKALLINIYK >moevan_st2 (SEQ ID NO: 30) 8.89 EAAAAARDESAYL
LQ
M
IDA
AAALSETRTIEELDTFKL
VA
FVT TVVQLAEELEHRFGRNRRGRTEIYKIVKEVDRKLLDLTDAVLAKEKKGED ILNMVAEIKALLINIYK GGGGSS EAAAAARDESAYL
LQ
M
IDA
AAALSETRTIEELDTFKL
VA
FVT TVVQLAEELEHRFGRNRRGRTEIYKIVKEVDRKLLDLTDAVLAKEKKGED ILNMVAEIKALLINIYK >sohair2_15r (SEQ ID NO: 31) 228 MTSDYIIEQIQRKQEEARL
VE
E
LEAVKEASKRGVSSDQLLNLIL
L A
IITTLIQIIEESNEAIKELIKNQ GGGGSGGGGSGGGGS DYIIEQIQRKQEEARLK
E
E
LEAVKEASKRGVSSDQLLNLIL
LA
II TTLIQIIEESNEAIKELIKNQ >bika1 (SEQ ID NO: 32) 63 SKEVLEQSLFL
LD
V
LLA
IHAIKIDRITGNMDKQKLDTAYL
VA
IE TTLYQLIEVSH >bika2 (SEQ ID NO: 33) 98 SKEVLEQSLFL
LD
V
LLA
IHAIKIDRITGNMDKQKLDTLYL
VA
IE TTLYQLIEVSH
TABLE-US-00008 TABLE 8 CoMAND ensemble structure statistics R-factors.sup.1 R.sub.ens 0.33 R.sub.mean 0.36 ± 0.11 Coverage.sup.2 89/115 Structure Quality Bonds (Å × 10.sup.−3) 1.94 ± 0.10 Angles (°) 0.52 ± 0.02 Impropers (°) 0.83 ± 0.12 Ramachandran Map (%) 97.2/2.0/0.8 Sidechain Regularity (%) 98.1 Clash Score 0 Number of Structures 12 Ordered Residues 2-53, 60-118 Backbone Heavy Atom 1.34 ± 0.44 All Heavy Atom 1.67 ± 0.42 .sup.1R-factors averaged across the sequence (±SD) are given for the final ensemble compiled by global optimization (Rmean). .sup.2The coverage refers to the number of residue used in factorization analysis, versus the total number expected from the sequence, excluding purification tags. .sup.3Determined by MOLPROBITY. The Ramachandran statistic lists the percentage of residues in favored/allowed/disfavored regions of the map (percentiles 98.0/99.8/>99.8). Sidechain regularity lists the percentage in allowed sidechain rotamers (percentile 98.0). The clash score lists steric overlaps > 0.4 Å per 1000 atoms. .sup.4The RMSD to the average structure based on superimposition over ordered residues, as defined in the table.
TABLE-US-00009 TABLE 9 SPR binding parameters 1:1 binding model.sup.1 Analyte k.sub.a (M.sup.−1s.sup.−1) k.sub.d (s.sup.−1) K.sub.d (M) X.sup.2 (R.U..sup.2) rhG-CSF 7.9 × 10.sup.5 2.8 × 10.sup.−4 3.6 × 10.sup.−10 4.3 6.4 × 10.sup.5 2.6 × 10.sup.−4 4.1 × 10.sup.−10 2.8 Boskar3 1.3 × 10.sup.5 1.5 × 10.sup.−3 1.2 × 10.sup.−8 2.0 1.2 × 10.sup.5 1.9 × 10.sup.−3 1.6 × 10.sup.−8 1.7 Boskar4 5.2 × 10.sup.5 4.5 × 10.sup.−3 8.5 × 10.sup.−9 2.1 8.9 × 10.sup.5 1.5 × 10.sup.−3 1.7 × 10.sup.−9 1.6 2.sup.nd order kinetics model.sup.2 Analyte k.sub.a (M.sup.−1s.sup.−1) k.sub.d (s.sup.−1) K.sub.d (M) X.sup.2 (R.U..sup.2) rhG-CSF 1.1 × 10.sup.4 9.0 × 10.sup.−4 8.2 × 10.sup.−8 2.5 1.0 × 10.sup.4 8.4 × 10.sup.−4 8.8 × 10.sup.−8 2.3 Boskar3 7.8 × 10.sup.2 4.7 × 10.sup.−3 6.0 × 10.sup.−6 1.0 2.2 × 10.sup.3 5.8 × 10.sup.−3 2.6 × 10.sup.−6 1.1 Boskar4 1.5 × 10.sup.3 9.5 × 10.sup.−3 6.4 × 10.sup.−6 1.8 1.4 × 10.sup.3 8.0 × 10.sup.−3 5.8 × 10.sup.−6 1.0 .sup.1Analysis was done using the Biacore ×100 evaluation software v.2.0.1. .sup.2Analysis was done using a second-order model.
TABLE-US-00010 TABLE 10 SPR binding parameters 1:1 binding model.sup.1 Analyte k.sub.a (M.sup.−1s.sup.−1) k.sub.d (s.sup.−1) K.sub.d (M) X.sup.2 (R.U..sup.2) rhG-CSF (3.0 ± 0.3) × (4.9 ± 2.8) × (1.1 ± 1.6) × 4.9 10.sup.5 10.sup.−4 10.sup.−9 Moevan (2.9 ± 0.4) × (5.9 ± 0.4) × (2.1 ± 0.4) × 0.7 10.sup.5 10.sup.−3 10.sup.−8 Moevan_t2 (3.1 ± 0.3) × (3.0 ± 3.2) × (1.1 ± 1.1) × 0.1 10.sup.5 10.sup.−4 10.sup.−9 diSohair2 (2.1 ± 0.1) × (9.5 ± 0.1) × (4.5 ± 0.3) × 1.9 10.sup.3 10.sup.−3 10.sup.−6 .sup.1Analysis was done using the Biacore ×100 evaluation software v.2.0.1.
[0621] While aspects of the invention are illustrated and described in detail in the Figures and in the foregoing tables and description, such Figures, tables and description are to be considered illustrative or exemplary and not restrictive. Also reference signs in the claims should not be construed as limiting the scope.
[0622] It will also be understood that changes and modifications may be made by those of ordinary skill within the scope and spirit of the claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above. It is also to be noted in this context that the invention covers all further features shown in the figures individually, although they may not have been described in the previous or following description. Also, single alternatives of the embodiments described in the figures and the description and single alternatives of features thereof can be disclaimed from the subject matter according to aspects of the invention.
[0623] Whenever the word “comprising” is used in the claims, it should not be construed to exclude other elements or steps. It should also be understood that the terms “essentially”, “substantially”, “about”, “approximately” and the like used in connection with an attribute or a value may define the attribute or the value in an exact manner in the context of the present disclosure. The terms “essentially”, “substantially”, “about”, “approximately” and the like could thus also be omitted when referring to the respective attribute or value. The terms “essentially”, “substantially”, “about”, “approximately” when used with a value may mean the value ±10%, preferably ±5%.
[0624] A number of documents including patent applications, manufacturer's manuals and scientific publications are cited herein. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.