Fusion Proteins Comprising Cytokines and Scaffold Proteins

Abstract

The present invention relates to the field of structural biology. More specifically, the present invention relates to novel fusion proteins, their use and methods in three-dimensional structural analysis of macromolecules, such as X-ray crystallography and high-resolution cryo-EM, and their use in structure-based drug design and screening. Even more specifically, the present invention relates to functional fusion proteins of a cytokine and a scaffold protein, where the scaffold is a folded protein that interrupts the topology of the cytokine to form a rigid fusion protein that retains its receptor binding and activation ability. More specifically, functional fusion proteins based on chemokines and interleukins, and their production and use are disclosed herein.

Description

[Technical field]

[0001] The present invention relates to the field of structural biology. More specifically, the present invention relates to novel fusion proteins, their use and methods in three-dimensional structural analysis of macromolecules, such as X-ray crystallography and high resolution cryo-EM, and their use in structure-based drug design and screening. Even more specifically, the present invention relates to functional fusion proteins of cytokines and scaffold proteins, where the scaffold is a folded protein that blocks the topology of the cytokine to form a rigid fusion protein that retains its receptor binding and activation capabilities. More specifically, functional fusion proteins based on chemokines and interleukins, and their production and use are disclosed herein. [Background technology]

[0002] The 3D structure analysis of many proteins and complexes in certain conformational states remains challenging. Macromolecular X-ray crystallography inherently has several drawbacks, such as the prerequisite of high-quality purified protein, the need for relatively large amounts of protein, and the preparation of diffraction-quality crystals. The application of crystallization chaperones in the form of antibody fragments or other proteins has been proven to facilitate obtaining well-ordered crystals by minimizing the conformational heterogeneity of the target. In addition, chaperones can provide initial, model-based phase information (Koide, 2009). Furthermore, single-particle cryo-electron microscopy (cryo-EM) has recently been developed as an alternative, versatile technique for the structural analysis of macromolecular complexes at atomic resolution (Nogales, 2016). Although instruments and methods for data analysis are steadily improving, the best achievable solution for 3D reconstruction depends mainly on the homogeneity of a given sample and the ability to repeatedly refine the orientation parameters of each individual particle to high accuracy. Preferential particle orientation due to surface properties of polymers that preferentially attach certain regions to the air-water interface or substrate support is a recurring problem in cryo-EM, and thus, in this embodiment as well, we are still searching for tools such as next-generation chaperones to overcome these hurdles.

[0003] Cytokines are a class of small proteins (5-20 kDa) that act as cell signaling molecules at picomolar or nanomolar concentrations to regulate inflammation and modulate cellular activities such as migration, growth, survival, and differentiation. Cytokines are a very large and diverse group of pro- or anti-inflammatory factors that are classified into families based on the homology of their structure or the homology of their receptors. Cytokines may include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, hormones, or growth factors. Interleukins (ILs) form a group of cytokines that have complex immunoregulatory functions including cell proliferation, maturation, migration, and adhesion, and play a key role in immune cell differentiation and activation. ILs can also have pro- and anti-inflammatory actions and are under constant pressure to evolve due to the ongoing competition between the host immune system and infectious organisms. For this reason, ILs have undergone significant evolution, which results in little amino acid conservation between orthologous proteins, complicating the organization of the gene family. However, crystallography data and identification of common structural motifs have led to a classification into four major groups, including genes encoding IL1-like cytokines, class I helical cytokines (IL4-like, gamma chain and IL6 / 12-like), class II helical cytokines (IL10-like and IL28-like), and IL17-like cytokines, with IL17F being structurally unrelated to other IL subfamilies and forming a cysteine ​​knot fold.

[0004] Chemokines are a group of small secreted globular proteins within the cytokine family whose general function is to induce cell migration. Binding of a cytokine or chemokine ligand to its cognate receptor results in receptor activation, which in turn triggers a cascade of signaling events that regulate a variety of cellular functions, including cell adhesion, phagocytosis, cytokine secretion, cell activation, cell proliferation, cell survival and death, apoptosis, angiogenesis, and proliferation.

[0005] Chemokines accumulate in gradients on the cell surface and extracellular matrix and are interpreted as directional signals by chemokine receptors on migrating cells. Most chemokine receptors are seven-transmembrane (7TM) G protein-coupled receptors (GPCRs) that activate Gαi-dependent intracellular pathways in response to chemokine binding. Some chemokine receptors transport or clear chemokines via other mechanisms and are therefore called atypical chemokine receptors (ACKRs). These "chemotactic cytokines" are involved in the chemoattraction of leukocytes and the migration of immune cells to locations throughout the body. The chemokine system is involved in many disease areas, including inflammatory pathologies such as asthma, atherosclerosis, and rheumatoid arthritis, and also in autoimmune diseases. Cytokines and chemokines play important roles in mediating neuroinflammation and neurodegeneration in various types of inflammatory neurodegenerative diseases, including bacterial meningitis, brain abscess, Lyme disease neuroborreliosis, and HIV encephalitis (for review, see Ramesh et al., 2013). Understanding the system is therefore crucial for the selection of appropriate therapeutic targets and resulting specificity.

[0006] Chemokines are small proteins of approximately 7-12 kDa and are classified into four subfamilies based on a characteristic pattern of cysteine ​​residues near the amino terminus of the mature ligand (CC, CXC, CX3C, and C). All chemokines exhibit homologous tertiary structures and interact with cell surface glycosaminoglycans (GAGs) as well as chemokine receptors in different oligomerization states. To date, approximately 45 human chemokines and 22 chemokine receptors are known, and chemokines within the same subfamily often bind to multiple receptors of the same class. Although chemokines appear in dimeric forms, it is their monomeric forms that bind and activate chemokine receptors. The two-site model of receptor binding and activation involves the N-terminus of the chemokine, which is essential for receptor activation, and the chemokine core domain, which mediates receptor binding. Natural chemokines have different receptor specificities, and variants of known chemokines have been shown to define different conformational states of these receptors, resulting in different signaling and responses. This allows some chemokines to act as agonists of a given receptor, while others may act as antagonists or inverse agonists. To fully understand this recognition and activation mechanism, high-resolution structures of chemokines or variants complexed with intact receptors are required. For example, structural studies of several CCL5 (or RANTES) variants, known as agonists and antagonists, are under examination for their potential as antibacterial agents in protecting against HIV (Kufareva et al., 2015). Some structures of chemokines are known, and structures have been solved for more tractable GPCRs that are reproduced as soluble complexes (β2-adrenergic receptor, rhodopsin). However, structural insight into chemokine / receptor complexes and interactions is still limited, and the conformational flexibility of the receptors as transmembrane proteins poses difficult problems.Crystal structures have been determined for the chemokine receptors CXCR4 and CCR5 GPCRs in complex with small molecules, and for CCR5 in complex with the antagonist chemokine variant 5P7-CCL5, CXCR4 in complex with the viral antagonist chemokine vMIP-II, and the viral receptor US28 in complex with human CX3CL1. Moreover, available crystal structures of GPCRs in complex with G-proteins and β-arrestins show no obvious notable differences in the conformation of the receptors when compared to each other, indicating that novel insights in ligand-receptor pairs are essential to assess their druggability (Proudfoot et al. 2015). Alternative methods of elucidating structural information, such as radiolysis footprinting, disulfide trapping, and mutagenesis, have been applied to map the structures of, for example, ACKR3:CXCL12 and ACKR3:small molecule complexes (Gustavsson et al., 2017). Such techniques will allow the incorporation, using molecular modeling, of dynamic regions revealed to be indecipherable by X-ray crystallography in homologous receptors, generating complete, cohesive, and experimentally driven models to expand existing knowledge of the assembly of receptor:chemokine and receptor:small molecule complexes. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Koide, 2009 [Non-Patent Document 2] Nogales, 2016 [Non-Patent Document 3] Ramesh et al., 2013 [Non-Patent Document 4] Kufareva et al., 2015 [Non-Patent Document 5] Proudfoot et al. 2015 [Non-Patent Document 6] Gustavsson et al., 2017 Summary of the Invention [Problem to be solved by the invention]

[0008] However, to explore novel pathways and discover novel mechanisms of ligand-induced conformational changes across GPCRs as well as other chemokine, interleukin, or "cytokine receptors," universal prototype chaperones are needed to facilitate X-ray crystallography or cryo-EM analysis of such complexes with their ligands, ligand analogs, or variants. [Means for solving the problem]

[0009] Summary of the Invention This application relates to the design and generation of novel functional fusion proteins and their use, for example, their role as next-generation chaperones in structural analysis. The fusion proteins described herein are based on the discovery that the expansion of cytokine ligands into rigid fusion proteins can facilitate structural analysis of ligand / receptor complexes in certain conformational states. Indeed, the present disclosure provides fusion proteins based on the assumption that the cytokine superfamily shares sequence similarity and exhibits structural homology and some disorder in their reciprocal receptor systems, but not functional similarity. Since cytokines are classified according to their structure, one can start from the similarity in structural elements within a subgroup of cytokines when designing a universal fusion scheme. Interleukins are a subgroup of cytokines, for example the IL-1 superfamily, that adopt a conserved signature β-trefoil fold that includes antiparallel β-strands arranged in a three-fold symmetric pattern with a conserved β-barrel hydrophobic core motif with significant flexibility in the loop regions. Chemokines are another subgroup of cytokines that exhibit a very similar basic tertiary structure, with the chemokine core domain comprising a β-sheet with at least three β-strands. The structural conservation of the subfamily makes cytokinins ideally a versatile approach and prototype for next-generation chaperones in the structural analysis of ligand / receptor complexes. The tertiary structure is homologous in these subfamilies, e.g., "IL-1 receptor-type interleukins," or "IL-1 family," which are used interchangeably herein, and chemokines, which have a conserved core that includes a secondary β-structure (β-sheet or β-barrel) that provides interconnection of their β-strands through exposed turns or loops, and thus the physical location in their core domain that is exposed and accessible for fusion with scaffold proteins can be applied as an example to form a ligand-integrating chaperone for the structural analysis of β-strand domain-containing cytokines in cytokine / receptor complexes in general.Interleukin-1 or chemokine ligands were used to construct rigid, larger ligands known as MegaKines™, and unexpectedly the enlarged ligand fusion proteins retained their receptor binding and activation capabilities. These novel functional fusion proteins provide a new route to capture receptors such as GPCRs in different conformational states and to facilitate their structural analysis. The novel fusions formed by rigidly inserting a scaffold protein into a cytokine core domain, such that it blocks the topology of this core domain of the cytokine without disturbing its folding or functionality, allow a new approach in structure-based drug discovery. The resulting functional fusion proteins are obtained via expression of genetic fusions between the cytokine and the scaffold protein (as demonstrated for chemokines and IL-1β), where the scaffold or a fragment thereof is designed to insert into the topology of the cytokine core domain. Surprisingly, the resulting novel fusion proteins are characterized by high rigidity in their fusion region and have been shown to unexpectedly retain their typical fold and functionality, i.e. they retained their binding affinity and, moreover, showed activation capabilities upon cytokine receptor binding. Indeed, genetic fusions formed between a cytokine's conserved core domain and a scaffold protein at accessible sites of exposed β-turns are selected by the skilled artisan so as not to interfere with or alter receptor binding. Thus, the present invention provides a novel and unique type of functional fusion protein by having perfectly selected sites in exposed β-turns or β-loops in the conserved core domain of a cytokine, e.g., the chemokine core domain, i.e., between β-strand β2 and β-strand β3, or in the IL-1β barrel core motif, i.e., between β-strand β6 and β-strand β7, allowing rigid, inflexible fusions with folded scaffold proteins that are not easy to design. The fusion proteins thereby provide novel tools to facilitate high-resolution cryo-EM and X-ray crystallography of chemokine ligand / receptor complexes by increasing mass and providing structural features.Thus, the design and generation of these next generation chaperones for structural analysis of cytokines, particularly chemokines or their variant ligands, or potentially any complex of interleukins, IL-1 or their variants with their receptors, allows for expanded ligands that add mass and / or defined features to the complex of interest to obtain high resolution structures without altering the conformational state. Thus, in practice, fusion proteins are useful as tools in structural analysis, as well as in structure-based drug design and screening, and are an added value for the discovery and development of novel biologics and small molecule agents.

[0010] A first aspect of the present invention relates to novel fusion proteins comprising a functional cytokine connected to a scaffold or fusion partner forming protein, said scaffold protein being a folded protein of at least 50 amino acids and binding to the cytokine at one or more accessible and thus surface exposed amino acid positions of said cytokine, thereby resulting in the blocking of the topology of said cytokine. Said fusion protein is further characterized in that it is functional, i.e. it retains its cytokine functionality compared to a cytokine ligand not fused to said scaffold protein. Another embodiment discloses a fusion protein of the present invention, in which the fusion of a scaffold protein with a cytokine protein results in the blocking of the primary topology of the cytokine, allowing the cytokine protein to retain its folding and typical tertiary structure compared to the folding of a cytokine ligand not fused to another protein. More specifically, the accessible amino acid positions are present in exposed regions of beta turns (β turns) or β loops interconnecting the conserved cytokine β-strand structures.

[0011] In certain embodiments of the invention, the fusion may be a direct fusion or a fusion created by a linker or linker peptide, said fusion site being perfectly designed to result in a rigid, inflexible fusion protein. Preferably, the linker comprises 5, 4, 3, or more preferably 2 amino acid residues, even more preferably 1 amino acid residue, or it may be a direct fusion (no linker).

[0012] Said fusion proteins with a cytokine or scaffold protein attached to a chemokine core domain at one or more accessible or exposed sites on the surface of the chemokine core domain are further characterized in that said accessible or exposed sites are not in regions involved or associated with receptor binding and receptor activation and retain their cytokine functionality in receptor binding and / or activation.

[0013] One embodiment of the present invention relates to a novel fusion protein, in which the cytokine is a functional chemokine connected to a scaffold or fusion partner forming protein, and the scaffold protein is attached to a chemokine core domain at one or more amino acid positions accessible and therefore exposed on the surface of the domain, thereby causing a blockage of the topology of the chemokine. The fusion protein is further characterized in that it retains its chemokine functionality compared to a chemokine ligand that is not fused to the scaffold protein. Another embodiment discloses a fusion protein of the invention, in which the fusion of a scaffold protein with a chemokine core domain results in a blockage of the primary topology of the chemokine core domain, allowing the chemokine core domain to retain its folding and typical tertiary structure compared to the folding of a chemokine ligand that is not fused to another protein. In one embodiment, the fusion protein comprises a chemokine core domain having an N-terminal loop, a β-sheet containing three β-strands, and a C-terminal helix. In certain embodiments, the exposed region in said chemokine core domain of the fusion protein specifically relates to the β-turn connecting β-strand β2 and β-strand β3, and thus the scaffold protein is inserted into the core domain at an accessible site present in the β-turn between these two β-strands.

[0014] An alternative embodiment relates to a fusion protein, wherein said cytokine is an interleukin, preferably an "IL-1 family" interleukin, and wherein said scaffold protein interrupts the topology of an interleukin β-barrel core motif at one or more accessible sites in the exposed β-turns of said β-barrel core motif. In a particular embodiment, the exposed region in said conserved β-barrel core motif of the fusion protein specifically relates to the β-turn connecting β-strand β6 and β-strand β7. Thus, the scaffold protein is inserted into the core motif at an accessible site present in the β-turn between these two β-strands.

[0015] In another embodiment of the invention, the scaffold protein used to generate the fusion protein is a circularly permuted protein, more specifically, a circular permutation can be made between the N-terminus and C-terminus of said scaffold protein. In certain embodiments, the circularly permuted scaffold protein is cleaved at another accessible site of said scaffold protein to provide a site for fusion to an accessible site of a chemokine core domain. Another embodiment of the invention relates to a fusion protein, in which the total molecular weight of the scaffold protein is at least 30 kDa.

[0016] A further aspect of the invention relates to a nucleic acid molecule encoding any of the fusion proteins described herein. Alternatively, in one embodiment, a chimeric gene is provided having at least a promoter, said nucleic acid molecule encoding a fusion protein, and a 3' terminal region containing a transcription termination signal. Another embodiment relates to an expression cassette encoding said fusion protein or comprising a nucleic acid molecule encoding said fusion protein. A further embodiment relates to a vector comprising said nucleic acid molecule encoding a fusion protein of the invention. In a particular embodiment, said vector is suitable for expression in E. coli, or an alternative host as provided herein, and suitable for yeast, phage, bacterial, or viral (surface) display. In another embodiment, a host cell comprising the fusion protein of the invention is disclosed. Alternatively, a host cell in which said fusion protein and a cytokine or chemokine receptor capable of binding to the cytokine portion of the fusion protein are co-expressed.

[0017] Another aspect of the invention relates to a complex comprising said fusion protein and a cytokine receptor. More specifically, it relates to a complex comprising a chemokine or interleukin receptor capable of binding to the cytokine portion of the fusion protein, or in particular the chemokine or interleukin portion of the fusion protein, and said fusion protein, said receptor protein specifically binding to said fusion protein. More specifically, said receptor protein binds to the cytokine portion or alternatively the chemokine or interleukin portion of the fusion protein, and even more specifically to a known receptor binding region of the fusion protein. In certain embodiments, the complex described herein comprises an activated receptor, which is activated when said receptor binds to the fusion protein at its cytokine receptor binding region, or in particular at its chemokine or interleukin receptor binding region.

[0018] Another aspect of the invention is a method for determining the three-dimensional structure of a cytokine receptor complex, comprising the steps of: (i) providing a fusion protein of the invention and a cytokine receptor (e.g., a chemokine / interleukin receptor) to form a complex, wherein the receptor protein specifically binds to the cytokine of the fusion protein (e.g., the chemokine or interleukin of the fusion protein, respectively), or alternatively providing a complex of the invention; and (ii) displaying the mixture or complex under conditions suitable for structural analysis; wherein the 3D structure of said ligand / receptor complex is determined at high resolution through said structural analysis.

[0019] Another aspect is a method of producing a functional fusion protein described herein, comprising: a. selecting a cytokine superfamily, e.g., a chemokine or an interleukin-like ligand, and a scaffold protein whose 3D structure reveals a folded protein of at least 10 kDa, wherein the cytokine has accessible sites in β-loops or β-turns exposed for interruption of the amino acid sequence without interrupting the primary topology of the conserved cytokine core domain; b. Designing a gene fusion construct in which a nucleic acid sequence is designed to encode a protein sequence, (i) the protein sequence of the cytokine ligand is interrupted at an amino acid position corresponding to a site between two β-strands of its conserved core domain structure that is a surface-exposed β-loop or β-turn; (ii) the most N-terminally blocked amino acid site of the cytokine (the C-terminus of the most N-terminal β-strand) is fused to the most N-terminally blocked site of the scaffold protein, and the most C-terminally blocked site of the cytokine (the N-terminus of the most C-terminal β-strand) is fused to the most C-terminally blocked site of the scaffold protein; Step, c. introducing the gene fusion construct into an expression system to obtain a fusion protein, wherein the chemokine is fused to a scaffold protein at two or more sites in its core domain. The present invention relates to a method comprising the steps of:

[0020] An alternative embodiment is a method of producing a fusion protein described herein, comprising: a. selecting chemokines and folded scaffold proteins with accessible loops or turns in their tertiary structure that are interrupted to create the protein sequence of the fusion protein without interrupting the primary topology of the chemokine or scaffold protein; b. Designing a gene fusion construct in which a nucleic acid sequence is designed to encode a protein sequence, (i) the protein sequence of the chemokine is interrupted by amino acids corresponding to an accessible site between β-strand β2 and β-strand β3 of the core domain; (ii) the scaffold protein is at least 10 kDa and is fused at its N-terminus and C-terminus to obtain a circularly permuted scaffold protein; (iii) the circularly permuted scaffold protein of ii) is further blocked in its amino acid sequence at accessible sites corresponding to exposed β-loops or β-turns that do not contain the amino acids fused in step ii); (iv) the C-terminal blocked portion of the chemokine of β-strand β2 is fused to the most N-terminal blocked amino acid residue, i.e., the N-terminus of the circularly permuted scaffold protein, and the N-terminal blocked portion of the chemokine of β-strand β3 is fused to the most C-terminal blocked amino acid residue, i.e., the C-terminus of the circularly permuted scaffold protein. Step, c. introducing the gene fusion construct into an expression system to obtain a fusion protein, wherein the chemokine is fused to a circularly permuted scaffold protein at two or more sites in its core domain. The present invention relates to a method comprising the steps of:

[0021] Another aspect relates to the use of the fusion protein or the nucleic acid molecule, vector, host cell or complex of the invention for structural analysis of a cytokine ligand / receptor protein, in particular the use of a fusion protein, wherein said cytokine receptor (or chemokine / interleukin / ··-receptor) protein is the protein bound to said fusion protein. In particular, one embodiment relates to the use of the fusion protein in structural analysis, including single particle cryo-EM or crystallography.

[0022] The drawings described are only schematic and non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. [Brief description of the drawings]

[0023] [Figure 1] Figure 1 is a diagram showing flexible fusion proteins compared to rigid chemokine chimeric proteins: (A) Flexible fusion or linker at the N- or C-terminus of a chemokine domain to a scaffold protein using only one direct fusion or linker, (B) Rigid fusion of a chemokine domain to a scaffold protein in which the chemokine domain is fused to the scaffold protein via at least two direct fusions or linkers connecting the chemokine domain to the scaffold. [Diagram 2] FIG. 2 is a diagram showing the operating principle of a chemokine fusion protein constructed from a circularly permuted variant of a scaffold protein inserted into the β-turn connecting β-strand β2 and β-strand β3 of a chemokine. The scheme shows how a chemokine can be grafted to a large scaffold protein via two peptide bonds or two short linkers connecting the chemokine domain to the scaffold. The scissors indicate which exposed turns must be cut in the chemokine and the scaffold. The dashed lines show how the chemokine and the remaining parts of the scaffold must be linked by the use of peptide bonds and short peptide linkers to construct a chemokine chimeric protein. [Diagram 3] Figure 3 shows a model 1 of a 50 kD 6P4-CCL5 fusion protein constructed from a circularly permuted variant of HopQ inserted into the β-turn connecting β-strand β2 and β-strand β3 of the 6P4-CCL5 chemokine. (A) A model of a chemokine fusion protein made by fusing the chemokine 6P4-CCL5 (top) with a circularly permuted variant of the adhesive domain of HopQ from H. pylori (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) A circularly permuted gene encoding the adhesive domain of type 1 HopQ from Helicobacter pylori strain G27 (bottom, PDB 5LP2, SEQ ID NO:2, c7HopQ) was inserted into the β-turn (β-turn β2-β3) connecting β-strand β2 and β-strand β3 of 6P4-CCL5 (top, PDB 5UIW, SEQ ID NO:1). (C) Amino acid sequence of the resulting chemokine chimeric protein (Mk6P4-CCL5 c7HopQ, SEQ ID NO:3). The sequence derived from the chemokine is shown in bold. The sequence derived from HopQ is in between. The C-terminal tag contains 6xHis and EPEA is underlined with a dashed line. [Figure 4]Figure 4 shows model 2 of a 50 kD 6P4-CCL5 fusion protein constructed from a circularly permuted variant of HopQ inserted into the β-turn connecting β-strand β2 and β-strand β3 of the 6P4-CCL5 chemokine. (A) Model of a chemokine fusion protein made by fusing the chemokine 6P4-CCL5 (top) with a circularly permuted variant of the adhesive domain of HopQ from H. pylori (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) A circularly permuted gene encoding the adhesive domain of type 1 HopQ from Helicobacter pylori strain G27 (bottom, PDB 5LP2, SEQ ID NO:2, c7HopQ) was inserted into the β-turn (β-turn β2-β3) connecting β-strand β2 and β-strand β3 of 6P4-CCL5 (top, PDB 5UIW, SEQ ID NO:1). (C) Amino acid sequence of the resulting chemokine chimeric protein (Mk6P4-CCL5 c7HopQ, SEQ ID NO: 4). The sequence derived from the chemokine is shown in bold. The sequence derived from HopQ is in between. The C-terminal tag contains 6xHis and EPEA is underlined with a dashed line. [Diagram 5]Figure 5 shows a model 3 of a 50 kD 6P4-CCL5 fusion protein constructed from a circularly permuted variant of HopQ inserted into the β-turn connecting β-strand β2 and β-strand β3 of the 6P4-CCL5 chemokine. (A) Model of a chemokine fusion protein made by fusing the chemokine 6P4-CCL5 (top) with a circularly permuted variant of the adhesive domain of HopQ from H. pylori (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) A circularly permuted gene encoding the adhesive domain of type 1 HopQ from Helicobacter pylori strain G27 (bottom, PDB 5LP2, SEQ ID NO:2, c7HopQ) was inserted into the β-turn (β-turn β2-β3) connecting β-strand β2 and β-strand β3 of 6P4-CCL5 (top, PDB 5UIW, SEQ ID NO:1). (C) Amino acid sequence of the resulting chemokine fusion protein (Mk6P4-CCL5 c7HopQ, SEQ ID NO:5). The sequence derived from the chemokine is shown in bold. The sequence derived from HopQ is in between. The C-terminal tag contains 6xHis and EPEA is underlined with a dashed line. [Figure 6]Figure 6 shows a model 4 of a 50 kD 6P4-CCL5 fusion protein constructed from a circularly permuted variant of HopQ inserted into the β-turn connecting β-strand β2 and β-strand β3 of the 6P4-CCL5 chemokine. (A) Model of a chemokine fusion protein made by fusing the chemokine 6P4-CCL5 (top) with a circularly permuted variant of the adhesive domain of HopQ from H. pylori (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) A circularly permuted gene encoding the adhesive domain of type 1 HopQ from Helicobacter pylori strain G27 (bottom, PDB 5LP2, SEQ ID NO:2, c7HopQ) was inserted into the β-turn (β-turn β2-β3) connecting β-strand β2 and β-strand β3 of 6P4-CCL5 (top, PDB 5UIW, SEQ ID NO:1). (C) Amino acid sequence of the resulting chemokine fusion protein (Mk6P4-CCL5 c7HopQ, SEQ ID NO: 6). The sequence derived from the chemokine is shown in bold. The sequence derived from HopQ is in between. The C-terminal tag contains 6xHis and EPEA is underlined with a dashed line. [Figure 7]Figure 7 is a diagram showing yeast display vectors for optimizing the composition and length of the linker peptide connecting the scaffold protein HopQ to chemokines. (A) Schematic of the display vector. LS: appS4, an engineered secretion signal of yeast α-factor that directs extracellular secretion in yeast (Rakestraw et al., 2009). N: N-terminal part of 6P4-CCL5 chemokine up to β-strand β2 (1-43 in SEQ ID NO:1); a circularly permuted gene encoding the adhesive domain of type 1 HopQ of Helicobacter pylori strain G27 (bottom, PDB 5LP2, SEQ ID NO:2, c7HopQ); 6P4-CCL5 C-terminus from β-strand β3 of 6P4-CCL5 chemokine (47-69 in SEQ ID NO:1); a flexible linker connecting to the displayed protein Aga2p, the adhesive subunit of the yeast agglutinin protein that binds to the yeast cell wall via a disulfide bond to the Aga1p protein (Chao et al., 2006); ACP: acyl carrier protein for orthogonal labeling of the displayed chemokine fusion protein to observe its expression level (Johnsson et al., 2005). (B) Sequence diversity of displayed chemokine fusion proteins (SEQ ID NOs: 25-28): Megakine Mk6P4-CCL5 c7HopQ in bold with AppS4 leader sequence in regular font, (X)1-2 random linkers, short peptide linkers of variable length (1 or 2 amino acids) and mixed composition; flexible (GGGS)n polypeptide linker in italics, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag. (C) Four pools of chemokine fusion protein sequences (each representing 25% of the library) were generated encoding a total of 176400 AA sequence variants by using an equimolar mixture of two forward PCR primers (SEQ ID NOs: 29, 30) and two reverse PCR primers (SEQ ID NOs: 31, 32) to introduce short peptide linkers of variable length (1 and 2 amino acids) and mixed composition. [Figure 8]FIG. 8 is a diagram showing successive rounds of selection of chemokine fusion proteins by yeast display and two-dimensional flow cytometry. Selection was performed by yeast display and flow cytometry to optimize the composition and length of the linker peptide connecting the scaffold protein HopQ to the chemokine CCL5. Each dot represents two fluorescent signals of separate EBY100 yeast cells transformed with pCTCON2 derivatives encoding the chemokine fusion protein Mk6P4-CCL5 c7HopQ fused to Aga2p and ACP via linkers with different lengths and compositions. Yeast cells were orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM) to measure Megakine display levels (Y-axis). To determine whether the displayed Megakine contains a folded CCL5 moiety, the cells were supplementarily stained with Alexa Fluor® 647-labeled anti-human RANTES (CCL5) antibody (X-axis). In round 1, the library was incubated with 0.25 mg / ml of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody. 200,000 yeast cells displaying high fluorescence for Megakine expression (PE channel) and anti-human RANTES (CCL5) (647 nm channel) were selected. In round 2, the enriched library was incubated with 0.025 mg / mL of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody. 20,000 yeast cells displaying the highest fluorescence for Megakine expression (PE channel) and anti-human RANTES (CCL5) (647 nm channel) were selected and subjected to sequence analysis. [Figure 9]FIG. 9 shows a qualitative analysis by two-dimensional flow cytometry of the display of four different chemokine fusion proteins with different linkers on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the chemokine fusion protein Mk6P4-CCL5 c7HopQ fused to Aga2p and ACP (A-D, models 1-4, SEQ ID NOs: 7-10, respectively). Yeast cells displaying MegaBody MbNb207 cHopQ were used as a positive control (E, SEQ ID NO: 11). In this experiment, non-transformed EBY100 yeast cells were included as a negative control (F). Transformed and non-transformed yeast cells were equally orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM). [Figure 10] FIG. 10 is a diagram showing the qualitative analysis by flow cytometry of the display of four different chemokine fusion proteins with different linkers on the surface of EBY100 yeast cells. Single parameter histograms show the relative fluorescence intensity of EBY100 yeast cells transformed with pCTCON2 derivatives encoding the chemokine fusion protein Mk6P4-CCL5 c7HopQ (versions 1-4, SEQ ID NOs: 7-10) fused to Aga2p and ACP, compared to the positive control MbNb207 cHopQ (SEQ ID NO: 11) and the negative control non-transformed EBY100 yeast cells. Transformed and non-transformed yeast cells were orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM). Models 1, 2, 3, 4 refer to the actual clones and fusion proteins. [Figure 11-1]FIG. 11 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c7HopQ fusion protein variants 1 and 2 displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c7HopQ fusion protein models 1 and 2 (SEQ ID NO: 7 and SEQ ID NO: 8) as Aga2p and ACP fusions. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthetase (1 μM) and incubated with five different concentrations of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody (15, 31, 62, 125 and 250 ng / mL, respectively). The y-axis shows the mean fluorescence intensity of relative PE / CoA-547 fluorescence (Megakine display level). The x-axis shows the mean fluorescence intensity of relative Alexa Fluor® 647 anti-human fluorescent RANTES (CCL5) antibody binding. Models 1, 2 refer to the actual clones. [Figure 11-2] FIG. 11 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c7HopQ fusion protein variants 1 and 2 displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c7HopQ fusion protein models 1 and 2 (SEQ ID NO: 7 and SEQ ID NO: 8) as Aga2p and ACP fusions. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthetase (1 μM) and incubated with five different concentrations of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody (15, 31, 62, 125 and 250 ng / mL, respectively). The y-axis shows the mean fluorescence intensity of relative PE / CoA-547 fluorescence (Megakine display level). The x-axis shows the mean fluorescence intensity of relative Alexa Fluor® 647 anti-human fluorescent RANTES (CCL5) antibody binding. Models 1, 2 refer to the actual clones. [Figure 11-3]FIG. 11 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c7HopQ fusion protein variants 1 and 2 displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c7HopQ fusion protein models 1 and 2 (SEQ ID NO: 7 and SEQ ID NO: 8) as Aga2p and ACP fusions. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthetase (1 μM) and incubated with five different concentrations of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody (15, 31, 62, 125 and 250 ng / mL, respectively). The y-axis shows the mean fluorescence intensity of relative PE / CoA-547 fluorescence (Megakine display level). The x-axis shows the mean fluorescence intensity of relative Alexa Fluor® 647 anti-human fluorescent RANTES (CCL5) antibody binding. Models 1, 2 refer to the actual clones. [Figure 12-1] 12 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c7HopQ fusion protein variants 3 and 4 displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c7HopQ fusion protein models 3 and 4 (SEQ ID NO: 9 and SEQ ID NO: 10) as Aga2p and ACP fusions. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM) and incubated with five different concentrations of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody (15, 31, 62, 125 and 250 ng / mL, respectively). The y-axis shows the mean fluorescence intensity of relative PE / CoA-547 fluorescence (Megakine display level), and the x-axis shows the mean fluorescence intensity of relative Alexa Fluor® 647 fluorescence (RANTES (CCL5) antibody binding). Models 3 and 4 refer to actual clones. [Figure 12-2]12 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c7HopQ fusion protein variants 3 and 4 displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c7HopQ fusion protein models 3 and 4 (SEQ ID NO: 9 and SEQ ID NO: 10) as Aga2p and ACP fusions. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM) and incubated with five different concentrations of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody (15, 31, 62, 125 and 250 ng / mL, respectively). The y-axis shows the mean fluorescence intensity of relative PE / CoA-547 fluorescence (Megakine display level), and the x-axis shows the mean fluorescence intensity of relative Alexa Fluor® 647 fluorescence (RANTES (CCL5) antibody binding). Models 3 and 4 refer to actual clones. [Figure 12-3] 12 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c7HopQ fusion protein variants 3 and 4 displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c7HopQ fusion protein models 3 and 4 (SEQ ID NO: 9 and SEQ ID NO: 10) as Aga2p and ACP fusions. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM) and incubated with five different concentrations of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody (15, 31, 62, 125 and 250 ng / mL, respectively). The y-axis shows the mean fluorescence intensity of relative PE / CoA-547 fluorescence (Megakine display level), and the x-axis shows the mean fluorescence intensity of relative Alexa Fluor® 647 fluorescence (RANTES (CCL5) antibody binding). Models 3 and 4 refer to actual clones. [Figure 13-1]13 is a diagram showing flow cytometry analysis of the functionality of the antigen-binding chimeric protein MbNb207 cHopQ displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the antigen-binding chimeric protein MbNb207 cHopQ (SEQ ID NO: 11) as an Aga2p and ACP fusion. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthetase (1 μM) and incubated with five different concentrations of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody (15, 31, 62, 125 and 250 ng / mL, respectively). The y-axis shows the mean fluorescence intensity of relative PE / CoA-547 fluorescence (antigen-binding chimeric protein display level) and the x-axis shows the mean fluorescence intensity of relative Alexa Fluor® 647 fluorescence (RANTES (CCL5) antibody binding). [Figure 13-2] 13 is a diagram showing flow cytometry analysis of the functionality of the antigen-binding chimeric protein MbNb207 cHopQ displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the antigen-binding chimeric protein MbNb207 cHopQ (SEQ ID NO: 11) as an Aga2p and ACP fusion. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthetase (1 μM) and incubated with five different concentrations of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody (15, 31, 62, 125 and 250 ng / mL, respectively). The y-axis shows the mean fluorescence intensity of relative PE / CoA-547 fluorescence (antigen-binding chimeric protein display level) and the x-axis shows the mean fluorescence intensity of relative Alexa Fluor® 647 fluorescence (RANTES (CCL5) antibody binding). [Figure 14]14 is a diagram showing a qualitative analysis by flow cytometry of the binding of four different chimeric chemokines to Alexa Fluor® 647 fluorescent RANTES (CCL5). Chart representation of the calculated mean fluorescence intensity of relative Alexa Fluor® 647 fluorescence (RANTES (CCL5) antibody binding) of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c7HopQ fusion proteins models 1-4 (SEQ ID NOs: 7-10) as Aga2p and ACP fusions and the negative control antigen-binding chimeric protein MbNb207 cHopQ (SEQ ID NO: 11). Yeast clones were induced and incubated with five different concentrations of Alexa Fluor® 647 anti-human RANTES (CCL5) antibody (15, 31, 62, 125 and 250 ng / mL, respectively). [Figure 15] Figure 15 shows that displayed chemokine fusion proteins can be eluted from yeast membranes. (A) Schematic of chemokine fusion proteins displayed on yeast membranes and eluted using 1 mM DTT. (B) 12% SDS-PAGE of elution fractions of four different variants and the antigen-binding chimeric protein MbNb207 cHopQ as a control. Western blot analysis of the same gel using primary mouse anti-cMYC antibody and goat anti-mouse alkaline phosphatase conjugated antibody. The molecular mass of approximately 50 kDa for Mk6P4-CCL5 c7HopQ was confirmed by molecular mass markers (arrows). [Figure 16]16 shows SDS-PAGE and Western blot analysis of the expression of four different recombinant chemokine fusion protein variants secreted from S. cerevisiae EBY100. His-tagged fusion proteins Mk6P4-CCL5 cHopQ models 1-4 (SEQ ID NOs: 12-15) were expressed in S. cerevisiae EBY100 fused to the appS4 leader sequence directing extracellular secretion in yeast and purified by nickel affinity chromatography (IMAC). (A) IMAC-purified fusion protein Mk6P4-CCL5 c7HopQ eluted with 500 mM imidazole and loaded onto a 12% SDS-PAGE gel. (B) Western blot analysis of the same gel using primary mouse anti-His and goat anti-mouse alkaline phosphatase conjugated antibodies. The molecular mass of approximately 50 kDa for Mk6P4-CCL5 c7HopQ was confirmed by molecular mass markers (left line: M). [Figure 17] FIG. 17 shows SDS-PAGE and Western blot analysis of the expression of four different recombinant chemokine fusion protein variants in the periplasm of E. coli WK6. His-tagged fusion proteins Mk6P4-CCL5 c7HopQ models 1-4 (SEQ ID NOs: 3-6) were expressed in the periplasm of E. coli and purified by nickel affinity chromatography (IMAC). (A) Samples of fusion protein Mk6P4-CCL5 c7HopQ from E. coli periplasmic extracts and purified protein eluted with 500 mM imidazole after IMAC and loaded onto a 12% SDS-PAGE gel. (B) Western blot analysis of the same gel using primary mouse anti-His and goat anti-mouse alkaline phosphatase conjugated antibodies. The molecular mass of approximately 50 kDa for Mk6P4-CCL5 c7HopQ was confirmed by molecular mass markers (right line: M). [Figure 18]FIG. 18 shows the biological activity of Mk6P4c-CCL5 c7HopQV1-V4 fusion protein variants on the chemokine receptor CCR5. Recruitment of miniGi to CCR5 induced by chemokine fusion protein variants at different dilutions (A) produced in the periplasm of E. coli and by chemokine fusion protein variants after Ni-NTA purification (B) was observed in HEK293T cells using NanoLuc complementation assay. Recombinant soluble 6P4-CCL5 chemokine produced in HEK293T and diluted 100-fold was used as a positive control. Results are expressed as fold increase in luminescence over untreated samples. Recruitment of β-arrestin-1 to CCR5 induced by chemokine fusion protein variants at different dilutions produced in the periplasm of E. coli (C) and after Ni-NTA purification (D) was observed in HEK293T cells using NanoLuc complementation assay. Recombinant soluble 6P4-CCL5 chemokine produced in HEK293T and diluted 100-fold was used as a positive control. Results are expressed as fold increase in luminescence over untreated samples. [Figure 19]Figure 19 shows a model of a 50 kD CXCL12 fusion protein constructed from a circularly permuted variant of HopQ inserted into the β-turn connecting β-strand β2 and β-strand β3 of the CXCL12 chemokine. (A) Model of a chemokine fusion protein made by fusing CXCL12 (top) with a circularly permuted variant of the adhesive domain of HopQ from H. pylori (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) A circularly permuted gene encoding the adhesive domain of type 1 HopQ from Helicobacter pylori strain G27 (bottom, PDB 5LP2, SEQ ID NO:2, c7HopQ) was inserted into the β-turn connecting β-strand β2 and β-strand β3 (β-turn β2-β3) of CXCL12 (top, SEQ ID NO:22). (C) Amino acid sequence of the resulting CXCL12 chemokine fusion protein (MkCXCL12 c7HopQ, SEQ ID NO: 23). The chemokine-derived sequence is shown in bold. The HopQ-derived sequence is in normal type. The C-terminal tag contains 6xHis and EPEA is underlined with a dotted line. [Figure 20]20 is a diagram showing a model of Mk6P4-CCL5 c1YgjKV1, a 94 kD 6P4-CCL5 fusion protein constructed from a circularly permuted c1YgjK variant inserted into the β-turn connecting β-strand β2 and β-strand β3 of the 6P4-CCL5 chemokine. (A) Model of a chemokine fusion protein made by fusion of the chemokine 6P4-CCL5 (top) with a circularly permuted variant of the YgjK glycosidase of E. coli (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) The circularly permuted variant 1 gene encoding the YgjK glycosidase of E. coli (bottom, PDB 3W 7S, SEQ ID NO: 36, c1YgjK) was inserted into the β-turn (β-turn β2-β3) connecting β-strand β2 and β-strand β3 of 6P4-CCL5 (top, PDB 5UIW, SEQ ID NO: 1). (C) Amino acid sequence of the resulting chemokine chimeric protein (Mk6P4-CCL5 c1YgjKV1, SEQ ID NO: 38). The sequence derived from the chemokine is shown in bold. The two amino acid peptide linker is underlined. The sequence derived from c1YgjK is in between. [Figure 21]21 is a diagram showing a model of Mk6P4-CCL5 c1YgjKV2, a 94 kD 6P4-CCL5 fusion protein constructed from a circularly permuted c1YgjK variant inserted into the β-turn connecting β-strand β2 and β-strand β3 of the 6P4-CCL5 chemokine. (A) Model of a chemokine fusion protein made by fusion of the chemokine 6P4-CCL5 (top) with a circularly permuted variant of the YgjK glycosidase of E. coli (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) The circularly permuted variant 1 gene encoding the YgjK glycosidase of E. coli (bottom, PDB 3W7S, SEQ ID NO: 36, c1YgjK) was inserted into the β-turn (β-turn β2-β3) connecting β-strand β2 and β-strand β3 of 6P4-CCL5 (top, PDB 5UIW, SEQ ID NO: 1). (C) Amino acid sequence of the resulting chemokine chimeric protein (Mk6P4-CCL5 c1YgjKV2, SEQ ID NO: 39). The sequence derived from the chemokine is shown in bold. The one amino acid peptide linker is underlined. The sequence derived from c1YgjK is in between. [Figure 22]22 is a diagram showing a model of Mk6P4-CCL5 c1YgjKV3, a 94 kD 6P4-CCL5 fusion protein constructed from a circularly permuted c1YgjK variant inserted into the β-turn connecting β-strand β2 and β-strand β3 of the 6P4-CCL5 chemokine. (A) Model of a chemokine fusion protein made by fusion of the chemokine 6P4-CCL5 (top) with a circularly permuted variant of the YgjK glycosidase of E. coli (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) The circularly permuted variant 1 gene encoding the YgjK glycosidase of E. coli (bottom, PDB 3W7S, SEQ ID NO:36, c1YgjK) was inserted into the β-turn (β-turn β2-β3) connecting β-strand β2 and β-strand β3 of 6P4-CCL5 (top, PDB 5UIW, SEQ ID NO:1). (C) Amino acid sequence of the resulting chemokine chimeric protein (Mk6P4-CCL5 c1YgjKV3, SEQ ID NO:40). The sequence derived from the chemokine is shown in bold. The sequence derived from c1YgjK is in between. [Diagram 23]23 is a diagram showing a model of Mk6P4-CCL5 c2YgjKV1, a 94 kD 6P4-CCL5 fusion protein constructed from a circularly permuted c2YgjK variant inserted into the β-turn connecting β-strand β2 and β-strand β3 of the 6P4-CCL5 chemokine. (A) Model of a chemokine fusion protein made by fusion of the chemokine 6P4-CCL5 (top) with a circularly permuted variant of the YgjK glycosidase of E. coli (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) The circularly permuted variant B gene encoding the YgjK glycosidase of E. coli (bottom, PDB 3W7S, SEQ ID NO:37, c2YgjK) was inserted into the β-turn (β-turn β2-β3) connecting β-strand β2 and β-strand β3 of 6P4-CCL5 (top, PDB 5UIW, SEQ ID NO:1). (C) Amino acid sequence of the resulting chemokine chimeric protein (Mk6P4-CCL5 c2YgjKV1, SEQ ID NO:41). The sequence derived from the chemokine is shown in bold. The two amino acid peptide linker is underlined. The sequence derived from c2YgjK is in between. [Figure 24]24 is a diagram showing a model of Mk6P4-CCL5 c2YgjKV3, a 94 kD 6P4-CCL5 fusion protein constructed from a circularly permuted c2YgjK variant inserted into the β-turn connecting β-strand β2 and β-strand β3 of the 6P4-CCL5 chemokine. (A) Model of a chemokine fusion protein made by fusion of the chemokine 6P4-CCL5 (top) with a circularly permuted variant of the YgjK glycosidase of E. coli (bottom) via two peptide bonds or linkers connecting the chemokine to the scaffold. (B) The circularly permuted variant 2 gene encoding the YgjK glycosidase of E. coli (bottom, PDB 3W7S, SEQ ID NO: 37, c2YgjK) was inserted into the β-turn (β-turn β2-β3) connecting β-strand β2 and β-strand β3 of 6P4-CCL5 (top, PDB 5UIW, SEQ ID NO: 1). (C) Amino acid sequence of the resulting chemokine chimeric protein (Mk6P4-CCL5 c2YgjKV3, SEQ ID NO: 42). The sequence derived from the chemokine is shown in bold. The sequence derived from c2YgjK is in between. [Figure 25-1]25 is a diagram showing a qualitative analysis by two-dimensional flow cytometry of the display of four different chemokine fusion proteins with different linkers and topologies on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the chemokine fusion proteins Mk6P4-CCL5 c1YgjKV1-V3 fused to Aga2p and ACP (A-C, SEQ ID NOs: 43-45, respectively) and Mk6P4-CCL5 c2YgjKV1 / V3 fused to Aga2p and ACP (D-E, SEQ ID NOs: 46-47, respectively). Yeast cells displaying megakine Mk6P4-CCL5 c7HopQV4 (SEQ ID NO: 10) were used as a positive control (F, SEQ ID NO: 11). Yeast cells displaying MegaBody MbNb207 cHopQ (G, SEQ ID NO: 11) and non-transformed EBY100 yeast cells (H) were included as negative controls in this experiment. Transformed and non-transformed yeast cells were orthogonally stained equally with CoA-547 (2 μM) using SFP synthase (1 μM). [Figure 25-2]25 is a diagram showing a qualitative analysis by two-dimensional flow cytometry of the display of four different chemokine fusion proteins with different linkers and topologies on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the chemokine fusion proteins Mk6P4-CCL5 c1YgjKV1-V3 fused to Aga2p and ACP (A-C, SEQ ID NOs: 43-45, respectively) and Mk6P4-CCL5 c2YgjKV1 / V3 fused to Aga2p and ACP (D-E, SEQ ID NOs: 46-47, respectively). Yeast cells displaying megakine Mk6P4-CCL5 c7HopQV4 (SEQ ID NO: 10) were used as a positive control (F, SEQ ID NO: 11). Yeast cells displaying MegaBody MbNb207 cHopQ (G, SEQ ID NO: 11) and non-transformed EBY100 yeast cells (H) were included as negative controls in this experiment. Transformed and non-transformed yeast cells were orthogonally stained equally with CoA-547 (2 μM) using SFP synthase (1 μM). [Figure 25-3]25 is a diagram showing a qualitative analysis by two-dimensional flow cytometry of the display of four different chemokine fusion proteins with different linkers and topologies on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the chemokine fusion proteins Mk6P4-CCL5 c1YgjKV1-V3 fused to Aga2p and ACP (A-C, SEQ ID NOs: 43-45, respectively) and Mk6P4-CCL5 c2YgjKV1 / V3 fused to Aga2p and ACP (D-E, SEQ ID NOs: 46-47, respectively). Yeast cells displaying megakine Mk6P4-CCL5 c7HopQV4 (SEQ ID NO: 10) were used as a positive control (F, SEQ ID NO: 11). Yeast cells displaying MegaBody MbNb207 cHopQ (G, SEQ ID NO: 11) and non-transformed EBY100 yeast cells (H) were included as negative controls in this experiment. Transformed and non-transformed yeast cells were orthogonally stained equally with CoA-547 (2 μM) using SFP synthase (1 μM). [Figure 25-4]25 is a diagram showing a qualitative analysis by two-dimensional flow cytometry of the display of four different chemokine fusion proteins with different linkers and topologies on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding the chemokine fusion proteins Mk6P4-CCL5 c1YgjKV1-V3 fused to Aga2p and ACP (A-C, SEQ ID NOs: 43-45, respectively) and Mk6P4-CCL5 c2YgjKV1 / V3 fused to Aga2p and ACP (D-E, SEQ ID NOs: 46-47, respectively). Yeast cells displaying megakine Mk6P4-CCL5 c7HopQV4 (SEQ ID NO: 10) were used as a positive control (F, SEQ ID NO: 11). Yeast cells displaying MegaBody MbNb207 cHopQ (G, SEQ ID NO: 11) and non-transformed EBY100 yeast cells (H) were included as negative controls in this experiment. Transformed and non-transformed yeast cells were orthogonally stained equally with CoA-547 (2 μM) using SFP synthase (1 μM). [Figure 26-1]26 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c1 / 2YgjK fusion protein variants displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c1YgjKV1-V3 fused to Aga2p and ACP (A-C, SEQ ID NOs: 43-45, respectively) and Mk6P4-CCL5 c2YgjKV1 / V3 fused to Aga2p and ACP (D-E, SEQ ID NOs: 46-47, respectively). Yeast cells displaying megakine Mk6P4-CCL5 c7HopQV4 (SEQ ID NO: 10) were used as a positive control (F, SEQ ID NO: 11). Yeast cells displaying MegaBody MbNb207 cHopQ (G, SEQ ID NO: 11) and non-transformed EBY100 yeast cells (H) were included as negative controls in this experiment. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM) and incubated with Alexa Fluor® 647 anti-human RANTES (CCL5) antibody at a concentration of 80 ng / mL. The y-axis shows relative CoA-547 fluorescence (Megakine display levels). The x-axis shows relative Alexa Fluor® 647 anti-human fluorescent RANTES (CCL5) antibody binding. [Figure 26-2]26 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c1 / 2YgjK fusion protein variants displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c1YgjKV1-V3 fused to Aga2p and ACP (A-C, SEQ ID NOs: 43-45, respectively) and Mk6P4-CCL5 c2YgjKV1 / V3 fused to Aga2p and ACP (D-E, SEQ ID NOs: 46-47, respectively). Yeast cells displaying megakine Mk6P4-CCL5 c7HopQV4 (SEQ ID NO: 10) were used as a positive control (F, SEQ ID NO: 11). Yeast cells displaying MegaBody MbNb207 cHopQ (G, SEQ ID NO: 11) and non-transformed EBY100 yeast cells (H) were included as negative controls in this experiment. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM) and incubated with Alexa Fluor® 647 anti-human RANTES (CCL5) antibody at a concentration of 80 ng / mL. The y-axis shows relative CoA-547 fluorescence (Megakine display levels). The x-axis shows relative Alexa Fluor® 647 anti-human fluorescent RANTES (CCL5) antibody binding. [Figure 26-3]26 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c1 / 2YgjK fusion protein variants displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c1YgjKV1-V3 fused to Aga2p and ACP (A-C, SEQ ID NOs: 43-45, respectively) and Mk6P4-CCL5 c2YgjKV1 / V3 fused to Aga2p and ACP (D-E, SEQ ID NOs: 46-47, respectively). Yeast cells displaying megakine Mk6P4-CCL5 c7HopQV4 (SEQ ID NO: 10) were used as a positive control (F, SEQ ID NO: 11). Yeast cells displaying MegaBody MbNb207 cHopQ (G, SEQ ID NO: 11) and non-transformed EBY100 yeast cells (H) were included as negative controls in this experiment. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM) and incubated with Alexa Fluor® 647 anti-human RANTES (CCL5) antibody at a concentration of 80 ng / mL. The y-axis shows relative CoA-547 fluorescence (Megakine display levels). The x-axis shows relative Alexa Fluor® 647 anti-human fluorescent RANTES (CCL5) antibody binding. [Figure 26-4]26 is a diagram showing flow cytometry analysis of the functionality of Mk6P4-CCL5 c1 / 2YgjK fusion protein variants displayed on the surface of EBY100 yeast cells. Dot plot representation of relative fluorescence intensity of individual EBY100 yeast cells transformed with pCTCON2 derivatives encoding Mk6P4-CCL5 c1YgjKV1-V3 fused to Aga2p and ACP (A-C, SEQ ID NOs: 43-45, respectively) and Mk6P4-CCL5 c2YgjKV1 / V3 fused to Aga2p and ACP (D-E, SEQ ID NOs: 46-47, respectively). Yeast cells displaying megakine Mk6P4-CCL5 c7HopQV4 (SEQ ID NO: 10) were used as a positive control (F, SEQ ID NO: 11). Yeast cells displaying MegaBody MbNb207 cHopQ (G, SEQ ID NO: 11) and non-transformed EBY100 yeast cells (H) were included as negative controls in this experiment. Yeast clones were induced and orthogonally stained with CoA-547 (2 μM) using SFP synthase (1 μM) and incubated with Alexa Fluor® 647 anti-human RANTES (CCL5) antibody at a concentration of 80 ng / mL. The y-axis shows relative CoA-547 fluorescence (Megakine display levels). The x-axis shows relative Alexa Fluor® 647 anti-human fluorescent RANTES (CCL5) antibody binding. [Figure 27] Figure 27 is a diagram showing the operating principle of an interleukin fusion protein constructed from a circularly permuted variant of a scaffold protein inserted into the β-turn connecting β-strand β6 and β-strand β7 of the IL-1β interleukin. The scheme shows how an interleukin can be grafted to a large scaffold protein via two peptide bonds or two short linkers connecting the chemokine domain to the scaffold. The scissors indicate which exposed turns must be cut in the interleukin and the scaffold. The dashed lines show how the interleukin and the remaining parts of the scaffold must be linked by the use of peptide bonds and short peptide linkers to construct an interleukin chimeric protein. [Figure 28] Figure 28 is a drawing showing the crystal structure of IL-1β bound to the ectodomains of IL-1RII and IL-1RAcP. The IL-1β·IL-1RI·IL-1RAcP complex is presented in two views rotated 90° around the vertical axis. IL-1RII and IL-1RAcP are shown as surfaces and IL-1β is shown as a ribbon structure. The β-turn connecting β-sheet β6 and β-sheet β7 is highlighted with an arrow. [Figure 29] 29 is a diagram showing a model of MkIL-1β c7HopQV1, a 58 kD IL-1β fusion protein constructed from a circularly permuted HopQ variant inserted into the β-turn connecting β-strand β6 and β-strand β7 of IL-1β interleukin. (A) Model of a chemokine fusion protein made by fusing human IL-1β interleukin (top) with a circularly permuted variant of the adhesive domain of H. pylori HopQ (bottom) via two peptide bonds or linkers connecting the interleukin to the scaffold. (B) A circularly permuted gene encoding the adhesive domain of H. pylori HopQ (bottom, PDB 5LP2, SEQ ID NO:2, c7HopQ) was inserted into the β-turn (β-turn β6-β7) connecting β-strand β6 and β-strand β7 of IL-1β interleukin (top, PDB 3O4O, SEQ ID NO:48). (C) Amino acid sequence of the resulting interleukin chimeric protein (MkIL-1β c7HopQV1, SEQ ID NO: 49). The sequence derived from the interleukin is shown in bold. The two amino acid peptide linker is underlined. The sequence derived from HopQ is in between. [Diagram 30]30 shows a model of MkIL-1β c7HopQV2, a 58 kD IL-1β fusion protein constructed from a circularly permuted HopQ variant inserted into the β-turn connecting β-strand β6 and β-strand β7 of IL-1β interleukin. (A) Model of a chemokine fusion protein made by fusing human IL-1β interleukin (top) with a circularly permuted variant of the adhesive domain of H. pylori HopQ (bottom) via two peptide bonds or linkers connecting the interleukin to the scaffold. (B) A circularly permuted gene encoding the adhesive domain of H. pylori HopQ (bottom, PDB 5LP2, SEQ ID NO:2, c7HopQ) was inserted into the β-turn (β-turn β6-β7) connecting β-strand β6 and β-strand β7 of IL-1β interleukin (top, PDB 3O4O, SEQ ID NO:48). (C) Amino acid sequence of the resulting interleukin chimeric protein (MkIL-1β c7HopQV2, SEQ ID NO:50). The sequence derived from the interleukin is shown in bold. The one amino acid peptide linker is underlined. The sequence derived from HopQ is in between. [Diagram 31]31 is a diagram showing a model of MkIL-1β c7HopQV3, a 58 kD IL-1β fusion protein constructed from a circularly permuted HopQ variant inserted into the β-turn connecting β-strand β6 and β-strand β7 of IL-1β interleukin. (A) Model of a chemokine fusion protein made by fusing human IL-1β interleukin (top) with a circularly permuted variant of the adhesive domain of H. pylori HopQ (bottom) via two peptide bonds or linkers connecting the interleukin to the scaffold. (B) A circularly permuted gene encoding the adhesive domain of H. pylori HopQ (bottom, PDB 5LP2, SEQ ID NO:2, c7HopQ) was inserted into the β-turn (β-turn β6-β7) connecting β-strand β6 and β-strand β7 of IL-1β interleukin (top, PDB 3O4O, SEQ ID NO:48). (C) Amino acid sequence of the resulting interleukin chimeric protein (MkIL-1β c7HopQV3, SEQ ID NO: 51). The interleukin-derived sequences are shown in bold. The HopQ-derived sequences are in between. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention will be described with respect to certain embodiments and with reference to certain drawings, but the present invention is not limited thereto, but only by the claims. Any reference signs in the claims should not be interpreted as limiting the scope. Of course, it is understood that not all aspects or advantages can be achieved according to any particular embodiment of the present invention. Thus, for example, a person skilled in the art will recognize that the present invention can be embodied or implemented to achieve or optimize one or a group of advantages taught herein, without necessarily achieving other aspects or advantages that may be taught or suggested herein.

[0025] The present invention, both in terms of its construction and method of operation, together with its features and advantages, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the present invention will be apparent and made clear by reference to the embodiments described below. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification may, but do not necessarily, all refer to the same embodiment. Similarly, in describing exemplary embodiments of the present invention, it should also be recognized that various features of the invention are sometimes grouped together in a single embodiment, figure, or description for the purpose of streamlining the disclosure and for the purpose of aiding in the understanding of one or more various aspects of the present invention. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, aspects of the invention lie in less than all features of a single foregoing disclosed embodiment.

[0026] definition When an indefinite or definite article is used to refer to a singular noun, such as "a", "an", "this", it includes a plurality of that noun unless specifically stated otherwise. When the term "comprises" is used in the description and claims of this specification, it does not exclude other elements or steps. Furthermore, the terms first, second, third, etc. in the description and claims are used to distinguish similar elements and not necessarily to describe them sequentially or chronologically. It should be understood that the terms used in this manner are interchangeable under appropriate conditions and that the embodiments of the invention described herein are capable of operating in other sequences than those described or illustrated herein. The following terms or definitions are provided to aid in the simple understanding of the present invention. Unless otherwise defined herein, all terms used herein have the same meaning as they would have to a person skilled in the art of the invention. The practitioner should refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). The definitions provided herein should not be construed to have a scope narrower than understood by a person of ordinary skill in the art.

[0027] As used herein, "about," when used in reference to a measurable value such as an amount, duration, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, where such variations are appropriate for carrying out the methods of the present disclosure. "Similar," as used herein, is interchangeable with resembling, similar, equivalent, corresponding, etc., means having the same or common characteristics, and / or exhibiting comparable results in a quantifiable manner, i.e., with a variation of up to 20%, 10%, more preferably 5%, or even more preferably 1% or less.

[0028] As used herein, "nucleotide sequence", "DNA sequence", or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double-stranded and single-stranded DNA, as well as RNA. The term also includes known types of modifications, such as methylation, "cap" substitution of one or more of the naturally occurring nucleotides with an analog. "Nucleic acid construct" refers to a nucleic acid sequence that has been constructed to contain one or more functional units that are not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes containing non-naturally occurring nucleic acid sequences, and the like.

[0029] A "coding sequence" is a nucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, and can also contain introns under certain circumstances.

[0030] As used herein, a "promoter region of a gene" refers to a functional DNA sequence unit that is operably linked to a coding sequence and sufficient to promote transcription of said coding sequence when possibly placed under appropriate inducing conditions. "Operably linked" refers to the juxtaposition of the components thus described in a relationship that allows them to function in their intended manner. A promoter sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the promoter sequence. "Gene" as used herein includes both the promoter region of a gene as well as the coding sequence. It refers to both genomic sequences (including possible introns) and cDNA derived from spliced ​​messengers operably linked to the promoter sequence. The term "terminator" or "transcription termination signal" encompasses control sequences that are DNA sequences at the end of a transcription unit that signal the 3' processing and polyadenylation of the primary transcript and the termination of transcription. Terminators can be derived from natural genes, a variety of other plant genes, or T-DNA. The added terminator may, for example, be derived from the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or preferably then any other eukaryotic gene.

[0031] "Genetic construct", "chimeric gene", "chimeric construct" or "chimeric gene construct" refers to a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably linked or associated with a nucleic acid sequence encoding an mRNA, whereby the regulatory nucleic acid sequence can regulate the transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of a chimeric gene is not operably linked to the associated nucleic acid sequence found in nature. In particular, the term "gene fusion construct" as used herein refers to a genetic construct that encodes an mRNA that is translated into the fusion protein of the invention disclosed herein.

[0032] The terms "vector", "vector construct", "expression vector" or "gene transfer vector" as used herein are intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked, and include any vector known to those skilled in the art, including any suitable type, including, but not limited to, plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenovirus, AAV, or baculovirus vectors, or artificial chromosome vectors, such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Expression vectors include plasmids as well as viral vectors, and generally contain the desired coding sequence and appropriate DNA sequences necessary for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in vitro expression system. Expression vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors that have an origin of replication that functions in the host cell). Other vectors can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating along with the host genome. Suitable vectors contain regulatory sequences, such as promoters, enhancers, terminator sequences, etc., if desired and according to the particular host organism (e.g., bacterial cells, yeast cells). Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment and may lack functional sequences necessary for expression of the desired DNA fragment. The construction of expression vectors used to transfect prokaryotic cells is also well known in the art and can thus be accomplished through standard techniques (see, for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 4, for definitions and terms in the art). thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016).

[0033] A "host cell" can be either a prokaryotic or eukaryotic cell. The cells can be transiently or stably transfected. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including, but not limited to, standard bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-, DEAE-dextran-, polycation-, or viral-mediated transfection. For all standard techniques, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). A recombinant host cell in the context of the present invention is a cell that has been genetically modified to contain an isolated DNA molecule, nucleic acid molecule, or expression construct or vector of the present invention. DNA can be introduced by any means known in the art that is appropriate for the particular type of cell, including but not limited to transformation, lipofection, electroporation, or viral-mediated transduction. DNA constructs capable of allowing expression of the chimeric proteins of the present invention can be readily prepared by techniques known in the art, such as cloning, hybridization screening, and polymerase chain reaction (PCR). Standard techniques for cloning, DNA isolation, amplification, and purification, standard techniques for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, etc., as well as various molecular techniques are known to those skilled in the art and are commonly used techniques. Several standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993) and Ausubel et al. (2016). Representative host cells that may be used with the present invention include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells, and animal cells. Bacterial host cells suitable for use with the present invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells.Animal host cells suitable for use with the present invention include insect and mammalian cells, most particularly cells derived from Chinese hamsters (e.g., CHO), and human cell lines such as HeLa. Yeast host cells suitable for use with the present invention include Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g., Pichia pastoris), Hansenula (e.g., Hansenula polymorpha), and the like. Examples of genera and species within the genus Saccharomyces polymorpha, Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces, etc. Saccharomyces cerevisiae, S. carlsbergensis, and K. lactis are the most commonly used yeast hosts, and convenient fungal hosts. The host cells may be provided as suspension or flask cultures, tissue cultures, organ cultures, etc. Alternatively, the host cells may also be transgenic animals.

[0034] The terms "protein", "polypeptide" and "peptide" are further used interchangeably herein to refer to polymers of amino acid residues and their variants and synthetic analogs. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The terms also include post-translational modifications of the polypeptide, such as glycosylation, phosphorylation, and acetylation. Based on the amino acid sequence and modifications, the atoms or molecular weights or weights of the polypeptide are expressed in (kilo)daltons (kDa). "Recombinant polypeptide" refers to a polypeptide that has been produced using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide. When the chimeric polypeptide or a biologically active portion thereof is produced recombinantly, it is also preferably substantially free of culture medium, i.e., the culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. "Isolated" refers to a material that is substantially or essentially free of components with which it is normally associated in its natural state. For example, an "isolated polypeptide" refers to a polypeptide that has been purified from molecules that flank it in nature, such as a fusion protein disclosed herein that has been removed from molecules that flank the polypeptide and are present in the production host. An isolated chimera can be produced by chemical synthesis of amino acids or by recombinant production. The term "heterologous protein" can mean that the protein is not derived from the same genus, species or strain used to display or express the protein.

[0035] A "homolog" or "homologs" of a protein includes peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the unmodified protein in question and that have similar biological and functional activity as the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the degree to which sequences are identical on an amino acid-by-amino acid basis over a comparison window. Thus, the "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met, also residues designated herein by their single letter code) are present in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. As used herein, a "substitution" or "mutation" results from the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid or nucleotide sequence of the parent protein or fragment thereof. It is understood that a protein or fragment thereof may have conservative amino acid substitutions that do not substantially affect the activity of the protein.

[0036] The term "wild type" refers to a gene or gene product that has been isolated from a naturally occurring source. A wild type gene is the gene that is most frequently observed in a population and is thus an arbitrarily designed "normal" or "wild type" form of the gene. In contrast, the terms "altered," "mutant," or "variant" refer to a gene or gene product that exhibits alterations in sequence, post-translational modifications, and / or functional properties (i.e., altered characteristics) when compared to the wild type gene or gene product. It should be noted that naturally occurring mutants can be isolated. These are identified by the fact that they have altered characteristics compared to the wild type gene or gene product. Alternatively, variants can also include synthetic molecules, for example chemokine ligand variants may be similar in structure and / or function to naturally occurring chemokines, but may relate to small molecules, or synthetic peptides or proteins made by the hand of man. Said variants with different functional properties may relate to superagonists, superantagonists, among other functional differences, as known to those skilled in the art.

[0037] A "protein domain" is a distinct functional and / or structural unit in a protein. Usually, a protein domain is involved in a specific function or interaction, which contributes to the overall role of the protein. Domains can exist in a variety of biological contexts, where similar domains can be found in proteins with different functions. Secondary structure elements (SSEs) in proteins typically form spontaneously as intermediates before a protein is folded into its three-dimensional tertiary structure. The two most common secondary structure elements in proteins are alpha helices and beta (β) sheets, although β turns and omega loops exist as well. A beta-barrel is a beta-sheet made up of tandem repeats that form a twist and coil to form a closed toroidal structure in which the first strand is connected (hydrogen bonds) to the last strand. The beta strands in many beta-barrels are arranged in an antiparallel fashion. Beta sheets consist of beta sheets (also called β strands) that are joined at the sides by at least two or three backbone hydrogen bonds, generally forming a twisted, pleated sheet. A β-strand is a stretch of polypeptide chain, typically 3-10 amino acids long, with an extended conformational backbone. A β-turn is a type of unconventional secondary structure in a protein that causes a change in the orientation of the polypeptide chain. A beta-turn (β-turn, β-turn, β-bend, tight turn, reverse turn, or β-loop (also referred to herein as loop) is a very common motif in proteins and polypeptides, and serves primarily to connect β-strands.

[0038] The term "circular permutation of a protein" or "circularly permuted protein" refers to a protein in which the order of amino acids in its amino acid sequence has been altered compared to the wild-type protein sequence, resulting in a protein structure with different binding strength but an overall similar three-dimensional (3D) shape. Circular permutation of a protein is similar to the mathematical concept of circular permutation in the sense that the sequence of a first part of the wild-type protein (adjacent to its N-terminus) is related to the sequence of a second part of the resulting circularly permuted protein (near its C-terminus), as described, for example, in Bliven and Prlic (2012). Circular permutation of a protein is obtained through genetic or artificial manipulation of a protein sequence compared to this wild-type protein, whereby the N-terminus and C-terminus of the wild-type protein are "connected" and the protein sequence is interrupted at another site, creating a new N-terminus and C-terminus of said protein. The circularly permuted scaffold protein of the present invention is the result of connecting the N-terminus and C-terminus of a wild-type protein sequence and truncating or blocking the sequence at an accessible or exposed site (preferentially a β-turn or loop) of the scaffold protein, thereby maintaining or similarly folding the circularly permuted scaffold protein compared to the folding of the wild-type protein. The connection of the N-terminus and C-terminus in the circularly permuted scaffold protein can be the result of peptide bond linkage, or the result of introduction of a peptide linker, or the result of deletion of a series of peptides near the original N-terminus and C-terminus when the wild-type protein is followed by a peptide bond or remaining amino acids.

[0039] The term "fused," as used herein, and when used interchangeably with "connected," "conjugated," and "ligated," refers specifically to "genetic fusion," e.g., by recombinant DNA techniques, as well as "chemical and / or enzymatic conjugation" resulting in a stable covalent bond.

[0040] The terms "chimeric polypeptide", "chimeric protein", "chimera", "fusion polypeptide", "fusion protein" or "non-naturally occurring protein" are used interchangeably herein and refer to a protein comprising at least two separate and distinct polypeptide components that may or may not be derived from the same protein. The term also refers to a non-naturally occurring molecule, meaning that it is made by the hand of man. The term "fused" and other grammatical equivalents, such as "covalently linked", "connected", "linked", "ligated", "conjugated", when referring to a chimeric polypeptide (as defined herein), refer to any chemical or recombinant mechanism for linking two or more polypeptide components. The fusion of two or more polypeptide components can be a direct fusion of sequences or can be an indirect fusion, for example, by an intervening amino acid sequence or linker sequence, or a chemical linker. The fusion of two polypeptides or the fusion of a cytokine, such as a chemokine, with a scaffold protein as described herein can also refer to a non-covalent fusion obtained by chemical linkage. For example, both the C-terminus of the β2β strand and the N-terminus of the β3β strand of a chemokine core domain can be linked to complementary chemical units or chemical units capable of binding to binding pockets linked or fused to a partial or full-length (circularly permuted) scaffold protein at exposed or accessible sites thereof.

[0041] As used herein, the term "protein complex" or "complex" refers to a group of two or more related macromolecules whereby at least one of the macromolecules is a protein. Protein complex, as used herein, refers to an association of macromolecules that can typically form under physiological conditions. The individual members of a protein complex are linked by non-covalent interactions. A protein complex can be a non-covalent interaction of proteins only, and thus can be referred to as a protein-protein complex; e.g., a non-covalent interaction of two proteins, three proteins, four proteins, etc. More specifically, it can be a complex of a fusion protein and a cytokine receptor, or a complex of a ligand protein (e.g., a fusion protein) that includes a cytokine or chemokine and its specifically bound interacting agent, e.g., a cytokine or chemokine receptor that can bind to a cytokine or chemokine ligand. A protein complex of a fusion protein based on a chemokine bound to a chemokine receptor that is known to bind to said chemokine ligand, a chemokine receptor, by its chemokine receptor interacting region (at its N-terminus) is a formed complex as used herein. Alternatively, a protein complex of a fusion protein based on a ligand of the interleukin-1 type bound by its IL-1 receptor may be a complex as used herein. For example, it is used in 3D structural analysis, where the aim is to elucidate the structure and interaction between a cytokine ligand receptor and a cytokine interaction site that is part of the fusion protein. More specifically, the interaction or binding site of a chemokine and a chemokine receptor are structurally analyzed in them. It is not relevant whether the complete structure of the fusion protein is determined or not. It is understood that the protein complex may be multimeric. The protein complex assembly may result in the formation of a homomultimeric or heteromultimeric complex. Moreover, the interaction may be stable or transient. The term "multimer", "multimeric complex", or "multimeric protein" includes a plurality of identical or heterogeneous polypeptide monomers.

[0042] As used herein, the terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

[0043] The term "suitable conditions" refers to environmental factors such as temperature, motion, other components, among others, and / or "buffer conditions," which specifically refer to the composition of the solution in which the assay is carried out. Said composition includes buffer solutions and / or solutes, such as pH buffer substances, water, saline, physiological salt solutions, glycerol, preservatives, etc., that one skilled in the art would know are suitable for optimal assay performance.

[0044] "Bind" refers to any interaction, direct or indirect. Direct interaction refers to contact between binding partners. Indirect interaction refers to any interaction where the interacting partners interact in a complex of more than two molecules. The interaction can be completely indirect with the aid of one or more bridging molecules, or partially indirect where there is still direct contact between the partners stabilized by the additional interaction of one or more molecules. In general, the binding domain can be immunoglobulin-based or immunoglobulin-like, or based on domains present in proteins, including but not limited to microbial proteins, protease inhibitors, toxins, fibronectins, lipocalins, single-stranded antiparallel coiled-coil proteins, or repeat motif proteins. Binding also includes the interaction of a ligand with this receptor, with respect to chemokines and chemokine receptor interactions. The term "specifically binds" as used herein refers to a binding domain that recognizes a specific target but does not substantially recognize or bind other molecules in the sample. In the case of chemokines, it is known to be a ligand for specifically binding to the chemokine receptor, and thus the binding to this receptor is specific. However, in many cases, chemokines of one subfamily can bind to receptors of the same family, so specific binding does not exclude binding to another chemokine receptor. Thus, specific binding does not mean exclusive binding. However, specific binding means that such a ligand or such a receptor inversely has a certain increased affinity or preference for one or a few chemokine receptors or ligands inversely. The term "affinity" as used herein generally refers to the degree to which a ligand (as further defined herein) binds to a target protein, so as to shift the equilibrium between the target protein and the ligand towards the presence of a complex formed by their binding. Thus, for example, when the receptor and the ligand are combined at relatively equal concentrations, a high affinity ligand binds to the receptor, so as to shift the equilibrium towards a higher concentration of the resulting complex.

[0045] Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. The term "conformation" or "conformational state" of a protein generally refers to the range of structures that a protein can adopt at any given instant in time. Those skilled in the art will recognize that determinants of conformation or conformational state include the amino acid sequence of the protein (including modified amino acids) and the primary structure of the protein as reflected in the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as the secondary structure (e.g., alpha helix, beta sheet, beta barrel, among others), tertiary structure (e.g., three-dimensional folding of the polypeptide chain), and quaternary structure (e.g., interactions of the polypeptide chain with other protein subunits) of the protein. Post-translational and other modifications to the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, among others, can affect the conformation of a protein. Additionally, environmental factors such as the pH, salt concentration, ionic strength, and osmolality of the surrounding solution, among others, as well as interactions with other proteins and cofactors, among others, can affect the conformation of a protein. The conformational state of a protein can be determined by functional assays for activity or binding to another molecule, or by physical methods such as X-ray crystallography, NMR, or spin labeling, among others. For a general discussion of protein conformation and conformational states, see Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules, WH Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, WH Freeman and Company, 1993.

[0046] Finally, the term "functional fusion protein" or "conformation-selective fusion protein" in the context of the present invention refers to a fusion protein that is functional in binding, optionally conformationally selectively, to its cytokine, or in particular interleukin or chemokine receptor protein, and / or functional in activating / inactivating this receptor (depending on the known characteristics of the ligand: agonist, antagonist, inverse agonist). A binding domain that selectively binds to a specific conformation of a target protein refers to a binding domain that binds with higher affinity to the target in a subset of conformations than other conformations that the target can adopt. The skilled artisan will recognize that a binding domain that selectively binds to a specific conformation of a target stabilizes or retains the target in this specific conformation. For example, a binding domain that is selective for an active state conformation preferentially binds to a target in an active conformational state and does not bind or binds to a lower extent to a target in an inactive conformational state, thus having a higher affinity for said active conformational state, or vice versa. The terms "specifically bind," "selectively bind," "preferentially bind," and their grammatical equivalents are used interchangeably herein. The terms "conformation-specific" or "conformation-selective" are also used interchangeably herein.

[0047] Detailed Description A novel concept for the design of rigidly fused cytokine-containing functional fusion proteins is presented herein. The novel fusion proteins originate from the generation of fusions between cytokines and scaffold proteins, which are folded proteins that block the topology of the cytokine so that the cytokine remains in its typical fold and functions to specifically bind to its cognate receptor in a similar manner compared to the non-fused cytokine ligand. The novel fusion proteins are demonstrated herein as fusions originating from cytokines with a core domain or motif based on a conserved secondary β-strand, such as the chemokine cytokines or interleukin (IL-1) family. Blocking the "β-strand core domain-containing" or "β-strand-containing domain" cytokines, their amino acid sequence by the insertion of a scaffold protein, used interchangeably herein, results in a change in the topology of the cytokine protein, which, unexpectedly, remains in its typical fold and functions to specifically bind to its receptor in a similar manner compared to the non-fused cytokine ligand. Classical conjugation of polypeptide components that are typically not linked in their native state is performed by linking their respective amino (N-) and carboxy (C-) termini directly or through peptide bonds to form a single continuous polypeptide. These fusions are often made via flexible linkers or at least connected in a flexible manner, meaning that the fusion partners are not in a stable position or conformation relative to each other. As shown in Figure 1A, linking proteins via their N- and C-termini results in a simple linear linkage, facile fusion, but unstable and susceptible to degradation, thus resulting in non-functional ligand proteins in some cases. On the other hand, the rigid chimeric / fusion proteins presented herein, which have one or more fusion points or connections in the primary topology of two or more proteins, possess at least one inflexible fusion point (Figure 1B).The present invention uniquely includes a cytokine ligand protein in which the rotation or bending of the cytokine protein relative to its fusion partner, the scaffold protein, is prohibited through the creation of several fusions. Through the presence of several fusions within the same chimera, an improvement in the rigidity of the novel chimera of the present invention is obtained, which is the result of a perfect design of the fusion site to allow a fusion that can preserve the folding of its cytokine domain as well as its function of binding to its receptor. The rigidity of a protein is indeed inherent to the (tertiary) structure of the protein, in this case the novel chimera. It has been shown that an increase in rigidity can be obtained by changing the topology of a known protein fold (King et al., 2015). Thus, the rigidity of the fusion created in the fusion protein of the present invention provides a sufficiently strong rigidity to "orient" or "fix" the cytokine receptor to which the fused cytokine ligand specifically binds, but in most cases the rigidity is still lower than that of the target or antigen itself. However, the fact that the rigid fusion protein of the present invention still maintains its receptor binding and activation functionality is a surprising finding, since interruption of the primary topology can lead to changes in domains or protein folding, affecting the tertiary topology and receptor binding or activation. Although one skilled in the art can use structural information to design such fusions, the actual folding of fusion proteins translated from novel nucleic acid constructs exogenously introduced into cells remains unpredictable. It is demonstrated herein that this interruption of primary topology does not affect receptor binding or activation, which opens new avenues in the fields of cytokine receptor structural biology and drug discovery, specifically chemokine and IL receptors as shown herein. The present invention relates to a novel combination of providing a unique next-generation fusion technology to allow non-covalent attachment of proteins, and providing high affinity and / or conformationally selective chemokine / IL receptor binding capacity.This new type of fusion protein can be useful in several valuable applications depending on the type of cytokine family, e.g., chemokines or chemokine variants, and IL or IL-1 receptor type interleukins, or the type of scaffold protein used to generate the fusion protein. The advantages are numerous and easy to use in structural biology, facilitating cryo-EM and X-ray crystallography for difficult proteins, such as seven-transmembrane proteins, such as GPCRs. With rigid chaperone tools now available and their use to develop improved and more robust therapeutic and diagnostic molecules, e.g., by structure-based drug design and structure-based screening of novel compounds, it is foreseeable that this next-generation fusion technology will lead to great advances in the structural biology of GPCR and IL receptor complexes. Indeed, when used for conformation-selective recognition of cytokine receptors, these tools are equally applicable in binding modes that stabilize the receptor in functional conformations, e.g., active conformations, more specifically, agonist, partial agonist, or biased agonist conformations. Depending on the cytokine ligand or ligand variant, further applications of the fusion proteins of the present invention are found based on specific cytokine (chemokine or IL) ligands that are described to specifically stabilize druggable signaling conformations to enable screening of pathway-selective agonists. With the rapid advancement of such technologies in biotechnology, it is foreseeable that the present invention will have an impact on the creation of novel protein therapeutics and the improvement of the performance of current protein drugs.

[0048] In a first aspect, the present invention relates to a functional fusion protein comprising a cytokine fused to a scaffold protein, said scaffold protein being connected to a cytokine protein in such a way that it blocks the topology of said cytokine via fusion at at least one or more amino acid sites accessible in said cytokine structural fold. The fusion protein is "functional" in that it retains its receptor binding functionality in a similar manner compared to a cytokine ligand in its native or wild-type form that is not fused to said scaffold protein. In one embodiment, said fusion protein is a conformation-selective binding domain. Cytokines comprise highly diverse ligand superfamilies, preferred cytokine superfamilies are those that have conserved core domains or motifs based on or containing β-strands, revealing accessible amino acid sites in these exposed regions present in β-turns or loops that interconnect these β-strands. The new fusion should contain accessible sites sufficiently distant from the receptor binding site of the cytokine so as not to impair receptor binding in order to retain this functionality. The fact that cytokines are relatively small proteins increases the complexity in designing such functional fusions and therefore provides a surprising solution as presented herein, allowing the skilled artisan to derive accessible sites present in exposed turns of the conserved core domains of these β-strand based cytokines.

[0049] In a first embodiment, the present invention relates to a fusion protein comprising a cytokine belonging to the chemokine superfamily fused to a scaffold protein, said scaffold protein being a folded protein of at least 50 amino acids, which is connected to a chemokine core domain in such a way as to block the topology of said core domain via fusion at at least one or more amino acid sites accessible in the exposed β-turn of the fold of said chemokine core domain. The fusion protein is further characterized in that it retains its receptor binding functionality in a similar manner compared to the native or wild-type chemokine not fused to said scaffold protein. Thus, in one embodiment, said fusion protein is a conformation-selective binding domain.

[0050] Chemokine protein ligands have been classified into four subfamilies, CC, CXC, C, and CX3C, where X is any amino acid, according to a characteristic pattern of cysteine ​​residues proximal to the N-terminus of the mature protein. However, the basic tertiary structure or architecture of all chemokines contains a disordered N-terminal "signaling domain" followed by a structured "core domain," which contains an N-loop, a three-stranded β-sheet, and a C-terminal helix (Figure 2).

[0051] In each subfamily, many chemokines bind multiple receptors, and some receptors bind multiple chemokines. Chemokines are known to form dimers, and different dimerization motifs among different subfamilies were initially hypothesized to define the specificity of the receptor. However, it appears that it is rather oligomerization that is essential for binding to glycosaminoglycans, although functional assays have demonstrated that monomers do indeed bind and activate the receptor. Generally, the chemokine core domain forms an interaction site or chemokine recognition site (CRS1) with the N-terminus of the chemokine receptor, while the N-terminus of the chemokine interacts with the receptor-ligand binding pocket of the receptor (chemokine recognition site 2, CRS2). The first interaction is the binding of the receptor N-terminal to the chemokine core domain (CRS1), which can precisely position the chemokine N-terminal signaling domain and allow this interaction with the CRS2 TM pocket. Multiple structural studies have shown that receptor binding and activation can be at least partially separated. However, further high-resolution structural analysis of conformation-specific complexes with intact receptors is required, which so far has been extremely challenging due to the nature of transmembrane receptors and therefore the limitations of analyzing the more tractable soluble complexes, most likely using NMR approaches.

[0052] The structural role of sulfotyrosine in the receptor has been established, which allows salt bridge formation with homologous basic residues in the β2-β3 hairpin or loop of the chemokine. The chemokine-receptor interface is thought to involve the N-loop and β2-β3 strands of the β-sheet of the core domain. The fact that the structural rearrangements upon CRS1 binding differ from complex to complex underscores the point that the recognition and activation mechanisms cannot be simplified, but that better structural determination tools are needed. Indeed, several modified chemokines have also been applied to elucidate the role of specific receptors in disease, and the pharmacology of ligands in the field of cytokines and more specifically chemokines has shown that they retain high affinity for the receptor but benefit from minor manipulations that result in adapted functional outcomes, such as agonist, inverse agonist, antagonist, or superagonist / antagonist features. Indeed, the general prototype chaperones, such as the fusion proteins presented here, provide a solution for profiling the features of chemokine ligand / receptor interactions and activation mechanisms. Chaperone proteins such as nanobodies are known to help stabilize membrane receptor conformations (Manglik et al., 2017), but these types of chaperones do not force the receptor into a conformation that binds only a specific ligand in a specific conformation. Moreover, novel chemokine fusion proteins may also offer advantages in drug screening of a specific receptor conformational state of the intact receptor. So far, very few chemokine / receptor complex structures have been determined using intact receptors (CXCR4 / vMIP-II, US28 / CX3CL1), and in recent years, complexes of CCR5 receptor with protein inhibitors, e.g., 5P7-CCL5, have provided new insights in chemokine-receptor signaling leading to HIV inhibition. The latter demonstrated that the ligand 5P7-CCL5 interacted with CCR5 in a manner that could not be precisely predicted from the two-site model described herein above, since its N-loop, β1 strand, and 30s loop are the main interaction sites with the receptor.So far, more structural data have been obtained using, for example, N-terminal peptides of the receptors together with ligands (CXCL8 / CXCR1 peptides; CXCL12 / CXCR4 sulfopeptides, CCL11 / CCR3 peptides), but there is a risk that only a partial picture of the structure in its native context is obtained.

[0053] Another embodiment relates to novel fusion proteins, wherein the cytokine is an interleukin, and the scaffold protein blocks the topology of the interleukin β barrel core motif at one or more accessible sites in the exposed β-turns of the β barrel core motif. More specifically, the fusion protein relates to novel fusion proteins, wherein the cytokine is an IL-1 receptor interleukin. The interleukin (IL-1) superfamily of cytokines is a key regulator of innate and adaptive immunity and plays a key role in host defense against infection, inflammation, injury, and stress. The "IL-1 receptor type interleukin" superfamily or "IL-1 family" interleukins are used interchangeably herein and include interleukins IL-1, IL-1α, IL-1β, IL-18, IL18BP, IL1F5, IL1F6, IL1F7, IL1F8, IL1F10, IL-33, and IL-36, IL36B, and IL-37. These cytokines are related to each other by origin, receptor structure, and signaling pathway. The receptors of the IL-1 superfamily interleukins share a similar structure, containing three Ig-like domains in their ectodomain, and an intracellular Toll / IL-1R (TIR) ​​domain, which is also found in Toll-like receptors. Initiation of cytokine signaling requires two receptors: a primary specific receptor and a co-receptor, which may be shared in some cases. The primary receptor is responsible for specific cytokine binding, whereas the co-receptor does not bind the cytokine itself, but associates with a preassembled binary complex from the cytokine and the primary receptor. Binding of the cytokine to its respective receptor forms a signaling tertiary complex, leading to dimerization of the TIR domains of the two receptors. This initiates intracellular signaling by activating mitogen-activated protein kinases (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Signaling induces inflammatory responses, such as induction of cyclooxygenase type 2, increased expression of adhesion molecules, and synthesis of nitric oxide.

[0054] The three-dimensional structures of several interleukin cytokines of the IL-1 superfamily have been determined, demonstrating that these cytokines adopt a conserved signature β-trefoil fold that contains 12 antiparallel β-strands arranged in a three-fold symmetry pattern with limited sequence similarity. The β-barrel core motif is packed with variable amounts of helices in each cytokine structure. Superimposition of the Cα atoms of each of the human cytokines reveals a conserved hydrophobic core with significant flexibility in the loop regions. Surface residues and loops between the β-strands do not appear to be important for overall stability and differ significantly among the cytokines, consistent with their low sequence similarity and partially explaining their unique recognition by their respective receptors (with specific loops). For example, human IL-18 shares approximately 65% ​​sequence identity with mouse IL-18, but only 15% and 18% identity with human IL-1α and human IL-1β, respectively. Nevertheless, IL-18 shows a striking similarity in its three-dimensional structure to other IL-1 cytokines, thus providing a second example of a superfamily within the cytokines with a conserved structural core domain based on β-strands interconnected by flexible β-turns or loops, which may be partly involved in receptor recognition and partly involved in connecting with the folded scaffold proteins presented here to obtain novel extended fusion ligands.

[0055] One embodiment provides a cytokine fusion protein in which a conserved core domain based on β-strands is fused to a scaffold protein such that the scaffold protein "blocks" the topology of the core domain. In general, the "topology" of a protein refers to the orientation of regular secondary structures with respect to each other in three-dimensional space. A protein fold is defined primarily by the topology of the polypeptide chain (Orengo et al., 1994). Thus, at the most fundamental level, the "primary topology" is defined as the sequence of secondary structural elements (SSEs), which are involved in protein fold recognition motifs and thus in the folding of secondary and tertiary proteins / domains. Thus, in terms of the structure of a protein, the true or primary topology is the sequence of SSEs, i.e., given that the N- and C-termini of the protein chain can be retained, and simply considered, the topology does not change whatever the protein fold. A protein fold is then described as the tertiary topology as well as the primary and tertiary structures of a protein (see also Martin, 2000).

[0056] Specifically, as presented herein, the chemokine core domain of the chemokine functional fusion protein of the invention is thus able to retain its 3D folding, although this primary topology is interrupted by the introduction of a scaffold protein fusion between the β2 and β3 β strands of the chemokine core domain at an accessible site of an exposed β-turn or loop, and, unexpectedly, said chemokine was also able to retain its tertiary structure and its functional receptor binding ability. Similarly, the IL-1-like receptor interleukin IL-1β has a conserved β-barrel core motif, whose primary topology is interrupted by an exposed β-turn between the two β-strands of the core, conserved by the insertion of the folded scaffold protein presented herein, providing a correctly folded or functional fusion protein, particularly with retained binding ability.

[0057] "Scaffold protein" refers to any type of protein that has a structure or fold that allows it to be fused with another protein, particularly a cytokine or chemokine as described herein. The classical principle of protein folding is that all the information required for a protein to adopt a precise three-dimensional conformation is provided by its amino acid sequence, resulting in a specific folded protein that is held together by various molecular interactions. To be useful as a scaffold herein, a scaffold protein must be folded into a distinct three-dimensional conformation. For this reason, said scaffold protein is defined herein as a "folded" protein, and in the case of shorter peptides, the length of these amino acids is limited to a minimum, since it is generally known that these are very flexible and do not provide a folded structure. For this reason, the scaffold protein used in the novel functional fusion protein used herein is uniquely different from peptides or very small polypeptides, and for example, a scaffold protein composed of less than 40 amino acids is not considered to be a suitable scaffold protein for fusion as a Megakine. Thus, a "scaffold protein" as defined herein is a folded protein of at least 200 amino acids, or 150 amino acids, or at least 100 amino acids, or at least 50 amino acids, or more preferably at least 40 amino acids, at least 30 amino acids, at least 20 amino acids, at least 10 amino acids, at least 9 amino acids. Linkers or peptides, particularly linkers of less than 8 amino acids, are not suitable as scaffold proteins for the purposes of the present invention. Furthermore, such "scaffold", "junction" or "fusion partner" proteins preferably have at least one exposed region in their tertiary structure, providing at least one accessible site for cleavage as a fusion point for a cytokine or chemokine.When a scaffold polypeptide is used to assemble with a cytokine or chemokine core domain, this results in a fusion protein in a docked configuration, increasing mass, providing symmetry, and / or providing an extended ligand that induces a specific conformation of the equivalent receptor, and / or improving or increasing functionality for the receptor. Thus, depending on the type of scaffold protein used, different purposes of the resulting fusion protein are foreseen. The type and nature of the scaffold protein is irrelevant in that it can be any protein, and depending on its structure, size, function, or presence, the scaffold protein fused to said cytokine or chemokine core domain as well as the fusion protein of the invention will be useful in different fields of application. The structure of the scaffold protein will affect the final chimeric structure, and therefore the skilled person must implement known structural information on the scaffold protein and take reasonable expectations into account when selecting a scaffold. Examples of scaffold proteins are provided in the examples of the present application, and a non-limiting number of folded proteins, among others, enzymes, membrane proteins, receptors, adaptor proteins, chaperones, transcription factors, nuclear proteins, antigen-binding proteins themselves, e.g. nanobodies, may be applied as scaffold proteins to generate the fusion proteins of the present invention. In a preferred embodiment, the 3D structure of said folded scaffold protein is known or can be predicted by the skilled person, so that an accessible site for fusing a cytokine or its conserved core domain can be determined by said skilled person.

[0058] The novel chimeric or fusion proteins are uniquely fused to avoid the junctions being flexible, loose and weak links / regions within the chimeric protein structure. A convenient means of linking or fusing two polypeptides is by expressing them as a fusion protein from a recombinant nucleic acid molecule comprising a first polynucleotide encoding a first polypeptide operably linked to a second polynucleotide encoding a second polypeptide in a classically known manner. However, in the recombinant nucleic acid molecules of the present invention, the interruption of the topology of the cytokine or its conserved core domain by the scaffold is also reflected in the design of the gene fusion from which the fusion protein is expressed. Thus, in one embodiment, the fusion protein is encoded by a chimeric gene formed by recombining a part of a gene encoding a cytokine, or specifically a chemokine or IL, and a part of a gene encoding a scaffold protein, said encoded scaffold protein blocking the primary topology of the encoded cytokine, or specifically a conserved core domain of said chemokine or IL, at one or more accessible sites of said domain in its exposed β-turns, via at least two or more direct fusions or via fusions made by encoded peptide linkers. Thus, the polynucleotides encoding the polypeptides to be fused are fragmented and recombined to provide a fusion protein that provides a rigid, inflexible link, connection or fusion between said proteins. The novel chimeras are generated by fusing a scaffold protein with a cytokine, or specifically a conserved chemokine or IL core domain, such that the primary topology of the cytokine or conserved core domain is blocked, which means that the amino acid sequence of the cytokine core domain is blocked at accessible sites and linked to accessible amino acids of the scaffold protein, thus possibly blocking the sequence. The joints are made intramolecularly, in other words, within the amino acid sequence (see the examples and figures).Thus, the recombinant fusion of the present invention results in chimeras that are not simply fused at the N-terminus or C-terminus, but contain at least one internal fusion site, which is either directly fused or fused via a linker peptide. When applying a circularly permuted scaffold to produce a fusion protein, the amino acid sequence of the scaffold protein is changed by connecting the N-terminus and the C-terminus, followed by truncation or separation of the amino acid sequence at another site in the sequence of the scaffold protein corresponding to an accessible site in its tertiary structure, and fusing it to the amino acid sequence of a cytokine or chemokine / IL moiety. The N-terminus and C-terminus connection to obtain a circular permutation can be made through direct fusion, through a linker peptide, or even via a terminal peptide bond after short deletion of the regions near the N-terminus and C-terminus.

[0059] The terms "accessible site", "fusion site", or "fusion point", or "connection site", or "exposed site" are used interchangeably herein and all refer to amino acid sites of a protein sequence that are structurally accessible, preferably located on or exposed to the surface of the protein, more preferably an exposed region of a β-turn or loop. Those skilled in the art can derive these sites of cytokines from the disclosure provided herein. Receptor binding or activation sites of cytokines, such as chemokines or ILs, are often associated with such exposed regions, for example the disordered N-terminal signaling domain or N-loop of chemokines, or the β-turn between β-strand 4 and β-strand 5 of IL-1. However, blocking these sites to fuse chemokines to scaffold proteins may result in loss of receptor binding or activation ability, which is not suitable for the fusion proteins of the present invention and is therefore not intended to apply herein as an accessible fusion site. Thus, "accessible sites" as "loops" or "beta turns" as defined herein, and "exposed regions" refer to those sites and regions that are not receptor sites or regions, or that do not (e.g. sterically) interfere with the receptor binding site. The binding sites may vary with respect to the targeted receptor, but generally involve the N-terminal signaling domain and N-loop of chemokines and the corresponding β-turn between β4 and β5 of IL-1 type receptor interleukins. The N- or C-termini of proteins are also in most cases the "loose" ends of the protein 3D structure and therefore accessible from the surface. These can be considered as accessible sites in the chimeras of the present invention, except when receptor binding or activation requires such ends to be free, and under conditions that result in blocking / insertion at this accessible site, blocking the topology, since at least one other accessible site in the cytokine / chemokine core domain is used for fusion, and fusion of this latter accessible site provides rigidity to the new chimera.Thus, accessible sites may include amino- and / or carboxy-terminal sites of the protein, but chimeras cannot be based solely on fusion from accessible sites composed of the N- or C-terminus. At least one or more sites of the chemokine / IL core domain are used for fusion with the scaffold protein, resulting in the interruption of the topology of the known conventional domain fold. Thus, in one embodiment, the at least one accessible site is not the N- and / or C-terminal site of said domain, if there is one, and does not include the N- or C-terminal site of said domain. In a particular embodiment, the at least one site is not the N- or C-terminal amino acid of said domain. In another embodiment, the accessible site may be the N- or C-terminal site of the conserved core domain, if at least one or more sites are used for fusion with the scaffold protein. The scaffold protein is fused via an accessible site that is also visible from its tertiary structure, in one embodiment, the at least one site is not the N- or C-terminus of the scaffold protein, and in an alternative embodiment, the at least one site is the N- or C-terminus of the folded scaffold.

[0060] In some embodiments, the fusion protein comprises an N-terminal fragment of the scaffold protein fused at a block in an exposed region of the conserved core domain, and a C-terminal fragment of the scaffold protein fused at its C-terminal end near the conserved core domain.

[0061] In some embodiments of the invention, the fusion may be a direct fusion or a fusion created by a linker peptide, said fusion site being fully designed to result in a rigid, inflexible fusion protein. In addition to the location of the selected accessible site, the length and type of the linker peptide contribute to the rigidity and possibly functionality of the resulting fusion protein. In the context of the present invention, the polypeptides constituting the fusion protein are directly fused to each other by connection via a peptide bond or indirectly linked, which assembles the two polypeptides through a connection via a short peptide linker. Preferred "linker molecules", "linkers" or "short polypeptide linkers" are peptides of up to 10 amino acids in length, more preferably 4 amino acids in length, typically only 3 amino acids in length, preferably only 2 amino acids in length, or even more preferably only 1 amino acid in length, to provide the desired rigidity to the fusion junction at the accessible site. Non-limiting examples of suitable linker sequences are described in the Examples section, which can be randomized, and linkers have been successfully selected to maintain a fixed distance between the structural domains and to allow the fusion partners to maintain their independent functions (e.g., receptor binding). In embodiments involving the use of rigid linkers, these are generally known to exhibit unique conformations, either by adopting an alpha-helical structure or by containing multiple proline residues, and in many circumstances these separate functional domains more efficiently than flexible linkers, and may be equally suitable with short lengths, preferably only 1-4 amino acids.

[0062] In an alternative embodiment, the fusion protein is described as a rigid fusion protein comprising i) an N-terminal amino acid sequence of a cytokine (e.g., a chemokine or IL), ii) a functional scaffold protein, and iii) a cytokine (e.g., a chemokine or IL) sequence lacking said N-terminal amino acid sequence of i), where i) and iii) are linked to said scaffold protein of ii). In a preferred embodiment, said rigid fusion protein comprises an N-terminal amino acid sequence corresponding to a chemokine N-terminal signaling domain, followed by a portion of a chemokine core domain containing the first two β-strands of a β-sheet fused to the amino acid sequence of a scaffold protein or a circularly permuted scaffold protein, which is blocked and fused at an accessible site corresponding to a site in an exposed surface loop or turn, and finally fused to the remainder of the chemokine containing the β3 strand of the core domain and the C-terminal helix of said domain. Thus, insertion of the scaffold protein into the chemokine protein sequence is obtained at a single blocked amino acid sequence site that corresponds to an accessible site in this β2-β3 turn or loop of the chemokine core domain, which is also called the 40s loop in chemokine structural terminology.

[0063] In one embodiment, the accessible site of the chemokine core domain is present in an exposed region of the domain fold. The exposed region is identified as a series of less fixed amino acids located mainly on the surface of the protein, at the edge of the structure. Preferably, the exposed region is present as a loop or β-turn of the protein structure. The simplest identification of an "exposed region" of the chemokine core domain is an exposed loop, preferably a β-turn, which is an exposed loop located at the edge of the β-sheet 3D structure. In the case of a three-stranded β-sheet structure, possibilities include a β1-β2 turn or loop, also called the 30s loop, or a β2-β3 turn or loop, also called the 40s loop. In certain chemokine receptor complexes, the 30s loop is known to be involved in receptor binding and is therefore less preferred for blocking upon fusion of the scaffold compared to the 40s loop.

[0064] In another embodiment, the scaffold protein has a circular permutation. In a preferred embodiment, said circular permutation of the scaffold protein is at the N-terminus and / or C-terminus of the scaffold protein, or most preferably between the N-terminus and C-terminus of the scaffold protein. Another embodiment provides a scaffold protein comprising at least two antiparallel β-strands.

[0065] In one embodiment, a fusion protein (with two peptide bonds or two short linkers) is obtained by connecting a cytokine or chemokine core domain to a scaffold through fusion with a circularly permuted scaffold protein at its truncated accessible site in its sequence corresponding to an exposed region of its structure (said exposed or accessible site is not N- or C-terminal), via interruption of the cytokine or chemokine core domain primary topology at a truncated accessible site in this sequence corresponding to a β2-β3 turn. Thus, in a particular embodiment where the circular permutation of the scaffold protein is present at the N- and C-terminus (as in FIG. 2), the scaffold protein sequence can be fused by recombination with the cytokine or chemokine fragment as a whole (as in FIG. 7). In a particular embodiment, said fusion protein increases its rigidity through the additional generation of a reinforcing disulfide bridge formed by a cysteine ​​residue located in the cytokine or chemokine, preferably near the accessible N-terminal end.

[0066] A further aspect of the invention relates to novel functional fusion proteins comprising a cytokine, such as a cytokine comprising a chemokine or IL core domain, fused to a scaffold protein, said scaffold protein interrupting the topology of the conserved core domain of said cytokine chemokine / IL, the total mass or molecular weight of the scaffold protein being at least 30 kDa, and the increase in mass and structural properties upon binding of the fusion to a target, such as a ligand's receptor, is significant and sufficient to allow three-dimensional structural analysis of the target when non-covalently bound to said chimera. In another embodiment, the total mass or molecular weight of the scaffold protein is at least 40, at least 45, at least 50, or at least 60 kDa. This particular size or mass increase has the effect of decreasing the signal-to-noise ratio in the image. The chimera then provides a structural guide by providing suitable features for accurate image registration of small or difficult to crystallize proteins to reach sufficiently high resolution using cryo-EM and X-ray crystallography.

[0067] A further aspect of the invention relates to a nucleic acid molecule encoding said fusion protein of the invention. Said nucleic acid molecule comprises the coding sequence of said cytokine, chemokine or interleukin and said scaffold protein and / or fragment thereof, and said domain interrupted topology is reflected by the fact that said domain sequence contains an insertion of a scaffold protein sequence (or a circularly permuted sequence or fragment thereof), whereby an N-terminal cytokine, chemokine or IL fragment and a C-terminal cytokine, chemokine or IL conserved core domain fragment are separated by a scaffold protein sequence or fragment thereof within said nucleic acid molecule. In another embodiment, a chimeric gene is described having at least a promoter, said nucleic acid molecule encoding a fusion protein, and a 3'-terminal region containing a transcription termination signal. Another embodiment relates to an expression cassette encoding said fusion protein of the invention or comprising a nucleic acid molecule or chimeric gene encoding said fusion protein. Said expression cassette is in certain embodiments applied in a universal format as a library containing a large set of cytokine fusions, e.g. chemokine or interleukin fusions, to select for the most suitable binders of receptors or antibodies, or alternatively targets or interaction partners. Further embodiments relate to vectors comprising said expression cassettes or nucleic acid molecules encoding the fusion proteins of the invention. In certain embodiments, vectors for expression in E. coli allow the production of fusion proteins in the presence or absence of their target and their purification. Alternative embodiments relate to host cells comprising the fusion proteins of the invention or the nucleic acid molecules or expression cassettes or vectors encoding the fusion proteins of the invention. In certain embodiments, said host cells further co-express a target protein or a receptor that specifically binds to the cytokine, e.g. a chemokine or IL, of the fusion protein. Another embodiment discloses the use of said host cells, or isolated membrane preparations thereof, or proteins isolated therefrom, for ligand screening, drug screening, protein capture and purification, or biophysical testing.The present invention further encompasses an option for high throughput cloning in generic fusion vectors. The generic vectors are described in additional embodiments, where the vectors are particularly suitable for surface display in yeast, phage, bacteria or viruses. Furthermore, the vectors have application in the selection and screening of large sets of different ligands, especially immune libraries that contain such generic vectors, e.g. with different linkers, or expression cassettes. Thus, differential sequences in the libraries constructed to screen novel fusion proteins for specific receptors are provided by differences in the linker sequence, or alternatively in other regions.

[0068] In one embodiment, the vectors of the invention are suitable for use in methods involving displaying a collection of cytokine fusion proteins on the extracellular surface of a population of cells. Surface display methods are reviewed in Hoogenboom, (2005; Nature Biotechnol 23, 1105-16) and include bacterial display, yeast display, and (bacteri)phage display. Preferably, the population of cells is yeast cells. The different yeast surface display methods all provide a means to tightly link each fusion protein encoded by the library to the extracellular surface of yeast cells that carry the plasmid encoding that protein. Most yeast display methods described to date use the yeast Saccharomyces cerevisiae, although other yeast species, such as Pichia pastoris, can also be used. More specifically, in some embodiments, the yeast strain is derived from a genus or species selected from the group consisting of Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, and Candida. In some embodiments, the yeast species is selected from the group consisting of S. cerevisiae, P. pastoris, H. polymorpha, S. pombe, K. lactis, Y. lipolytica, and C. albicans. Most yeast expressed fusion proteins are based on GPI (glycosyl-phosphatidyl-inositol) anchored proteins that play a key role in the surface expression of cell surface proteins and are essential for yeast viability. One such protein, alpha-agglutinin, consists of a core subunit encoded by AGA1, linked through a disulfide bridge to a small binding subunit encoded by AGA2. Proteins encoded by the nucleic acid library can be introduced into the N-terminal region of AGA1 or the C-terminal or N-terminal regions of AGA2. Either fusion pattern results in the display of the polypeptide on the yeast cell surface.

[0069] The vectors disclosed herein may also be suitable for prokaryotic host cells to display proteins on their surfaces. Suitable prokaryotic cells for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41, disclosed in DD 266,710 published April 12, 1989). Examples of suitable cell surface proteins include Pseudomonas, such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative and not limiting. When the host cell is a prokaryotic cell, examples of suitable cell surface proteins include suitable bacterial outer membrane proteins. Such outer membrane proteins include fimbriae and flagella, lipoproteins, ice nucleation proteins, and autotransporters. Exemplary bacterial proteins used for heterologous protein display include LamB (Charbit et al., EMBO J, 5(11):3029-37 (1986)), OmpA (Freudl, Gene, 82(2):229-36 (1989)), and intimin (Wentzel et al., J Biol Chem, 274(30):21037-43, (1999)).Additional exemplary outer membrane proteins include, but are not limited to, FliC, pullulanase, OprF, OprI, PhoE, MisL, and cytolysin. A comprehensive list of bacterial membrane proteins that have been used for surface display is detailed in Lee et al., Trends Biotechnol, 21(1):45-52 (2003), Jose, Appl Microbiol Biotechnol, 69(6):607-14 (2006), and Daugherty, Curr Opin Struct Biol, 17(4):474-80 (2007).

[0070] Furthermore, to allow detailed screening selection, the vectors can be applied to yeast and / or phage display after FACS and panning, respectively. For example, display of cytokine or chemokine fusion proteins on yeast cells combined with the resolving power of fluorescence-activated cell sorting (FACS) provides a preferred selection method. In yeast display, each cytokine or chemokine fusion protein is displayed as a fusion to the Aga2p protein, for example, at about 50,000 copies on the surface of a single cell. In the case of selection by FACS, labeling with different fluorescent dyes determines the selection procedure. The yeast library displaying the fusion proteins can then be stained with a mixture of the fluorescent proteins used. Two-color FACS can then be used to analyze the properties of each fusion protein displayed on a particular yeast cell and to reveal the individual cell populations. Yeast cells displaying a fusion protein that is well suited for binding the protein of interest, such as a receptor or antibody, bind and can be sorted along a diagonal in two-color FACS. The use of vectors for such selection methods is most preferred when screening of fusion proteins specifically targets, for example, transient protein-protein interactions or conformation-selective binding states. Similarly, vectors for phage display can be applied and used to display fusion proteins on bacteriophages, followed by panning. Display can be performed on M13 particles, for example, by fusion of cytokine or chemokine fusion proteins with phage coat protein III in the same universal vector (Hoogenboom, 2000; Immunology today. 5699: 371-378). When selecting fusion proteins and / or transient protein-protein interactions that specifically bind to a particular conformation, for example, only one of the interacting promoters is immobilized on a solid phase. Then, biological selection by panning of the fusion proteins displayed on phage is performed in the presence of an excess of the remaining soluble promoters. Optionally, it can start with a panning round of cross-linked complexes or proteins immobilized on a solid phase.

[0071] Another aspect of the invention relates to a complex comprising said fusion protein and a receptor protein, said receptor protein specifically binding to a cytokine, for example a chemokine or interleukin, among other types of cytokines, and their cognate receptors. More particularly, an embodiment relates to a protein complex in which said receptor protein is bound to the cytokine portion of said fusion protein. An embodiment discloses a complex as described herein, in which the cytokine or chemokine or IL of said fusion protein is a conformation-selective ligand. More particularly, a complex is disclosed in which the cytokine or chemokine or IL portion of the fusion protein stabilizes the receptor protein in a functional conformation. More particularly, said functional conformation may involve an agonist conformation, may involve a partial agonist conformation, or may involve a biased agonist conformation, among others. Alternatively, complexes of the invention are disclosed in which the cytokine or chemokine or IL of the fusion protein stabilizes the receptor protein in a functional conformation, said functional conformation being an inactive conformation or said functional conformation involving an inverse agonist conformation. Another embodiment relates to said cytokine fusion protein, or a chemokine or IL fusion protein in complex with said receptor, which is activated upon binding of the receptor to the fusion protein. As already described herein, several cytokine receptors, including chemokine and / or IL receptors, require several interfaces to bind ligands to achieve an activated state.

[0072] Another embodiment of the invention relates to a method for producing a cytokine functional fusion protein according to the invention, comprising the steps of: (a) culturing a host comprising the vector, expression cassette, chimeric gene, or nucleic acid sequence of the invention under conditions conducive to expression of the fusion protein, and (b) optionally recovering the expressed polypeptide.

[0073] A more particular embodiment is a method of producing a chemokine fusion protein as described herein, comprising the steps of: (a) selecting a chemokine ligand and a scaffold protein whose 3D structure reveals accessible sites in exposed regions as loops or turns for interruption of the amino acid sequence without interrupting the primary topology; (b) designing a gene fusion construct, wherein the nucleic acid sequence is selected from the following nucleic acid sequence molecules: 1. The chemokine sequence interruption is located at a position corresponding to an accessible site between β-strand β2 and β-strand β3 of the conserved core domain structure of a chemokine protein; 2. A scaffold sequence (as a whole) for insertion by fusing its 5' and 3' nucleic acid sequence ends, or a scaffold protein for insertion by fusing alternative blocked sites of this sequence of said scaffold protein, present in an accessible site of said scaffold, such as a loop or β-turn; 3. The 5'-most blocked sequence of the 3'-end of the chemokine (corresponding to the amino acid residues C-terminal of β-strand β2) is fused to the 5'-start of the 5'-most (blocked) site of the scaffold protein, and the 5'-start of the C-most blocked site of the chemokine (corresponding to the amino acid residues N-terminal of β-strand β3) is fused to the 3'-end of the C-most blocked site of the scaffold protein. designed to encode a protein sequence encoded by the nucleic acid sequence molecule; (c) introducing the gene fusion construct into an expression system to obtain a fusion protein, wherein the chemokine is fused to a scaffold protein at two or more sites in its core domain. The present invention relates to a method comprising the steps of:

[0074] An alternative embodiment is a method of producing or purifying a fusion protein as described herein, comprising the steps of: (a) selecting a chemokine ligand and a scaffold protein that have accessible loops or turns in their tertiary structure that can be interrupted to create a fusion protein without interrupting the primary topology of the chemokine and / or the primary topology of the scaffold protein; (b) designing a gene fusion construct, the nucleic acid sequence of which is designed to encode a protein in an expression host, comprising: 1. The protein sequence of the chemokine is interrupted by amino acids corresponding to an accessible site between β-strand β2 and β-strand β3 of the core domain; 2. A scaffold protein is fused at its N-terminus and C-terminus to obtain a circularly permuted scaffold protein; 3. The circularly permuted scaffold protein of 2. is blocked in its amino acid sequence corresponding to an accessible site in an exposed loop or turn of its tertiary sequence, the blocking site being different from the amino acids fused in step 2. 4. The C-terminal end of the N-terminal portion of the chemokine (i.e., the blocked portion of the chemokine on the C-terminal side of β-strand β2) is fused to the N-terminus of the circularly permuted scaffold protein, and the N-terminal start of the C-terminal portion of the chemokine (i.e., the blocked portion of the chemokine on the N-terminal side of β-strand β3) is fused to the C-terminus of the circularly permuted scaffold protein; (c) introducing the gene fusion construct into an expression system to obtain a fusion protein, wherein the chemokine is fused to a circularly permuted scaffold protein at two or more sites in its core domain. A method is disclosed that includes:

[0075] Another aspect relates to the use of the cytokine functional fusion protein of the invention or the nucleic acid molecule, chimeric gene, expression cassette, vector, or complex in the structural analysis of its cognate receptor protein. In particular, the use of a cytokine fusion protein based on a β-strand-core domain in the structural analysis of a receptor protein, said receptor protein being a protein specifically bound to said cytokine portion of said fusion protein. "Analyzing a structure" or "structural analysis" as used herein refers to determining the arrangement of atoms or atomic coordinates of a protein, often performed by biophysical methods such as X-ray crystallography or cryo-electron microscopy (cryo-EM). In particular, embodiments relate to use in structural analysis, including single particle cryo-EM or crystallography. A major advantage of the use of such cytokine fusion proteins of the invention in structural biology is that they act as aids in crystallography, i.e. they serve as crystal contacts, increasing symmetry and can even be applied as rigidity tools in cryo-EM, which are invaluable for solving large structures of tricky proteins such as membrane receptors, reducing the size barriers that are dealt with today, as well as for increasing symmetry and for stabilizing and visualizing specific conformational states of receptors in complex with said cytokine or chemokine fusion proteins.

[0076] The use of cryo-EM for structure determination has several advantages over traditional approaches such as X-ray crystallography. In particular, cryo-EM has less stringent requirements on the samples analyzed in terms of purity, homogeneity, and quality. Importantly, cryo-EM can be applied to targets that do not form suitable crystals for structure determination. Suspensions of purified or non-purified proteins, alone or in complex with other proteinaceous molecules, such as the cytokine fusion proteins of the invention, or in complex with non-proteinaceous molecules, such as nucleic acids, can be applied to carbon grids for imaging by cryo-EM. The coated grids are typically flash frozen in liquid ethane, preserving the particles in suspension in a frozen hydrated state. Larger particles can be vitrified by freeze fixation. The vitrified samples can be cut into thin sections (typically 40-200 nm thick) in a cryo-ultramicrotome, and the sections can be placed on electron microscope grids for imaging. The quality of the data obtained from the images can be improved by using parallel illumination and good axial alignment of the microscope, allowing resolutions as high as about 3.3 Å to be obtained. At such high resolution, initial model building of the complete atomic structure is possible. However, if structural data at atomic resolution for the selected or closely related target protein and selected heterologous proteins or closely related homologues are available for constrained comparative modeling, lower resolution imaging may be sufficient. To further improve the quality of the data, the microscope is adjusted to a resolution of 1 / 3 Å in the Fourier transform of carbon film images recorded under the same conditions used for imaging. -1 The defocus value for each micrograph can then be determined using software such as CTFFIND.

[0077] Further disclosed herein is a method for determining the three-dimensional structure of a ligand / receptor complex, comprising the steps of: (i) providing a fusion protein according to the present invention, and providing a receptor to form a complex, wherein said receptor protein binds to the cytokine portion of the fusion protein of the present invention, or providing a complex as described herein; (ii) displaying said complex under conditions suitable for structural analysis, wherein the 3D structure of said protein complex is determined at high resolution.

[0078] In certain embodiments, the structural analysis is performed via X-ray crystallography. In another embodiment, the 3D analysis includes cryo-EM. More specifically, the methodology of cryo-EM analysis is described herein below. The sample (e.g., the selected fusion protein in complex with the receptor of interest) is applied to the best performing discharge grid selected (carbon-coated copper grid, C-Flat, 1.2 / 1.3 200-mesh: Electron Microscopy Sciences; gold R1.2 / 1.3 300 mesh UltraAuFoil grid: Quantifoil; etc.) before blotting, and then flash-frozen in liquid ethane (Vitrobot Mark IV (FEI), or other plunger selected). The data of a single grid is collected on a 300 kV electron microscope (e.g., Krios 300 kV with a selected auxiliary phase plate) equipped with a selected detector (e.g., Falcon 3EC direct detector). Micrographs are collected in electronic measurement mode at an appropriate magnification appropriate for the expected size of the ligand / receptor complex. Collected micrographs are manually checked before further image processing. Drift correction, sample motion, dose weighting, CTF fitting and phase shift estimation are applied by the selected software (RELION, SPHIRE package as an example). Particles are collected by the selected software and used for 2D classification. 2D classes are manually inspected and false positives are removed. Particles are binned according to data collection settings. An initial 3D reference model is generated by applying appropriate low pass filters and multiple 3D classes (6 as an example) are generated. Original particles are used for 3D refinement (use soft mask if necessary). Fourier shell correlation (FSC) = 0.143 criterion is used to estimate the resolution of the reconstruction. Local resolution can be calculated by MonoRes implementation in Scipion. Reconstructed cryo-EM maps can be analyzed using UCSF Chimera and Coot software.The design model can be first fitted using UCSF Chimera and analyzed by the software of choice (UCSF Chimera, PyMOL, or Coot).

[0079] Another advantage of the method of the present invention is that structural analysis, which is conventionally possible only with highly pure proteins, becomes less stringent in terms of purity requirements thanks to the use of cytokine fusion proteins. Such cytokine-ligand fusion proteins, more particularly cytokine fusion proteins based on the conserved core domain of such β-strands, such as chemokine or IL-1 fusion proteins, are specifically filtered out from the receptor of interest via their high affinity binding sites in the complex mixture. The receptor protein can thus be captured, frozen and analyzed via cryo-EM.

[0080] In an alternative embodiment, the method is also suitable for 3D analysis, where the receptor protein is a transient protein-protein complex or in a transient specific conformational state.In addition, the fusion protein molecule can also be applied to methods for determining the three-dimensional structure of receptors to stabilize transient protein-protein interactions as targets to enable these structural analyses.

[0081] Another embodiment relates to a method of selecting or screening for a panel of fusion proteins that bind to different conformations of the same receptor protein, comprising the steps of: (i) designing a ligand library of fusion proteins that bind to a receptor protein; and (ii) selecting the fusion proteins via surface yeast display, phage display, or bacteriophage to obtain a fusion protein panel comprising proteins that bind to several related conformational states of said receptor protein, thereby resulting in separate images of several conformations of the receptor protein that are analyzed, for example, in cryo-EM. To obtain specific or certain conformational states, the inventors can utilize cell-based systems where the receptor is on a membrane and the cells can be treated or manipulated according to the experimental objectives.

[0082] In another embodiment, the method and the fusion protein of the invention are used for structure-based drug design and structure-based drug screening. The iterative process of structure-based drug design often proceeds through multiple cycles before an optimized lead enters Phase I clinical trials. The first cycle involves cloning, purification, and structure determination of the receptor protein or nucleic acid by one of three basic methods: X-ray crystallography, NMR, or homology modeling. A computer algorithm is used to place compounds or fragments of compounds from a database into selected regions of the structure. The fusion protein of the invention can be used to fix or stabilize the conformation of a particular structure of the receptor. The selected compounds are scored and ranked based on their steric and electrostatic interactions with the target site, and the best compounds are tested by biochemical assays. In the second cycle, structure determination of the target in complex with promising leads from the first cycle that have at least micromolar inhibition in vitro reveals sites on the compound that can be optimized to increase potency. Similarly, at this point, the fusion protein of the invention begins to act by facilitating the structural analysis of the target receptor protein in a particular conformational state. Additional cycles include synthesis of optimized leads, structure determination of novel target:lead complexes, and further optimization of the lead compounds. After several cycles of the drug design process, optimized compounds usually show significant improvement in binding, and often in target specificity. Once a hit is obtained by screening the library, it is further developed into a lead, and this structural information as well as medicinal chemistry is essential for structure-activity relationship analysis.

[0083] Another embodiment is a method for identifying a (stereoselective) compound comprising the steps of: i) providing a target receptor protein of the invention and a fusion protein that specifically binds to said target receptor protein; ii) Providing a test compound iii) evaluating the selective binding of the test compound to the target receptor protein. The present invention relates to a method comprising the steps of:

[0084] According to a particularly preferred embodiment, the above method of identifying a conformation-selective compound is carried out by a ligand binding or competition assay, even more preferably by a radioligand binding or competition assay. Most preferably, the above method of identifying a conformation-selective compound is carried out in a comparative assay, more particularly in a comparative ligand competition assay, even more particularly in a comparative radioligand competition assay.

[0085] Although specific embodiments, specific configurations, and materials and / or molecules have been discussed herein with respect to the engineered cells and methods according to the present disclosure, it is understood that various changes or modifications in form and details can be made without departing from the scope of the present invention. The following examples are provided to better illustrate certain embodiments and should not be considered as limiting the present application. The present application is limited only by the claims. EXAMPLES

[0086] General A novel type of functional rigid fusion protein, also called "Megakine™" (Mk), consisting of a cytokine and a scaffold protein, in which a conserved core domain or motif based on β-strands of a cytokine or a specific subfamily of cytokines is connected to the scaffold protein via two or three short linkers or via two or three direct bonds, has been designed. The principle is illustrated here for two superfamilies of cytokines, including chemokines (specifically by CCL5 and CXCL12) and interleukins, more specifically IL-1 type receptor interleukins, both of which represent cytokines that contain such a conserved core domain based on β-strands. Depending on the mechanism of action and binding mode of the chemokine or interleukin to its receptor, these rigid fusion proteins bind to and fix specific and different conformational states of the chemokine or interleukin receptor. These fusion proteins, in effect, represent an extended chemokine or interleukin ligand and are useful for determining the protein structure of a chemokine or interleukin complex (e.g., in complex with its receptor), serving several applications including X-ray crystallography and cryo-EM applications. Megakine functions as a next-generation crystallization chaperone by reducing the conformational flexibility of the bound cognate cytokine receptor, extending the surface that is more amenable to forming crystal contacts, as well as providing additional topological information. By mixing a specific Megakine protein with a chemokine or interleukin-specific receptor, their specific binding interaction results in an added "mass" and locks in a particular conformational state of the receptor.

[0087] As a proof of concept for this approach, a circularly permuted variant (c7HopQ) of the gene encoding the adhesive domain of HopQ (a periplasmic protein from H. pylori, PDB 5LP2) was inserted as a folded scaffold protein into the β-turn between β-strand 2 (β2) and β-strand 3 (β3) of the chemokine core domain of the chemokine CCL5 variant 6P4 (superagonist) FIG. 2 (Example 1) and the chemokine core domain of the chemokine CXCL12 (FIG. 19) (Example 7). Alternatively, the c7HopQ scaffold was inserted into the β-turn between β-strand 6 (β6) and β-strand 7 (β7) of the β-barrel core motif or domain of interleukin IL-1β (FIG. 27) (Example 10). Furthermore, for the CCL5 chemokine, a larger scaffold protein, E. coli Ygjk (PDB code 3W7S; Kurakava et al., 2008), was used to generate alternative Megakines, for which two circularly permuted variants (C1Ygjk and C2Ygjk) were designed to test fusions of the Megakines with CCL56P4 (Example 8).

[0088] Constructs were designed using Modeller software (https: / / salilab.org / modeller / ) and different short linkers were used to generate the different fusions.

[0089] Yeast surface display of several different fusion protein constructs containing different linkers was performed (Examples 6, 8, 10), which demonstrated that all of the different constructs for cytokine-based Megakines were able to bind to cytokine ligand-specific monoclonal antibodies (Examples 2, 9, and 11). These fusion proteins were expressed as secreted proteins in yeast (Example 3) and in the periplasm of E. coli (Example 4). Furthermore, Example 5 shows that purified proteins or periplasmic extracts applied in cell-based assays can activate the CCR5 receptor, and in some cases to the level observed for the 6P4-CCL5 chemokine agonist itself.

[0090] Example 1: Design and generation of a 50 kDa fusion protein constructed from a c7HopQ scaffold inserted into the β-strand β2-β3 connecting β-turn of the 6P4-CCL5 chemokine.

[0091] As a first proof of concept to obtain a rigid fusion protein “Megakine”, an improved CCL5 chemokine, called 6P4-CCL5 chemokine, was grafted onto a large scaffold protein via two peptide bonds connecting 6P4-CCL5 to the scaffold according to Figure 2 to construct a rigid Megakine.

[0092] The 50 kDa Megakine described herein is a chimeric polypeptide linked from a part of a chemokine and a part of a scaffold protein connected according to Figures 2-6. The chemokine used here is 6P4-CCL5, derived from the natural CCL5 ligand, belonging to the subfamily of CC chemokines, modified to a superagonist of the CCR5 GPCR shown in SEQ ID NO:1 (6P4-CCL5 is an analogue of the antagonist CCL5-5P7; Zheng et al., 2017; PDB code CCL5-5P7:5UIW). The β-turn connecting β-strand 2 and β-strand 3 of 6P4-CCL5 was blocked for fusion to the scaffold protein. The scaffold protein is an adhesin domain of Helicobacter pylori strain G27 (PDB 5LP2; SEQ ID NO:2) called HopQ (Javaheri et al., 2016). The N- and C-termini of HopQ were connected after truncation of seven amino acids (called c7HopQ) in the circularly permuted region, which otherwise appears as a loop that is not completely visible in the electron density of the crystal structure. This truncated fusion generated a circularly permuted variant of HopQ, called c7HopQ, in which the cut in the amino acid sequence was made elsewhere in the sequence (i.e., at a position corresponding to an accessible site in the exposed region of the scaffold protein). In silico molecular modeling using Modeler software (https: / / salilab.org / modeller) and custom Python scripts were used to design functional Megakine fusion protein variants. As a result, four low free energy Mk 6P4-CCL5 c7HopQ HopQ models were generated in which all moieties were connected to each other by peptide bonds from the amino (N-) terminus to the carboxy (C-) terminus in the order shown: Mk 6P4-CCL5 c7HopQV1 (SEQ ID NO: 3): the N-terminus up to β-strand 2 of 6P4-CCL5 chemokine (residues 1 to 43 of SEQ ID NO: 1), the C-terminal portion of HopQ (residues 193 to 411 of SEQ ID NO: 2), the N-terminal portion of HopQ (residues 18 to 185 of SEQ ID NO: 2), the C-terminal portion from β-strand 3 to the end of 6P4-CCL5 chemokine (residues 47 to 69 of SEQ ID NO: 1), 6xHis tag and EPEA tag (U.S. Patent No. 9,518,084 B2; SEQ ID NO: 21). Mk 6P4-CCL5 c7HopQ V2 (SEQ ID NO: 4): the N-terminus up to β-strand 2 of 6P4-CCL5 chemokine (residues 1 to 44 of SEQ ID NO: 1), a one amino acid linker of Thr, the C-terminal portion of HopQ (residues 194 to 411 of SEQ ID NO: 2), the N-terminal portion of HopQ (residues 18 to 185 of SEQ ID NO: 2), the C-terminal portion from β-strand 3 to the end of 6P4-CCL5 chemokine (residues 47 to 69 of SEQ ID NO: 1), 6×His tag, and EPEA tag. Mk 6P4-CCL5 c7HopQ V3 (SEQ ID NO: 5): the N-terminus up to β-strand 2 of 6P4-CCL5 chemokine (residues 1 to 45 of SEQ ID NO: 1), the C-terminal portion of HopQ (residues 192 to 411 of SEQ ID NO: 2), the N-terminal portion of HopQ (residues 18 to 185 of SEQ ID NO: 2), the C-terminal portion from β-strand 3 to the end of 6P4-CCL5 chemokine (residues 47 to 69 of SEQ ID NO: 1), 6xHis tag and EPEA tag (U.S. Patent No. 9,518,084 B2). Mk 6P4-CCL5 c7HopQ V4 (SEQ ID NO: 6): The N-terminus up to β-strand 2 of 6P4-CCL5 chemokine (residues 1 to 44 of SEQ ID NO: 1), the C-terminal portion of HopQ (residues 193 to 411 of SEQ ID NO: 2), the N-terminal portion of HopQ (residues 18 to 185 of SEQ ID NO: 2), the C-terminal portion from β-strand 3 to the end of 6P4-CCL5 chemokine (residues 47 to 69 of SEQ ID NO: 1), 6×His tag, and EPEA tag.

[0093] Example 2 Yeast display of a 50 kDa fusion protein constructed from a c7HopQ scaffold inserted into the β-strand β2-β3 connecting β-turn of the 6P4-CCL5 chemokine.

[0094] Four Mk 6P4-CCL5 c7HopQ To demonstrate that the V1-V4 Megakine variants (SEQ ID NOs: 3-6) can be expressed as well-folded functional proteins, the proteins were displayed on the surface of yeast (Boder, 1997). Proper folding of the 6P4-CCL5 chemokine moiety was examined using a fluorescently conjugated monoclonal antibody (Alexa Fluor® 647 anti-human RANTES (CCL5) antibody from Biolegend, ref. 515506; anti-CCL5-mAb647) that binds to the functional 6P4-CCL5 chemokine. Mk 6P4-CCL5 c7HopQ To display the V1-V4 Megakine variants in yeast, open reading frames encoding Megakine fused to several accessory peptides and proteins were constructed using standard methods (SEQ ID NOs: 7-10): the appS4 leader sequence, which directs extracellular secretion in yeast (Rakestraw, 2009), Mk 6P4-CCL5 c7HopQ The Megakine variant, an adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond to the ga1p protein, an acyl carrier protein for orthogonal fluorescent staining of the displayed fusion protein (Johnsson, 2005), followed by a cMyc tag. This open reading frame was placed into the pCTCON2 vector (Chao, 2006), under the transcriptional control of the galactose-inducible GAL1 / 10 promoter, and introduced into the yeast strain EBY100.

[0095] EBY100 yeast cells harboring this plasmid were grown and induced overnight in high galactose medium to produce the Mk 6P4-CCL5 c7HopQThe expression and secretion of the -Aga2p-ACP fusion was induced. For orthogonal staining of ACP, cells were incubated for 1 h in the presence of a fluorescently labeled CoA analog (CoA-547, 2 μM) and catalytic amounts of SFP synthase (1 μM). To analyze the functionality of the displayed Megakine, its ability to be recognized by an Alexa Fluor® 647 fluorescently labeled anti-CCL5 monoclonal antibody (anti-CCL5-mAb647) was examined by flow cytometry. Thus, EBY100 yeast cells were induced and orthogonal fluorescently stained with CoA547 to express Mk 6P4-CCL5 c7HopQ The orthogonally stained yeast cells were then incubated for 1 h in the presence of different concentrations of anti-CCL5-mAb647 (15, 31, 62, 125 and 250 ng / mL). In these experiments, the induced yeast cells were washed and subjected to flow cytometry to measure CoA547 fluorescence levels using the MegaBody Mb Nb207 cHopQ -Aga2p-ACP fusion (SEQ ID NO: 11; MegaBody is similar to Megakine, but instead of a chemokine, a Nanobody (Nb) is fused to a scaffold protein, here referred to as GFP-specific Nb 207 The level of Megakine display in each cell was measured by comparing it with yeast cells displaying Mb (having Nb207 cHopQ The binding of CCL5-mAb647 (high 647 fluorescence level) to yeast cells with significant Megakine display levels (high CoA547 fluorescence level) was analyzed by examining the 647 fluorescence level, which should be linearly correlated with the expression level of CCL5. Indeed, two-dimensional flow cytometry analysis confirmed that anti-CCL5-mAb647 (high 647 fluorescence level) bound only to yeast cells with significant Megakine display levels (high CoA547 fluorescence level) (Figure 9 and Figures 10-14). In contrast, anti-CCL5-mAb647 bound only to yeast cells with MegaBody Mb Nb207 cHopQ -Aga2p-ACP fusion (SEQ ID NO: 11) and does not bind to yeast cells stained in the same way.

[0096] From these experiments, four Mk 6P4 c7HopQ We conclude that the V1 to V4 Megakine variants (SEQ ID NOs: 3 to 6) can be expressed on the surface of yeast as well-folded, functional chimeric proteins.

[0097] Example 3 Yeast expression and purification of a 50 kDa fusion protein constructed from a c7HopQ scaffold inserted into the β-strand β2-β3 connecting β-turn of the 6P4-CCL5 chemokine.

[0098] Having been able to display functional Megakines on the yeast surface, we then addressed the expression of these 50 kDa fusion proteins as soluble secreted proteins in EBY100 cells, which we purified to homogeneity and characterized.

[0099] 4 Megakine, Mk 6P4-CCL5 c7HopQ To express the V1 to V4 Megakine variants (SEQ ID NOs: 3 to 6), standard methods were used to construct open reading frames encoding Megakine fused to several accessory peptides and proteins (SEQ ID NOs: 12 to 15): the appS4 leader sequence, which directs extracellular secretion in yeast (Rakestraw, 2009), Mk 6P4-CCL5 c7HopQ The open reading frame was placed in the pCTCON2 vector (Chao, 2006) under the transcriptional control of the galactose-inducible GAL1 / 10 promoter and introduced into the yeast strain EBY100. EBY100 yeast cells harboring this plasmid were grown and induced overnight in high galactose medium to express Mk 6P4 c7HopQExpression and secretion of V1 to V4 variants (SEQ ID NOs: 12 to 15) was induced at 30°C. Recombinant Megakine fusion proteins were collected from the medium on a HisTrap (NiNTA) FF 5mL prepacked column. The proteins were then eluted from the NiNTA resin by applying 500mM imidazole and concentrated by centrifugation using a NMWL (Nominal Molecular Weight Limit) filter with a cutoff of 10kDa (Figures 15 to 16).

[0100] From these experiments, several Mk 6P4 c7HopQ We conclude that the V1 to V4 Megakine variants (SEQ ID NOs: 3 to 6) can be expressed as well-folded functional chimeric proteins and purified by conventional purification methods.

[0101] Example 4: Bacterial expression and purification of a 50 kDa fusion protein constructed from a c7HopQ scaffold inserted into the β-strand β2-β3 connecting β-turn of the 6P4-CCL5 chemokine.

[0102] Having successfully demonstrated that functional Megakine can be displayed on the yeast surface and expressed as a soluble protein in yeast, we then proceeded to express this 50 kDa fusion protein in the periplasm of E. coli, purified it to homogeneity, and characterized it. 6P4-CCL5 c7HopQTo express the V1-V4 Megakine variant proteins (SEQ ID NOs: 3-6), standard methods were used to construct a vector allowing the expression of 6P4-CCL5 megakine, in which a scaffold can be inserted into the β-turn connecting β-strand 2 (β2) and β-strand 3 (β3) of the 6P4-CCL5 chemokine. This vector is a derivative of pMESy4 (Pardon, 2014) and contains an open reading frame encoding the following polypeptides: a DsbA leader sequence directing secretion of Megakine into the periplasm of E. coli, an N-terminus up to β-strand β2 of the 6P4-CCL5 chemokine, a multiple cloning site into which in this example a circularly permuted variant of HopQ (c7HopQ) has been cloned, a C-terminus from β-strand β3 of the 6P4-CCL5 chemokine, a 6xHis tag, and an EPEA tag followed by an Amber stop codon. Any other suitable scaffold can be cloned in the multiple cloning site of this vector.

[0103] To express Megakine in the periplasm of E. coli and purify the recombinant protein to homogeneity, four His-tagged and EPEA-tagged MkA vectors containing the DsbA leader sequence under the transcriptional control of the pLac promoter were transfected into the periplasm of E. coli using standard methods. 6P4-CCL5 c7HopQ Vectors directing the expression of V1 to V4 Megakine variants (SEQ ID NOs: 16 to 19) were constructed. WK6 bacterial cells (WK6 is a strain of su - The His-tagged and EPEA-tagged Mk 6P4-CCL5 c7HopQPeriplasmic expression of V1-V4 Megakine variants (SEQ ID NOs: 16-19) was continued overnight at 28°C. Cells were harvested by centrifugation and recombinant Megakine was released from the periplasm using osmotic shock (Pardon et al., 2014). Recombinant Megakine was then separated from the cytoplasm by centrifugation and recovered from the clarified supernatant on a HisTrap FF 5mL prepacked column. Protein was then eluted from the NiNTA resin by applying 500mM imidazole and concentrated by centrifugation using a NMWL (nominal molecular weight limit) filter with a 10kDa cutoff (Figure 17). Expressed and purified to homogeneity MegaBody Mb Nb207 c7HopQ (SEQ ID NO: 20) was used as a control for functional experiments.

[0104] From these experiments, several Mk 6P4-CCL5 c7HopQ We conclude that the V1 to V4 Megakine variants (SEQ ID NOs: 3 to 6) can be expressed as well-folded functional chimeric proteins and purified by conventional purification methods.

[0105] Example 5: Cell-based assay confirming functionality of a 50 kDa fusion protein constructed from a c7HopQ scaffold inserted into the β-strand β2-β3 connecting β-turn (40s loop) of the 6P4-CCL5 chemokine.

[0106] The preservation of functionality / proper folding of 6P4-CCL5 when presented on the C7HopQ scaffold was confirmed by Mk 6P4-CCL5 c7HopQThe V1–V4 Megakine variants were assessed by their ability to activate CCR5, the cognate receptor for CCL5, in a cell-based assay that monitors the recruitment of β-arrestin-1 or miniGi (an engineered GTPase domain of the Gα subunit; Wan et al., 2018) to CCR5 after agonist stimulation, based on split NanoLuciferase (NanoBiT-Promega) complementation (Dixon AS et al., 2016, ACS Chem Biol.).

[0107] 5×10 6 HEK293T cells were seeded in 10 cm culture dishes and, after 24 h, co-transfected with pNBe2 vectors (Promega) encoding human CCR5 C-terminally fused to SmBiT (VTGYRLFEEIL) (nanoluciferase subunit I) separated by a 15Gly / Ser linker (GSSGGGGSGGGGSSG) and pNBe3 vectors (Promega) encoding human β-arrestin-1 or miniGi N-terminally fused to LgBiT (nanoluciferase subunit II, residues 1-156) followed by a 15Gly / Ser linker. Cells were harvested 24 h after transfection and incubated with 100-fold diluted Nano-Glo Live Cell substrate for 25 min at 37 °C and 5 × 10 4 Cells were distributed in white 96-well plates at 100 cells / well. Recruitment of β-arrestin-1 or miniGi to CCR5 upon addition of Megakine was assessed via NanoLuciferase complementation, thus assessing the recovery of catalytic activity as measured in a Mithras LB940 luminometer (Berthold Technologies). Purified Mk selected from unpurified periplasmic extracts and yeast display were 6P4-CCL5 c7HopQ The activity of the V1 to V4 Megakine variants (SEQ ID NO: 16 to 19) was compared with that of non-purified recombinant soluble 6P4-CCL5 chemokine (SEQ ID NO: 33) produced in mammalian cells (HEK293T) under the CMV promoter dependency using the pIRES-puromycin vector.

[0108] The 6P4-CCL5 chemokine is Mk 6P4-CCL5 c7HopQ The V1-V4 Megakine variants retain their functionality upon insertion into the β2-β3 connecting β-strand of the c7HopQ scaffold, as demonstrated by their ability to induce the recruitment of β-arrestin-1 and miniGi to CCR5 in a concentration-dependent manner (Figure 18).

[0109] Example 6: Design and generation of another 50 kDa fusion protein constructed from a c7HopQ scaffold inserted into the β-strand β2-β3 connecting β-turn of the 6P4-CCL5 chemokine by in vivo selection.

[0110] Since the folding ability, as well as the stability and rigidity, of Megakines may depend on the composition and length of the polypeptide bonds connecting the chemokine to the scaffold, in vitro evolution techniques were introduced to fine-tune specific Megakine formats as needed. Starting from the Megakines described in Example 1, a library suitable for in vivo selection was constructed in which two short peptides of variable length and mixed amino acid composition code for Megakines with a similar design connecting the chemokine to the scaffold according to Figure 2.

[0111] The 50 kDa Megakine described herein is a chimeric polypeptide linked from a portion of a chemokine and a portion of a scaffold protein connected according to Figures 2 and 3. The chemokine used here is 6P4-CCL5, an agonist of the CCR5 GPCR, shown in SEQ ID NO: 1 (6P4-CCL5 is an analogue of the antagonist CCL55P7; Zheng et al., 2017; PDB code CCL55P7:5UIW). The β-turn connecting β-strand 2 and β-strand 3 of 6P4-CCL5 was blocked for fusion to the scaffold protein. The scaffold protein is an adhesin domain of Helicobacter pylori strain G27 (PDB:5LP2; SEQ ID NO: 2), called HopQ (Javaheri et al., 2016). The N- and C-termini of HopQ were connected after truncation of seven amino acids (termed c7HopQ) in the circularly permuted region, which otherwise appears as a loop that is not completely visible in the electron density of the crystal structure. This truncated fusion generated a circularly permuted variant of HopQ, termed c7HopQ, in which the cut in the amino acid sequence was made elsewhere in the sequence (i.e., at a position that corresponds to an accessible site in an exposed region of the scaffold protein). All moieties were connected to each other by peptide bonds from amino terminus to carboxy terminus in the following order: N-terminus up to β-strand 2 of 6P4-CCL5 chemokine (residues 1-44 of SEQ ID NO:1), a peptide linker of one or two amino acids with random composition, C-terminal portion of HopQ (residues 195-411 of SEQ ID NO:2), N-terminal portion of HopQ (residues 18-183 of SEQ ID NO:2), a peptide linker of one or two amino acids with random composition, C-terminal portion of β-strand 3 to the end of 6P4-CCL5 chemokine (residues 47-69 of SEQ ID NO:1).

[0112] To display and select functional variants of Megakine described in Examples 1 to 5 with different linker composition and length connecting the chemokine to the scaffold on yeast, a library of open reading frames encoding various Megakines fused to several accessory peptides and proteins was constructed according to Figure 7 using standard methods (SEQ ID NOs: 25 to 28). The appS4 leader sequence directing extracellular secretion in yeast (Rakestraw, 2009), the N-terminus up to β-strand 2 of 6P4-CCL5 chemokine (1 to 44 in SEQ ID NO: 1), a peptide linker of 1 or 2 amino acids with random composition, the C-terminal part of HopQ (residues 195 to 411 in SEQ ID NO: 2), the N-terminal part of HopQ (residues 18 to 183 in SEQ ID NO: 2), a peptide linker of 1 or 2 amino acids with random composition, the C-terminal part of β-strand 3 to the end of 6P4-CCL5 chemokine (residues 47 to 69 in SEQ ID NO: 1), flexible (GGSG) n A peptide linker, the adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond to the Aga1p protein, an acyl carrier protein for orthogonal fluorescent staining of the displayed fusion protein (Johnsson, 2005), followed by a cMyc tag. This open reading frame was placed into the pCTCON2 vector (Chao, 2006), under the transcriptional control of the galactose-inducible GAL1 / 10 promoter, to construct a yeast display library encoding 176,400 different variants of Megakine (see Figure 7).

[0113] The library was introduced into yeast strain EBY100 for in vitro selection. Transformed cells were grown and induced overnight in high-galactose medium. Induced cells were orthogonally stained with coA-547 (2 μM) using SFP synthase (1 μM) and incubated with 0.25 μg / mL of Alexa Fluor® 647 fluorescently labeled anti-CCL5 monoclonal antibody (anti-CCL5-mAb647). These cells were then washed and subjected to two-parameter FACS analysis to identify yeast cells that displayed high levels of Megakine expression (high CoA-547 fluorescence) and bound anti-CCL5-mAb647 (high Alexa Fluor® 647 fluorescence). Cells displaying high levels of anti-CCL5-mAb647 binding were selected and amplified in high-glucose medium for subsequent rounds of selection by yeast display and two-parameter FACS analysis (Figure 8).

[0114] After two rounds of selection, several representative highly fluorescent cells in the CoA-547 and Alexa Fluor® 647 channels were grown as single colonies and subjected to DNA sequencing to determine the sequences of several representative peptide linkers connecting the chemokines to the scaffold proteins. Two representative clones for each type of linker with 1-1, 1-2, 2-1 and 2-2 amino acid short linker variants are shown in Table 1.

[0115] [Table 1]

[0116] This demonstrates that different short peptide connections between chemokines and scaffold proteins can be selected from the Megakine library by in vivo selection using yeast display and displayed as functional chemokine chimeric proteins on the surface of yeast cells.

[0117] Example 7. Bacterial expression and purification of a 50 kDa fusion protein constructed from a c7HopQ scaffold inserted into the β-strand β2-β3 connecting β-turn of the CXCL12 chemokine.

[0118] As a second proof of concept to obtain a rigid fusion protein “Megakine”, the CXCL12 chemokine, which belongs to the subfamily of CXC chemokines, was grafted onto a large scaffold protein via two peptide bonds connecting CXCL12 to the scaffold according to Figure 2 to construct a rigid Megakine.

[0119] The 50 kDa Megakine described herein is a chimeric polypeptide linked from parts of a chemokine and parts of a scaffold protein connected according to Figures 2 and 3. The chemokine used here is CXCL12, also called SDF-1, which binds and activates CXCR4 GPCR and ACKR3 GPCR, as shown in SEQ ID NO: 22 (PDB code: 3HP3). The scaffold protein was inserted into the β-turn connecting β-strand 2 and β-strand 3 of CXCL12. The scaffold protein is an adhesin domain of Helicobacter pylori strain G27 (PDB 5LP2; SEQ ID NO: 2) called HopQ (Javaheri et al., 2016). The N- and C-termini of HopQ were connected after truncation of seven amino acids (called c7HopQ) in the circularly permuted region, which otherwise appears as a loop that is not completely visible in the electron density of the crystal structure. This truncated fusion generated a circularly permuted variant of HopQ, called c7HopQ, in which the cut in the amino acid sequence was made elsewhere in the sequence, i.e., at a position corresponding to an accessible site in an exposed region of the scaffold protein. As in Example 1, the low free energy Mk CXCL12 c7HopQ(SEQ ID NO:23) was produced containing: the N-terminus up to β-strand 2 of the CXCL12 chemokine (residues 1-43 of SEQ ID NO:22), the C-terminal portion of HopQ (residues 192-411 of SEQ ID NO:2), the N-terminal portion of HopQ (residues 18-184 of SEQ ID NO:2), the C-terminal portion of β-strand 3 to the end of the CXCL12 chemokine (residues 45-68 of SEQ ID NO:22), a 6xHis tag and an EPEA tag (U.S. Pat. No. 9,518,084 B2).

[0120] We expressed this 50 kDa fusion protein in the periplasm of E. coli, purified it to homogeneity, and characterized it. CXCL12 c7HopQ To express CXCL12, a vector was constructed using standard methods that allows the expression of CXCL12 Megakine, in which a scaffold can be inserted into the β-turn connecting β-strand 2 (β2) and β-strand 3 (β3) of the CXCL12 chemokine. This vector is a derivative of pMESy4 (Pardon, 2014) and contains an open reading frame encoding the following polypeptides: a DsbA leader sequence directing secretion of Megakine into the periplasm of E. coli, an N-terminus up to β-strand β2 of the CXCL12 chemokine, a multiple cloning site into which in this example a circularly permuted variant of HopQ (c7HopQ) has been cloned, a C-terminus from β-strand β3 of the CXCL12 chemokine, a 6xHis tag, and an EPEA tag followed by an Amber stop codon (SEQ ID NO: 24). Any other suitable scaffold can be cloned in the multiple cloning site of this vector. Mk CXCL12 c7HopQ Expression is as described in Example 4.

[0121] Example 8: Design and generation of a 94 kDa fusion protein constructed from a YgjK scaffold inserted into the β-strand β2-β3 connecting β-turn of the 6P4-CCL5 chemokine.

[0122] Based on the successful design of the first Megakine from the 6P4-CCL5 chemokine grafted onto c7HopQ (Examples 1-6), we also sought to develop other Megakine designs constructed from chemokines attached to larger scaffold proteins.

[0123] The 94 kDa Megakine described herein is a chimeric polypeptide linked from a portion of a chemokine and a portion of a scaffold protein connected according to FIG. 2. The chemokine used here is 6P4-CCL5, used in the previous examples and shown in SEQ ID NO: 1. The β-turn connecting β-strand 2 and β-strand 3 of 6P4-CCL5 was blocked for fusion to the scaffold protein. The scaffold protein is an 86 kDA periplasmic protein of E. coli called YgjK (PDB code 3W7S, SEQ ID NO: 34) (Kurakava et al., 2008). In the tertiary structure of YgjK, two antiparallel β-strands with surface-accessible β-turns, β-turn A'S1-A'S2 and β-turn NS6-NS7, were identified. To generate distinct Megakines of 94 kDa MW with (differently) blocked topologies, these two β-turns were truncated and additional circular permutations were introduced to generate two scaffold proteins: c1YgjK (SEQ ID NO: 36): the C-terminal portion of YgjK (residues 464 to 760 of SEQ ID NO: 34), a short peptide linker (SEQ ID NO: 35) that connects the C-terminus and N-terminus of YgjK to prepare a circularly permutant of the scaffold protein, and the N-terminus of YgjK (residues 1 to 461 of SEQ ID NO: 34), c2YgjK (sequence number 37): the C-terminal portion of YgjK (residues 105 to 760 of sequence number 34), a short peptide linker (sequence number 35) that connects the C-terminus and N-terminus of YgjK to create a circular permutation product of the scaffold protein, and the N-terminal portion of YgjK (residues 1 to 102 of sequence number 34).

[0124] To design functional Megakine fusion protein variants, in silico molecular modeling was performed using accessible crystal structures (PDB codes CCL5-5P7:5UIW and YgjK:3W7S). 6P4-CCL5 c1YgjK Model and two Mk 6P4-CCL5 c2YgjK Models were generated in which all the moieties were connected to each other by peptide bonds from the amino (N-) terminus to the carboxy (C-) terminus in the order shown: Mk 6P4-CCL5 c1YgjK V1 (SEQ ID NO: 38, FIG. 20): N-terminus up to β-strand 2 of 6P4-CCL5 chemokine (1 to 45 of SEQ ID NO: 1), 2 amino acid linker of Gly-Gly, c1YgjK scaffold protein (SEQ ID NO: 36), 2 amino acid linker of Gly-Gly, C-terminal portion from β-strand 3 to the end of 6P4-CCL5 chemokine (47 to 69 of SEQ ID NO: 1), Mk 6P4-CCL5 c1YgjK V2 (SEQ ID NO: 39, FIG. 21): N-terminus of β-strand 2 of 6P4-CCL5 chemokine (1-45 of SEQ ID NO: 1), 1 amino acid linker of Gly, c1YgjK scaffold protein (SEQ ID NO: 36), 1 amino acid linker of Gly, C-terminal portion of β-strand 3 to the end of 6P4-CCL5 chemokine (47-69 of SEQ ID NO: 1), Mk 6P4-CCL5 c1YgjK V3 (SEQ ID NO: 40, FIG. 22): N-terminus of 6P4-CCL5 chemokine to β-strand 2 (1-45 in SEQ ID NO: 1), c1YgjK scaffold protein (SEQ ID NO: 36), C-terminal portion of β-strand 3 to the end of 6P4-CCL5 chemokine (47-69 in SEQ ID NO: 1), Mk 6P4-CCL5 c2YgjKV1 (SEQ ID NO: 41, FIG. 23): N-terminus of β-strand 2 of 6P4-CCL5 chemokine (1-45 of SEQ ID NO: 1), 2 amino acid linker of Gly-Gly, c2YgjK scaffold protein (SEQ ID NO: 37), 2 amino acid linker of Gly-Gly, C-terminal portion of β-strand 3 to the end of 6P4-CCL5 chemokine (47-69 of SEQ ID NO: 1), Mk 6P4-CCL5 c2YgjK V3 (sequence number 42, Figure 24): N-terminus up to β-strand 2 of 6P4-CCL5 chemokine (1 to 45 in sequence number 1), c2YgjK scaffold protein (sequence number 37), C-terminal portion of β-strand 3 to the end of 6P4-CCL5 chemokine (47 to 69 in sequence number 1).

[0125] [Example 9] Yeast display of a 94 kDa fusion protein constructed from the c1YgjK and c2Ygjk scaffolds inserted into the β-strand β2-β3 connecting β-turn of the 6P4-CCL5 chemokine.

[0126] These five Mk 6P4-CCL5 c1YgjK V1~V3 variants and Mk 6P4-CCL5 c2YgjK To demonstrate that the V1 / V3 Megakine variants (SEQ ID NOs: 38 to 42) can be expressed as correctly folded functional proteins, these proteins were expressed using the Mk 6P4-CCL5 c7HopQ The V1-V4 Megakine variants were displayed on the yeast surface as was done for them (Example 2) (Boder, 1997). Proper folding of the 6P4-CCL5 chemokine moiety was examined using a fluorescently conjugated monoclonal antibody (Alexa Fluor® 647 anti-human RANTES (CCL5) antibody from Biolegend, ref. 515506; anti-CCL5-mAb647) that binds to the functional 6P4-CCL5 chemokine. Mk 6P4-CCL5 c1YgjK V1~V3 variants and Mk 6P4-CCL5 c2YgjKTo display the V1 / V3 Megakine variants on yeast, standard methods were used to construct an open reading frame encoding Megakine fused to several accessory peptides and proteins for yeast display (SEQ ID NOs: 43-47): the appS4 leader sequence, which directs extracellular secretion in yeast (Rakestraw, 2009), Mk 6P4-CCL5 c1YgjK Or Mk 6P4-CCL5 c2YgjK The Megakine variant, an adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond to the Aga1p protein, an acyl carrier protein for orthogonal fluorescent staining of the displayed fusion protein (Johnsson, 2005), followed by a cMyc tag. This open reading frame was placed into the pCTCON2 vector (Chao, 2006), under the transcriptional control of the galactose-inducible GAL1 / 10 promoter, and introduced into the yeast strain EBY100.

[0127] EBY100 yeast cells harboring this plasmid were grown and induced overnight in high galactose medium to produce the Mk 6P4-CCL5 c1 / 2YgjK Expression and secretion of the -Aga2p-ACP fusion was induced. For orthogonal staining of ACP, cells were incubated for 1 h in the presence of a fluorescently labeled CoA analog (CoA-547, 2 μM) and catalytic amounts of SFP synthase (1 μM). To analyze the functionality of the displayed Megakine, its ability to be recognized by Alexa Fluor® 647 fluorescently labeled anti-CCL5 monoclonal antibody (anti-CCL5-mAb647) was examined by flow cytometry. Thus, EBY100 yeast cells were induced and orthogonal fluorescently stained with CoA547 to express Mk 6P4-CCL5 c1 / 2YgjK -Aga2p-ACP fusion was observed. 6P4-CCL5 c7HopQYeast cells expressing V4 (SEQ ID NO: 10, Example 2) were used as an additional positive control. These orthogonally stained yeast cells were then incubated for 1 hour in the presence of anti-CCL5-mAb647 (at a concentration of 80 ng / mL). In these experiments, induced yeast cells were washed and subjected to flow cytometry to measure CoA547 fluorescence levels using the MegaBody Mb Nb207 cHopQ -Aga2p-ACP fusion (SEQ ID NO: 11; MegaBody is similar to Megakine, but instead of a chemokine, a Nanobody (Nb) is fused to a scaffold protein, here referred to as GFP-specific Nb 207 The level of Megakine display in each cell was measured by comparing it with yeast cells displaying the five Mk 6P4-CCL5 c1 / 2YgjK For all variants, Mk 6P4-CCL5 c1 / 2YgjK The quantified display level of the -Aga2p-ACP fusion was approximately 70% (Figure 25).

[0128] Next, binding of anti-CCL5-mAb647 was analyzed by measuring the Mk 6P4-CCL5 c1 / 2YgjK The expression levels of the variants were analyzed by examining the 647 fluorescence levels, which should be linearly correlated with the expression levels of the variants. Two-dimensional flow cytometry analysis confirmed that anti-CCL5-mAb647 (high 647 fluorescence levels) bound only to yeast cells with significant Megakine display levels (high CoA547 fluorescence levels). 6P4-CCL5 c2YgjK V1 (SEQ ID NO: 46) and Mk 6P4-CCL5 c2YgjK The linear fit for V3 (SEQ ID NO: 47) was the highest, likely due to the best accessibility of the epitope recognized by anti-CCL5-mAb647 (Figure 26). In contrast, anti-CCL5-mAb647 did not recognize the MegaBody Mb Nb207 cHopQ-Aga2p-ACP fusion (SEQ ID NO: 11, GFP-specific MegaBody as negative control) and does not bind to yeast cells stained in the same way.

[0129] From these experiments, five Mk 6P4-CCL5 c1YgjK V1~V3 and Mk 6P4-CCL5 c2YgjK We conclude that all of the V1 / V3 Megakine variants (SEQ ID NOs: 38-42) can be expressed on the surface of yeast as well-folded, functional chimeric proteins.

[0130] Example 10. Design and generation of a 58 kDa fusion protein constructed from a HopQ scaffold inserted into the β-strand β6-β7 connecting β-turn of IL-1β interleukin.

[0131] Based on the successful design of the first Megakines from 6P4-CCL5 and CXCL12 chemokines grafted onto c7HopQ (Examples 1-7) and c1YgjK / c2Ygjk (Examples 8 and 9) scaffolds, we also sought to develop other Megakine designs constructed from different classes of cytokines, specifically interleukins, connected to larger scaffolds.

[0132] The 58 kDa Megakine described herein is a chimeric polypeptide linked from a portion of an interleukin and a portion of a scaffold protein connected according to FIG. 27. The interleukin used here is human IL-1β (SEQ ID NO: 48) which belongs to the subfamily of interleukins that exert their effects through the IL-1 receptor type I (IL-1RI) and the IL-1 receptor accessory protein (IL-1RAcP) (PDB 3O4O, Wang et al., 2010). In the functional IL-1β·IL-1RI·IL-1RAcP complex, the β-turn connecting β-strand β6 and β-strand β7 of IL-1 is exposed to the solvent and is therefore accessible for scaffold protein fusion (FIG. 28). The scaffold protein is the c7HopQ scaffold used to generate Megakines based on the 6P4-CCL5 chemokine (Examples 1-6). To design functional MkIL-1βc7HopQ Megakine fusion protein variants, we performed in silico molecular modeling using accessible crystal structures (PDB code IL-1β:3O4O, PDB code HopQ:5LP2). IL-1β c7HopQ Models were generated in which all the moieties were connected to each other by peptide bonds from the amino (N-) terminus to the carboxy (C-) terminus in the order shown: Mk IL-1β c7HopQ V1 (SEQ ID NO: 49, FIG. 29): the N-terminus up to the β-strand β6 of human IL-1β interleukin (1 to 73 of SEQ ID NO: 48), a two amino acid linker of Gly-Gly, the C-terminal portion of HopQ (residues 193 to 411 of SEQ ID NO: 2), the N-terminal portion of HopQ (residues 18 to 185 of SEQ ID NO: 2), a two amino acid linker of Gly-Gly, the C-terminal portion derived from the β-strand β7 of human IL-1β interleukin (residues 78 to 153 of SEQ ID NO: 48), Mk IL-1β c7HopQV2 (SEQ ID NO: 50, FIG. 30): the N-terminus up to the β-strand β6 of human IL-1β interleukin (1 to 73 of SEQ ID NO: 48), one amino acid linker of Gly, the C-terminal portion of HopQ (residues 193 to 411 of SEQ ID NO: 2), the N-terminal portion of HopQ (residues 18 to 185 of SEQ ID NO: 2), one amino acid linker of Gly, the C-terminal portion derived from the β-strand β7 of human IL-1β interleukin (residues 78 to 153 of SEQ ID NO: 48), Mk IL-1β c7HopQ V3 (sequence number 51, Figure 31): The N-terminus up to β-strand β6 of human IL-1β interleukin (residues 1 to 73 of SEQ ID NO: 48), the C-terminal portion of HopQ (residues 193 to 411 of SEQ ID NO: 2), the N-terminal portion of HopQ (residues 18 to 185 of SEQ ID NO: 2), and the C-terminal portion derived from β-strand β7 of human IL-1β interleukin (residues 78 to 153 of SEQ ID NO: 48).

[0133] [Example 11] Yeast display of a 58 kDa fusion protein constructed from a HopQ scaffold inserted into the β-strand β6-β7 connecting β-turn of IL-1β interleukin.

[0134] Three Mk IL-1β c7HopQ To demonstrate that the Megakine variants (SEQ ID NOs: 49 to 51) can be expressed as correctly folded functional proteins, Mk 6P4-CCL5 c7HopQ Megakine variant (Example 2) and Mk 6P4-CCL5 cYgjkA / B Yeast surface display of these proteins (Boder, 1997) is required, as was performed for the Megakine variants (Example 9). Proper folding of the IL-1β interleukin moiety can be examined using a fluorescently conjugated monoclonal antibody (Alexa Fluor® 647 anti-human IL-1β antibody (CRM46) from Life Technologies, ref. 51-7018-42) that binds to functional IL-1β interleukin. Mk IL-1β c7HopQTo display the V1-V3 Megakine variants in yeast, standard methods are used to construct open reading frames encoding Megakines (SEQ ID NOs: 52-54) fused to several accessory peptides and proteins: the appS4 leader sequence, which directs extracellular secretion in yeast (Rakestraw, 2009), the Mk IL-1β c7HopQ The Megakine variant, an adhesive subunit of the yeast agglutinin protein Aga2p, which binds to the yeast cell wall via a disulfide bond to the Aga1p protein, an acyl carrier protein for orthogonal fluorescent staining of the displayed fusion protein (Johnsson, 2005), followed by a cMyc tag. This open reading frame is cloned into the pCTCON2 vector (Chao, 2006) under the transcriptional control of the galactose-inducible GAL1 / 10 promoter and introduced into the yeast strain EBY100. EBY100 yeast cells harboring this plasmid are grown and induced overnight in high galactose medium to express Mk IL-1β c7HopQ -Aga2p-ACP fusion expression and secretion are induced. For orthogonal staining of ACP, cells are incubated for 1 h in the presence of a fluorescently labeled CoA analog (CoA-547, 2 μM) and catalytic amounts of SFP synthase (1 μM) as shown in the previous example. To analyze the functionality of the displayed Megakine, its ability to be recognized by Alexa Fluor® 647 fluorescently labeled IL-1β monoclonal antibody (anti-human IL-1β antibody CRM46) is observed by flow cytometry. Thus, EBY100 yeast cells are induced and orthogonal fluorescently stained with CoA547 to express Mk IL-1β c7HopQThe display of the -Aga2p-ACP fusion is observed. Yeast cells displaying the IL-1β interleukin (SEQ ID NO: 55) form an additional positive control. These orthogonally stained yeast cells are then incubated for 1 hour in the presence of the anti-human IL-1β antibody CRM46 (at a concentration of 80 ng / mL). In these experiments, the induced yeast cells are washed and subjected to flow cytometry to measure CoA547 fluorescence levels using the MegaBody Mb Nb207 cHopQ -Aga2p-ACP fusion (SEQ ID NO: 11; MegaBody is similar to Megakine, but instead of an interleukin, a Nanobody (Nb) is fused to a scaffold protein, here referred to as a GFP-specific Nb 207 The level of Megakine display in each cell was measured by comparing it to yeast cells displaying Mk (having Mk) and orthogonally staining in the same way. The binding of the anti-human IL-1β antibody CRM46 was then measured by comparing it to Mk on the surface of the yeast. IL-1β c7HopQ The expression level of the variants can be analyzed by examining the 647 fluorescence level, which should be linearly correlated with the expression level of the variants. Two-dimensional flow cytometry analysis confirmed that the anti-human IL-1β antibody CRM46 (high 647 fluorescence level) only binds to yeast cells with significant Megakine display levels (high CoA547 fluorescence level). In contrast, the anti-human IL-1β antibody CRM46 binds only to yeast cells with significant Megakine display levels (high CoA547 fluorescence level). Nb207 cHopQ -Aga2p-ACP fusion (SEQ ID NO: 11) and does not bind to yeast cells stained in the same way.

[0135] Sequence Listing >SEQ ID NO: 1: 6P4-CCL5 chemokine >SEQ ID NO:2: Helicobacter pylori strain G27 HopQ adhesin domain protein (PDB 5LP2) >Sequence number 3: Mk 6P4-CCL5 c7HopQ V1 Megakine (N-terminus of 6P4-CCL5-chemokine, HopQ sequence underlined, C-terminus of 6P4-CCL5 chemokine in bold, 6xHis tag, EPEA tag)

[0136] [ka]

[0137] >Sequence number 4: Mk 6P4-CCL5 c7HopQ V2 Megakine (6P4-CCL5-chemokine N-terminus, T-short peptide linker, HopQ sequence underlined, 6P4-CCL5 chemokine C-terminus in bold, 6xHis tag, EPEA tag)

[0138] [ka]

[0139] >Sequence number 5: Mk 6P4-CCL5 c7HopQ V3 Megakine (N-terminus of 6P4-CCL5-chemokine, HopQ sequence underlined, C-terminus of 6P4-CCL5 chemokine in bold, 6xHis tag, EPEA tag)

[0140] [ka]

[0141] >Sequence number 6: Mk 6P4-CCL5 c7HopQ V4 Megakine (N-terminus of 6P4-CCL5-chemokine, HopQ sequence underlined, C-terminus of 6P4-CCL5 chemokine in bold, 6xHis tag, EPEA tag)

[0142] [ka]

[0143] >Sequence number 7: Mk 6P4-CCL5 c7HopQ V1_Aga2p_ACP protein sequence (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c7HopQ V1, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0144] [ka]

[0145] >Sequence number 8: Mk 6P4-CCL5 c7HopQ V2_Aga2p_ACP protein sequence >Sequence number 9: Mk 6P4-CCL5 c7HopQ V3_Aga2p_ACP protein sequence >Sequence number 10: Mk 6P4-CCL5 c7HopQ V4_Aga2p_ACP protein sequence >Sequence number 11: Mb Nb207 cHopQ _Aga2p_ACP protein sequence (appS4 leader sequence, MegaBody Mb shown in bold) Nb207 cHopQ , Flexibility (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0146] [ka]

[0147] >Sequence number 12: Mk 6P4-CCL5 c7HopQ V1 yeast secreted protein sequence (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c7HopQV1, 6xHis tag, EPEA tag)

[0148] [ka]

[0149] >Sequence number 13: Mk 6P4-CCL5 c7HopQ V2 yeast secreted protein sequence >Sequence number 14: Mk 6P4-CCL5 c7HopQ V3 yeast secreted protein sequence >Sequence number 15: Mk 6P4-CCL5 c7HopQ V4 Yeast Secreted Protein Sequence >Sequence number 16: DsbA_Mk 6P4-CCL5 c7HopQ V1 protein sequence (DsbA leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c7HopQ V1, 6xHis tag, EPEA tag)

[0150] [ka]

[0151] >Sequence number 17: DsbA_Mk 6P4-CCL5 c7HopQ V2 protein sequence >Sequence number 18: DsbA_Mk 6P4-CCL5 c7HopQ V3 protein sequence >Sequence number 19: DsbA_Mk 6P4-CCL5 c7HopQ V4 protein sequence >Sequence number 20: DsbA_Mb Nb207 c7HopQ MegaBody (DsbA leader sequence, MegaBody Mb shown in bold) Nb207 c7HopQ , 6×His tag, EPEA tag)

[0152] [ka] TIFF0007627910000011.tif16166

[0153] >SEQ ID NO: 21: Affinity tag (US Pat. No. 9,518,084 B2) >SEQ ID NO: 22: CXCL12 chemokine (human) >Sequence number 23: Mk CXCL12 c7HopQ Protein sequence (CXCL in bold, c7HopQ in normal type, 6×His tag, and EPEA tag underlined with dotted lines).

[0154] [ka]

[0155] >SEQ ID NO:24: DsbA-Mk CXCL12 c7HopQ Protein sequence (DsbA leader sequence underlined, Mk CXCL12 c7HopQ : CXCL12 in bold, c7HopQ in normal typeface, 6×His tag, and EPEA tag underlined with dotted lines).

[0156] [ka]

[0157] >Sequence number 25: Mk 6P4-CCL5 c7HopQ Random Linker (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c7HopQ YD1, X is a short peptide linker of random composition with 1AA, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0158] [ka]

[0159] >Sequence number 26: Mk 6P4-CCL5 c7HopQ Random Linker (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c7HopQ YD1, X is a short peptide linker of random composition with 1AA, XX is a short peptide linker of random composition with 2AA, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0160] [ka] TIFF0007627910000016.tif29166

[0161] >Sequence number 27: Mk 6P4-CCL5 c7HopQ Random Linker (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c7HopQ YD1, XX is a short peptide linker of random composition with 2AA, and X is a short peptide linker of random composition with 1AA, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0162] [ka]

[0163] >Sequence number 28: Mk 6P4-CCL5 c7HopQ Random Linker (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c7HopQYD1, XX is a short peptide linker of random composition with 2AA, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0164] [ka]

[0165] >Sequence number 29 / 31: Megakine Mk 6P4-CCL5 c7HopQ Forward / reverse primers for introducing a short peptide linker of 1 amino acid length into the yeast display library >Sequence number 30 / 32: Megakine Mk 6P4-CCL5 c7HopQ Forward / reverse primers for introducing a two-amino acid short peptide linker into the yeast display library >SEQ ID NO: 33: SS-6P4-CCL5 Recombinant soluble 6P4-CCL5 chemokine for production in mammalian cells (HEK293T) (Seq signals underlined, 6P4 sequence (SEQ ID NO: 1), CCL5)

[0166] [ka]

[0167] >SEQ ID NO: 34: Escherichia coli Ygjk protein (PDB 3W7S) >SEQ ID NO: 35: cYgjk circularly permuted linker peptide > SEQ ID NO: 36: c1YgjK scaffold protein (PDB 3W7S) (YgjK sequence underlined, circularly permuted linker in italics)

[0168] [ka]

[0169] > SEQ ID NO: 37: c2YgjK scaffold protein (PDB 3W7S) (YgjK sequence underlined, circularly permuted linker in italics)

[0170] [ka]

[0171] >Sequence number 38: Mk 6P4-CCL5 c1YgjK V1 Megakine (N-terminus of 6P4-CCL5 chemokine, GG short peptide linker, c1YgjK scaffold protein sequence underlined, GG short peptide linker, C-terminus of 6P4-CCL5 chemokine in bold)

[0172] [ka] TIFF0007627910000023.tif48166

[0173] >Sequence number 39: Mk 6P4-CCL5 c1YgjK V2 Megakine (N-terminus of 6P4-CCL5 chemokine, G short peptide linker, c1YgjK scaffold protein sequence underlined, G short peptide linker, C-terminus of 6P4-CCL5 chemokine in bold)

[0174] [ka]

[0175] >Sequence number 40: Mk 6P4-CCL5 c1YgjK V3 Megakine (N-terminus of 6P4-CCL5-chemokine, c1YgjK scaffold protein sequence underlined, C-terminus of 6P4-CCL5 chemokine in bold)

[0176] [ka] TIFF0007627910000026.tif12166

[0177] >Sequence number 41: Mk 6P4-CCL5 c2YgjK V1 Megakine (N-terminus of 6P4-CCL5 chemokine, GG short peptide linker, c2YgjK scaffold protein sequence underlined, GG short peptide linker, C-terminus of 6P4-CCL5 chemokine in bold)

[0178] [ka]

[0179] >Sequence number 42: Mk 6P4-CCL5 c2YgjK V3 Megakine (N-terminus of 6P4-CCL5-chemokine, c2YgjK scaffold protein sequence underlined, C-terminus of 6P4-CCL5 chemokine in bold)

[0180] [ka]

[0181] >Sequence number 43: Mk 6P4-CCL5 c1YgjK V1_Aga2p_ACP protein sequence (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c1YgjK V1, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0182] [ka]

[0183] >Sequence number 44: Mk 6P4-CCL5 c1YgjKV2_Aga2p_ACP protein sequence (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c1YgjK V2, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0184] [ka]

[0185] >Sequence number 45: Mk 6P4-CCL5 c1YgjK V3_Aga2p_ACP protein sequence (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c1YgjK V3, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0186] [ka]

[0187] >Sequence number 46: Mk 6P4-CCL5 c2YgjK V1_Aga2p_ACP protein sequence (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c2YgjK V1, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0188] [ka]

[0189] >Sequence number 47: Mk 6P4-CCL5c2YgjK V3_Aga2p_ACP protein sequence (appS4 leader sequence, Megakine Mk shown in bold) 6P4-CCL5 c2YgjK V3, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0190] [ka]

[0191] >SEQ ID NO: 48: Mature form of human IL-1β >Sequence number 49: Mk IL-1β c7HopQ V1 Megakine (N-terminus of IL-1β interleukin, GG short peptide linker, HopQ sequence underlined, GG short peptide linker, C-terminus of IL-1β interleukin in bold)

[0192] [ka]

[0193] >Sequence number 50: Mk IL-1β c7HopQ V2 Megakine (N-terminus of IL-1β interleukin, G short peptide linker, HopQ sequence underlined, G short peptide linker, C-terminus of IL-1β interleukin in bold)

[0194] [ka] TIFF0007627910000036.tif36166

[0195] >Sequence number 51: Mk IL-1β c7HopQ V3 Megakine (N-terminus of IL-1β interleukin, HopQ sequence underlined, C-terminus of IL-1β interleukin in bold)

[0196] [ka]

[0197] >Sequence number 52: Mk IL-1β c7HopQ V1_Aga2p_ACP protein sequence (appS4 leader sequence, Megakine Mk shown in bold) IL-1β c7HopQ V1, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0198] [ka]

[0199] >Sequence number 53: Mk IL-1β c7HopQ V2_Aga2p_ACP protein sequence (appS4 leader sequence, Megakine Mk shown in bold) IL-1β c7HopQ V2, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0200] [ka] TIFF0007627910000040.tif55166

[0201] >Sequence number 54: Mk IL-1β c7HopQ V3_Aga2p_ACP protein sequence (appS4 leader sequence, Megakine Mk shown in bold) IL-1βc7HopQ V3, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0202] [ka]

[0203] >SEQ ID NO: 55: IL-1β_Aga2p_ACP protein sequence (appS4 leader sequence, IL-1β shown in bold, flexible (GGGS) n Polypeptide linker, Aga2p protein sequence underlined, ACP sequence double underlined, cMyc tag)

[0204] [ka]

[0205] References

[0206] [Table 2] TIFF0007627910000044.tif49166

Claims

1. A fusion protein comprising a cytokine fused to a scaffold protein and retaining its cytokine functionality compared to a cytokine ligand not fused to said scaffold protein, said scaffold protein being a folded protein of at least 50 amino acids having at least one exposed region in its three-dimensional structure to provide a fusion point that interrupts the topology of said cytokine at one or more accessible sites in an exposed β-turn of a β-strand-containing core domain of said cytokine via at least two or more direct fusions or fusions made by linkers, said cytokine core domain comprising: a chemokine core domain comprising an N-terminal loop; a β-sheet comprising three β-strands; and a C-terminal helix, in which the scaffold protein is inserted into the exposed β-turn connecting β-strand β2 and β-strand β3 of the chemokine core domain; or an interleukin β barrel core motif of the IL-1 family, where the scaffold protein is inserted into the exposed β turn connecting β strand β6 and β strand β7 of the β barrel core motif; Fusion proteins.

2. The fusion protein of claim 1 , wherein the scaffold protein is a circularly permuted protein.

3. The fusion protein of claim 1 or 2, wherein the scaffold protein has a total molecular mass of at least 30 kDa.

4. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. 6. The vector of claim 5 for expression in E. coli or for surface display in yeast, phage, bacteria or viruses.

7. A host cell comprising the fusion protein according to any one of claims 1 to 3.

8. The host cell of claim 7 , wherein the fusion protein and the cytokine receptor are co-expressed.

9. (i) a fusion protein according to any one of claims 1 to 3, and (ii) Receptor Protein A complex comprising: A complex in which a receptor protein is bound to a cytokine in a fusion protein.

10. The complex of claim 9 , wherein the receptor is activated upon binding to the fusion protein.

11. 1. A method for determining the three-dimensional structure of a ligand / receptor complex, comprising: (i) providing a fusion protein according to any one of claims 1 to 3 and a receptor to form a complex, wherein the receptor protein is bound to the cytokine portion of the fusion protein, or providing a complex according to claim 9 or 10; (ii) displaying the complex under conditions suitable for structural analysis Including, A method in which the 3D structure of the ligand / receptor complex is determined using cryo-EM or X-ray crystallography.

12. Use of the fusion protein according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5 or 6, the host cell according to claim 7 or 8, or the complex according to claim 9 or 10 for structural analysis of a cytokine / receptor complex.

13. The use of a fusion protein according to claim 12, wherein the structural analysis comprises single particle cryo-EM or crystal structure analysis.

14. A method for producing the fusion protein of claim 1, comprising: (i) selecting chemokines and scaffold proteins with accessible β-turns to interrupt the chemokine protein sequence without interrupting the folding of the chemokine core domain; (ii) designing a gene fusion construct encoding: a) the protein sequence of a chemokine interrupted between β-strand β2 and β-strand β3 of the core domain; b) scaffold proteins fused at the N-terminus and C-terminus to obtain a circularly permuted scaffold protein; c) a circularly permuted scaffold protein of b), the amino acid sequence of which is interrupted at an accessible site, such as a loop or turn, that is distinct from the original N- or C-terminus. d) an amino acid at the C-terminal end of the interrupted site of a chemokine of β-strand β2 fused to an amino acid at the most N-terminal interrupted site of the circularly permuted scaffold protein, and an amino acid at the N-terminal end of the interrupted site of a chemokine of β-strand β3 fused to an amino acid at the most C-terminal end of the interrupted site of the circularly permuted scaffold protein; (iii) introducing the gene fusion construct into an expression system to obtain a fusion protein, in which the chemokine is fused to the circularly permuted scaffold protein at two sites in its core domain; The method includes:

Images