Therapeutic cytokines and methods

Abstract

Disclosed herein are single-chain dimeric cytokines with improved biological activity compared to wild-type cytokine monomers. The single-chain dimeric cytokines of the present disclosure may be useful in the treatment of various diseases or disorders associated with inflammatory or proliferative conditions. Also disclosed are delivery vectors or agents for delivering the single-chain dimeric cytokines to a subject. Such delivery vectors or agents include cells, bacteria, and bacteriophages.

Description

[Technical Field]

[0001] The present invention relates to novel therapeutic cytokines and methods for their use in therapy. In particular, the present invention relates to novel dimeric cytokines, more particularly to conformationally constrained / conformational restricted dimeric cytokines, and their use in therapy. [Background technology]

[0002] Cytokines are biomolecules of great interest with potential in human therapy. They regulate the immune system and play important roles in cancer, inflammation, immune responses, and tissue regeneration, among others. Despite their great potential, only a handful of cytokines have been approved for therapeutic use. This is because many of them have short serum half-lives, high production costs, pleiotropic effects, high toxicity, and potentially harmful side effects due to their typical targeting of various cell types (see Figure 2).

[0003] Various methods have been proposed to improve the pharmacodynamics and pharmacokinetics of selected cytokines (e.g., fusion of IL-7 to the IgG-Fc region). 2 and the use of the adjuvant polyethylene glycol for many cytokines. 3 Other properties that need improvement include affinity, resistance to proteolysis, and / or toxicity, which may be partially addressed by designing surface mutations that target interactions with one or more receptors, thereby maximizing therapeutic efficacy while reducing dosage. 4) .

[0004] One potential way to reduce the toxicity of cytokines is to deliver them locally by using therapeutic bacteria. The use of recombinant bacteria to produce and deliver therapeutic molecules of interest in situ has been well documented. 5While many such efforts have shown high efficacy in animal models, they have not achieved clinical success, partly due to the difficulty of achieving and maintaining sufficiently effective concentrations of therapeutic molecules at disease sites. Nevertheless, interest in the use of microorganisms as therapeutic agents continues to grow, driven by the promise of effective treatments that are inexpensive to produce, can be delivered orally or topically (e.g., Cutibacterium acnes for acne; https: / / pubmed.ncbi.nlm.nih.gov / 31981578 / ), and minimize systemic side effects. Several companies are developing bacteria for human therapeutics (e.g., Pulmobiotics, Eligo Bioscience, Synlogic, Azitra, Intrexon, Rebiotix) or are using bacteriophages containing genes of interest for expression in target bacteria (e.g., Armata Pharmaceuticals, Eligo Bioscience).

[0005] Other ways to reduce toxicity and improve efficacy could be to fuse the cytokine to a molecule (e.g., an antibody) that targets the cytokine to the desired site of action; to introduce mutations that change the relative affinity for one of the various components of the receptor assembly (e.g., IL2 and its receptor alpha (IL-2RA)), ultimately taking into account that different cells express different levels of receptor components; or to fuse the cytokine to other protein domains that prevent receptor recognition and are detached from the cytokine at the correct location.

[0006] Against the above background, it would be desirable to have new and / or improved therapeutic cytokines, therapies, and / or methods that improve the half-life, safety, efficacy, specificity, and / or local targeting of cytokines when administered using conventional methods or biological delivery vectors.

[0007] The present invention seeks to overcome, or at least mitigate, one or more of the deficiencies in the prior art. Summary of the Invention

[0008] The present invention relates to a single-chain dimeric cytokine polypeptide comprising (a) a first cytokine monomer domain or functional portion thereof and (b) a second cytokine monomer domain or functional portion thereof, wherein the sequence of one of the cytokine monomer domains or functional portions is not contiguous but is interrupted by the sequence of the other cytokine monomer domain or functional portion thereof.

[0009] In one embodiment, the sequence or functional portion of a first cytokine monomer domain is not contiguous and is interrupted by the sequence of a second cytokine monomer domain or functional portion thereof.

[0010] In some aspects of this embodiment, the first sequence portion of the first cytokine monomer domain corresponds to the N-terminal portion of a naturally occurring cytokine monomer domain, and the second sequence portion of the first cytokine monomer domain corresponds to the C-terminal portion of a naturally occurring cytokine monomer domain.

[0011] In other aspects of this embodiment, the first sequence portion of the first cytokine monomer domain corresponds to the C-terminal portion of a naturally occurring cytokine monomer domain, and the second sequence portion of the first cytokine monomer domain corresponds to the N-terminal portion of a naturally occurring cytokine monomer domain.

[0012] In another embodiment, the sequence or functional portion of the second cytokine monomer domain is not contiguous and is interrupted by the sequence or functional portion thereof of the first cytokine monomer domain.

[0013] In some aspects of this embodiment, the sequence of the first class I cytokine monomer domain or functional portion thereof is continuous in sequence, while the sequence of the second cytokine monomer domain is such that a first sequence portion of the second class I cytokine monomer located at the N-terminus of the first class I cytokine monomer or functional portion thereof corresponds to the N-terminal portion of a naturally occurring class I cytokine monomer, and a second sequence portion of the second class I cytokine monomer located at the C-terminus of the second class I cytokine monomer or functional portion thereof corresponds to the C-terminal portion of a naturally occurring class I cytokine monomer.

[0014] In another aspect of this embodiment, the sequence of the second Class II cytokine monomer domain or functional portion thereof is contiguous and in reverse order compared to its native sequence order, while the sequence of the first Class II cytokine monomer domain is such that a first sequence portion of the first Class II cytokine monomer domain located N-terminal to the second Class II cytokine monomer domain or functional portion thereof corresponds to the N-terminal portion of the native Class II cytokine monomer domain, and a second sequence portion of the first Class II cytokine monomer domain located C-terminal to the second Class II cytokine monomer domain or functional portion thereof corresponds to the C-terminal portion of the native Class II cytokine monomer domain.

[0015] In another embodiment, the invention relates to a single-chain dimeric cytokine polypeptide comprising a first cytokine monomer and a second cytokine monomer that form a swapped domain dimer, wherein a linker peptide bridges the N-terminus of the first cytokine monomer and the C-terminus of the second cytokine monomer.

[0016] The present invention further relates to polypeptides comprising the single-chain dimeric polypeptides of the present invention.

[0017] The present invention further relates to a nucleic acid encoding the single-chain dimeric polypeptide of the present invention or a polypeptide comprising same.

[0018] The present invention also relates to an expression vector comprising a nucleic acid of the present invention.

[0019] Further provided are host cells comprising the nucleic acids or expression vectors of the invention.

[0020] The present invention also relates to pharmaceutical compositions comprising the single-chain dimeric polypeptide of the present invention or a polypeptide comprising the same.

[0021] Further provided is a method for treating a disease, comprising administering the single-chain dimeric polypeptide of the present invention or a polypeptide comprising same to a subject in need thereof, optionally wherein the subject would benefit from a decrease or increase in the rate of inflammatory response or cell proliferation.

[0022] The present invention further relates to the single-chain dimeric polypeptide of the present invention or a polypeptide comprising same, for use in the treatment of a disease, optionally wherein the disease is a disease that would benefit from a decrease or an increase in the rate of inflammatory response or cell proliferation.

[0023] The present invention further relates to the single-chain dimeric polypeptide of the present invention or a polypeptide comprising same, for the manufacture of a medicament for the treatment of a disease, optionally a disease that would benefit from a decrease or increase in the rate of inflammatory response or cell proliferation.

[0024] Broadly, the present disclosure relates to the structural design of new cytokine polypeptide variants that can provide beneficial therapeutic effects. Such therapeutic polypeptides can be delivered to a target subject by any method available to those skilled in the art, for example, orally, by aerosol, injection, or by using biological vectors such as viruses, bacteriophages, and / or bacteria.

[0025] The novel cytokine polypeptide variants of the present disclosure comprise a pair of cytokine monomer sequences covalently linked to form a single-chain polypeptide. The cytokine monomer sequences may be derived from the same or different wild-type cytokine protein sequences. Advantageously, the single-chain polypeptides of the present disclosure fold to form a tertiary structure comprising two structural domains (e.g., left and right 3D domains), each of which may have the function and / or activity of the native cytokine monomer from which it is based / derived, or may have a different function or activity.

[0026] In one aspect, disclosed is a single-chain dimeric cytokine polypeptide comprising (a) a first cytokine monomer domain or functional portion thereof and (b) a second cytokine monomer domain or functional portion thereof, wherein the first cytokine monomer domain or functional portion thereof is fused to the second cytokine monomer domain or functional portion thereof via a peptide linker, the peptide linker sequence comprising five or fewer (preferably three or fewer) adjacent Gly and / or Ser residues. Preferably, the artificial peptide linker has a sequence of about 3 to about 20 amino acids. In embodiments, the artificial peptide linker has about 3 to about 16 amino acids, about 3 to about 12 amino acids, about 3 to about 8 amino acids, about 4 to about 8 amino acids, or about 4 to about 6 amino acids.

[0027] In a second aspect, disclosed is a single-chain dimeric cytokine polypeptide comprising (a) a first cytokine monomer domain or a functional portion thereof and (b) a second cytokine monomer domain or a functional portion thereof, wherein the sequence of the second cytokine monomer domain or a functional portion thereof is inserted within the sequence of the first cytokine monomer domain such that the first sequence portion of the first cytokine monomer is located at the N-terminus of the second cytokine monomer or a functional portion thereof and the second sequence portion of the first cytokine monomer is located at the C-terminus of the second cytokine monomer or a functional portion thereof. Advantageously, the single-chain dimeric cytokine polypeptide has a tertiary structure comprising a left 3D cytokine domain and a right 3D cytokine domain, wherein the left 3D cytokine domain is connected to the right 3D cytokine domain by two bridging linker peptides. In embodiments, the left 3D cytokine domain is preferably a split domain cytokine formed from first and second sequence portions of a first cytokine monomer domain or functional portion thereof, and the right 3D cytokine domain is preferably a continuous domain cytokine formed from a second cytokine monomer domain or functional portion thereof.

[0028] In a third aspect, disclosed is a single-chain dimeric cytokine polypeptide comprising (a) a first cytokine monomer domain or functional portion thereof and (b) a second cytokine monomer domain or functional portion thereof, wherein the sequence of the second cytokine monomer domain or functional portion thereof is inserted within the sequence of the first cytokine monomer domain such that the first sequence portion of the first cytokine monomer is located at the N-terminus of the second cytokine monomer or functional portion thereof and the second sequence portion of the first cytokine monomer is located at the C-terminus of the second cytokine monomer or functional portion thereof. In various embodiments, the first sequence portion of the first cytokine monomer domain corresponds to the C-terminal portion of a native cytokine monomer and the second sequence portion of the first cytokine monomer domain corresponds to the N-terminal portion of a native cytokine monomer.

[0029] In a fourth aspect, disclosed is a single-chain dimeric cytokine polypeptide comprising (a) a first cytokine monomer domain or a functional portion thereof and (b) a second cytokine monomer domain or a functional portion thereof, wherein the first cytokine monomer domain sequence is derived from a different cytokine than the second cytokine monomer domain sequence, the left cytokine domain is a split-domain cytokine formed from first and second sequence portions of the first cytokine monomer domain or a functional portion thereof, and the right 3D cytokine domain is a continuous-domain cytokine formed from the second cytokine monomer domain or a functional portion thereof. Suitably, the single-chain dimeric cytokine polypeptide has a tertiary structure comprising a left 3D cytokine domain formed from the first cytokine monomer domain or a functional portion thereof, and a right 3D cytokine domain formed from the second cytokine monomer domain or a functional portion thereof. Advantageously, the first cytokine monomer domain is connected to the second cytokine monomer domain by two bridging linker peptides. In embodiments, the first cytokine monomer domain is derived from a class II cytokine and the second cytokine monomer is derived from a class I cytokine, or the first cytokine monomer domain is derived from a class I cytokine and the second cytokine monomer is derived from a class II cytokine.

[0030] In a fifth aspect, disclosed is a single-chain dimeric cytokine polypeptide comprising (a) a first cytokine monomer domain or a functional portion thereof and (b) a second cytokine monomer domain or a functional portion thereof, wherein the first cytokine monomer domain sequence is derived from a different cytokine than the second cytokine monomer domain sequence, and wherein the single-chain dimeric cytokine polypeptide has a tertiary structure comprising a left 3D cytokine domain formed by the first cytokine monomer domain or a functional portion thereof, and a right 3D cytokine domain formed by the second cytokine monomer domain or a functional portion thereof. In various embodiments, (i) the first cytokine monomer domain sequence is derived from an IL-10 or IFNγ cytokine sequence and the second cytokine monomer domain sequence is derived from a Class I or Class II cytokine sequence, or (ii) the first cytokine monomer domain sequence is derived from a Class I or Class II cytokine sequence and the second cytokine monomer domain sequence is derived from an IL-10 or IFNγ cytokine sequence.

[0031] The present disclosure also includes isolated polynucleotides that include any of the polypeptides of the present disclosure. Also included are vectors, such as bacteriophage or viral vectors, that include such polynucleotides.

[0032] Additionally, the present disclosure encompasses cells comprising cells that express the polynucleotides of the present disclosure and / or the polypeptides of the present disclosure.

[0033] The present disclosure also relates to pharmaceutical compositions comprising the polypeptides, polynucleotides, vectors and / or cells of the present disclosure.

[0034] Furthermore, the present disclosure relates to therapeutic uses and methods of medical treatment involving the polypeptides, polynucleotides, vectors, cells and / or (pharmaceutical / therapeutic) compositions of the present disclosure.

[0035] Various aspects and embodiments of the present disclosure are defined in the clauses and appended claims. Further aspects and embodiments of the inventive concepts are described elsewhere herein.

[0036] It will be understood that any feature of one aspect or embodiment of the invention may be combined with any combination of features of other aspects or embodiments of the invention, unless otherwise stated, and such combinations are within the scope of the claimed invention. Accordingly, the invention includes all of the clauses and claims of this disclosure in any and all combinations except where such combinations are incompatible. [Brief explanation of the drawings]

[0037] The invention is further explained by the accompanying drawings.

[0038] [Figure 1]Figure 1 shows a schematic of the general concept of a foldikine. The basic foldikine concept is a single-chain molecule consisting of a folded cytokine domain (transparent ribbon; right-hand side) that is continuous in sequence and 3D space, linked via its N- and C-termini (a grey Xn linker; this can be the natural N- and C-termini, or can result from opening a loop in the natural sequence to form new N- and C-termini in combination with closing the natural N- and C-termini with an NtCt linker, also depicted in grey) to another cytokine-based domain, which is split into two parts of equal or unequal size (black and medium grey, left-hand side), and the N- and C-termini of the resulting foldikine molecule can be the natural N- and C-termini of the second (left-hand side) cytokine domain, or new N- and C-termini resulting from splitting the second cytokine domain. In this particular example, a foldkine is illustrated, based on a type II cytokine, in which the native N- and C-termini of the cytokine monomers forming a continuous 3D domain are linked with an engineered linker sequence (NtCt). To create the fused dimeric structure, the loop naturally bridging α-helices 3 and 4 in the two monomers is opened, and the newly engineered N- and C-termini of both domains are linked via two Xn-designed linkers (gray). In this way, the continuous 3D domain (helices 1B-6B, labeled in light gray) is connected to each of the two halves of the non-continuous 3D domain (helices 1A-3A, labeled in black, and helices 4A-6A, labeled in gray). The helices are numbered in the order they appear in the sequence for each domain (helices 1A-6A) of the native monomeric foldkine. In the continuous 3D domain of a foldikine, according to this embodiment, the order of the helices in the N- to C-terminal direction is 4B, 5B, 6B, 1B, 2B, 3B. The number of helices varies depending on the cytokine from which the foldikine is derived.For example, type I cytokines typically contain four conserved α-helices, while type II cytokines typically contain six conserved α-helices. The designed linker is shown as a ribbon, depicting the protein backbone (N, CA, CO atoms). [Figure 2] FIG. 2 is a flow diagram illustrating the cytokine problem and the solution using foldikines. [Figure 3] Figure 3 shows a schematic diagram of the process for forming a foldikine based on a dimeric swapped-domain cytokine type II. N-ter A and C-ter A indicate the N- and C-termini of the first cytokine monomer, and N-ter B and C-ter B indicate the N- and C-termini of the second cytokine monomer in the swapped-domain dimer. Swapped-domain foldikines, such as IL-10, are composed of two chains (dark and gray helices) that form two cytokine domains composed of the N- and C-regions of each molecule. The black loop represents a novel linker (NC linker) designed to join the N-terminus of one of the cytokine monomers (the B monomer in this example) to the C-terminus of the other cytokine monomer (the A monomer in this example) to create a "continuous foldikine domain" (see also Figure 25) connected by two linkers to the split domain (left 3D domain). [Figure 4]Figure 4 is a schematic diagram of the process for designing a foldikine according to Method B based on two monomeric class I short- or long-chain cytokines. N-ter A and C-ter A indicate the N- and C-termini of one of the original or wild-type cytokine monomers (A), and N-ter B and C-ter B indicate the N- and C-termini of the other original cytokine monomer (B) that forms the dimeric cytokine. The black loops in the A cytokine domain are exemplary and represent the loops that are opened in this example to create a foldikine by combining the original N-ter A and C-ter B of monomers A and B and the original N-ter B and C-ter A to create a single-chain foldikine (illustrated with a black linker; center). In this diagram, peptide loops in the A domain are opened to form the N- and C-termini of the foldikine (see also Figures 6 and 27). In other embodiments, different loops between helices may be opened in the A domain to form new N- and C-termini. In other embodiments, loops in B cytokines may be opened to form novel N- and C-termini. [Figure 5]Figure 5 is a schematic diagram of the process for designing foldikines according to Method A based on two monomeric class II cytokines. N-ter A and C-ter A indicate the N- and C-termini of one of the original or wild-type cytokine monomers (A), and N-ter B and C-ter B indicate the N- and C-termini of the other original cytokine monomer (B). The black loops (upper left panel) are exemplary and represent two loops that are opened to create new N- and C-termini (NN-ter A and B and NC-ter A and B; upper right panel). These new N- and C-termini can be connected with new peptide linkers (indicated by small black arrows; lower right panel). The original N-ter and C-ter of one of the two original / wild-type cytokine monomers (cytokine B in this example) are then joined with a novel peptide linker (small black arrow; bottom left panel) to form a single polypeptide with two foldikine domains sandwiched between two halves of the split domain (left): the continuous domain (right) (see also Figure 27). [Figure 6]Figure 6 is a schematic diagram of the process for designing chimeric foldikines based on monomeric class I and class II cytokines. According to method B, N-ter A and C-ter A refer to the N- and C-termini of the original / wild-type class II cytokine monomer (i), and according to method C, N-ter B and C-ter B refer to the N- and C-termini of the original / wild-type class I cytokine monomer (ii) (see upper left in panels A and B). Two possible strategies for creating foldikines composed of type I and type II monomers are illustrated: (i) the N-terminus of one monomer is covalently linked to the C-terminus of the other monomer, or vice versa, to create a single polypeptide (black line, see upper right panel). Additionally, the linker region between adjacent alpha helices in one of the original monomer domains (black line; see lower right panel) is opened to create new N- and C-termini in the foldikine (NN-ter and NC-ter; lower left panel). In the depicted embodiment, new termini are created in the original cytokine type I sequence of domain B (see also Figures 4 and 27). (ii) A linker region / loop is opened in the type II cytokine (cytokine A) to create new N- and C-termini (NN-ter and NC-ter; top right panel). The new N-terminus (NN-ter) of monomer A is then covalently linked to the native C-terminus (C-ter B) of monomer B, and the new C-terminus (NC-ter) of monomer A is covalently linked to the native N-terminus (N-ter B) of monomer B, joining the two molecules (black Xn-labeled line; see bottom right panel) to create foldikine II-I. Alternatively, the native N- and C-termini of monomer A are joined (NtCt) to form a circular permutant continuous domain of monomer A, and the linker region / loop is opened in the type I cytokine (monomer B) to generate novel N- and C-termini (lower left panel) creating foldikine I-II. [Figure 7]Figure 7 is a schematic diagram showing how the structure of a swapped-domain type II cytokine (SC-IL10) can be used as a reference for superimposing two type II cytokine monomers, A and B. In a first step, monomers A and B are superimposed onto the respective folded 3D domains of the swapped-domain template to create the desired relative positioning and orientation of the A and B monomers. In a second step, opposing loops between the helices of each monomer domain are opened to create new N- and C-termini on each monomer that are joined with compatible new termini on the opposing monomer to form a bridging linker pair joining the monomers (as illustrated in Figures 3-6). The length and composition of the bridging linker sequence may be determined by modeling an optimal linker sequence taking into account the sequence and structural conformation of each monomer to form a two-domain foldikine that maintains the optimal or desired structural conformation and geometric configuration between the two foldikine domains. In a separate step, the natural N- and C-termini of one of the monomers (A or B) are linked to close one of the monomers and create a single-chain foldikine polypeptide. Monomers A or B that are closed by linking their natural N- and C-termini result in a "continuous" domain in sequence and 3D space. [Figure 8]8 is a schematic representation of the structural confirmation of three different wild-type swapped-domain cytokine dimers when bound to their respective receptors, showing the angles between the two halves of the dimeric cytokine and their approximate orientation relative to the cell membrane: (A) IFNγ cytokine, with receptors R1 and R2 pointed out and exhibiting a V-angle between the left- and right-hand domains; (B) IL-10 cytokine, with receptors R1 and R2 pointed out and exhibiting a V-angle between the left- and right-hand domains; (C) IL-5 cytokine, with one receptor bound and exhibiting a flat 180-degree angle between the two structural 3D domains. Foldikines according to the present disclosure are preferably designed and constructed with covalent peptide linkers to constrain / promote a tertiary structure that mimics the quaternary structure of the swapped wild-type cytokine dimer. [Figure 9] Figure 9 shows the effect of recombinant IL-10 or bacterially secreted IL-10 on primary monocyte cells. (A), (B) Mean fluorescence intensity (MFI) of flow cytometry data of cells from human donors (n = 4) for anti-inflammatory markers (CD163, CD16, MerTK, CCR4, and PDL1) and pro-inflammatory markers (MHC2 and CD86) after addition of monocyte medium (control) or hIL-10 recombinant (hIL-10r) (A) or after addition of supernatant from wild-type (WT) M. pneumoniae (control) or M. pneumoniae expressing the IL-10 ORF (WT_IL10) (B). Light gray bars represent the mean fluorescence intensity (MFI) for the control condition; dark gray bars represent the MFI for the test condition. *p<0.01 (2-way ANOVA and post-hoc Bonferroni multiple comparison test). (C) Western blot results of monocytes isolated from two independent donors showing unstimulated cells (control) or cells stimulated with recombinant human IL-10 (hIL-10r), WT M. pneumoniae supernatant (WT control), or IL-10-expressing M. pneumoniae supernatant (WT-IL-10). [Figure 10]Figure 10 is a schematic diagram of the IL-10 mutations generated in this study to increase IL-10 R1 and R2 receptor binding affinity. Details for each of the positions mutated to improve interaction with R1 or R2 are shown in the individual panels. [Figure 11] Figure 11 shows the expression levels and apparent dissociation constants of selected IL-10 variants expressed in or by M. pneumoniae. (A) Apparent KD (molar) for the IL-10 ORFs MutM, hIL-10r, Mut1, Mut2, Mut3, MutSC1, and MutSC2. The average apparent KD (picomolar) is shown in gray above each mutant. n>3 biological replicates. *, p<0.05 (one-way ANOVA and post-hoc Bonferroni multiple comparison test, selected as reference IL-10 ORF condition). (B) Expression levels in femtograms (fg) of IL-10 secreted into the medium normalized by the number of bacterial colony-forming units (CFU) at 48 hours post-inoculation (48 hpi). *, p<0.05 (one-way ANOVA with Tukey post-hoc test). (C) Impact of linker composition on protein functionality for MutSC1 and MutSC1_controlGly. The x-axis shows the range of IL-10 concentrations analyzed, and the y-axis shows the absorbance at 630 nm measured in HEK-Blue™ cells (N=3, n=2). (D) Western blot of p-STAT3 activation after induction with a fixed IL-10 concentration (20 ng / mL) in two different cell lines: THP-1 and HAFTL. The ratio p-STAT3 / STAT3 was measured after 20 min of induction and is shown below the graph. (E and F) Average fold change in apparent Kd determined by flow cytometry between the IL-10 ORF and each of the single-chain mutants MutSC1 and MutSC2, determined in BlaER1 (E) and HAFTL cells (F). [Figure 12]Figure 12 is a schematic depicting the design of single-chain (SC) IL-10 foldikine. (A) Steps for generating two different fusion patterns: (1) deletion of fringe residues; (2) rewiring scheme; (3) structural rearrangement after rewiring the corresponding regions; and (4) final numbering in the SC, including a numerical gap long enough to accommodate peptide bridges of different lengths (up to 20) during linker search or bridge formation. Residue numbers for monomer 2 are marked with ' in the left-hand panel. (B) Construction of MutSC1 and MutSC2 from IL-10 fusion pattern 1. Linker sequences are shown as gray labels; post-rewiring mutations are included for MutSC2 using the monomer IL-10 numbering. [Figure 13]Figure 13. In vivo characterization of the Mycoplasma pneumoniae CV8 chassis. C57Bl / 6 mice were infected with 10 CFU of WT or CV8 strains and sacrificed 2 or 4 days postinfection (2 or 4 dpi) (n≥3 / group). (A) Similar bacterial burdens (Log10 CFU / lung homogenate) were found for the WT (circles) and CV8 (squares) strains at the analyzed time points. (B) Inflammatory profile of lungs inoculated with WT (center bar), CV8 (right bar), or PBS (left bar). Gene expression was analyzed by RT-qPCR using gapdh as an endogenous control (Methods). Data are presented as mean ± SD of fold change (FC) in mRNA expression (one-way ANOVA + Tukey post-hoc test) (*, p<0.05). (C) Histological findings of lung samples. Left: Plots represent the quantitative assessment (score: 0-5) of alveolar (upper panel) and peribronchiolar / peribronchiolar infiltrates (lower panel). Parameters were normalized using the mean of the PBS group (FC = sample value / mean PBS; FC control group, approximately 1). Data are presented as mean ± SEM of FC (one-way ANOVA + Tukey post-hoc test) (*, p<0.05). Right: Representative images of lungs stained with hematoxylin-eosin are shown (bars represent 250 μm). (D) Characterization of WT and CV8-expressing cells. IL-10 ORF production by the analyzed WT and CV8 strains was measured by ELISA and normalized by protein content (biomass). The functionality of IL-10 variants produced by WT or CV8 strains was verified in HEK-Blue™ cells using supernatants of WT or CV8 strains as negative controls (one-way ANOVA + Tukey post-hoc test) (*, p<0.05). [Figure 14]Figure 14. Immunomodulatory potential of IL-10 variants in vivo. (A) Schematic representation of the mouse model. (B) CFU of PAO1 (left) or mycoplasma strains (right) recovered from lung samples. Data are shown as mean ± SEM of Log10 CFU / lung homogenate. (C) Fold change (FC) of mRNA gene expression of inflammatory markers. Values ​​were normalized using gapdh as an endogenous control using the 2-ΔΔCt method. Data are shown as mean ± SEM of FC. Statistical analysis was performed using an unpaired t-test. (D) P values ​​for statistical comparison of the PAO1 + PBS group compared with uninfected animals or groups treated with PAO1 + CV8, IL-10 WT (CV8-IL-10 ORF), MutSC1 (CV8_MutSC1), MutSC2 (CV8_MutSC2), or CV8 encoding human IL-10 recombinant protein (hIL-10r). Dark gray = significant increase in gene expression; light gray = significant decrease. (E) Quantitative analysis of neutrophil elastase (NE) as demonstrated by immunohistochemistry (IHC) of lung samples. Data are expressed as the mean ± SEM of the percentage of positive cells, calculated as follows: % = 100 × (positive cells / positive cells + negative cells). (F) Representative images of IHC for NE are shown. Lines indicate NE-positive cells. [Figure 15] Figure 15 shows a multiple sequence alignment of IL-10 polypeptides from different vertebrate species performed using the ClustalX algorithm: IL-10 human (SEQ ID NO: 349); IL-10 mouse (SEQ ID NO: 350); IL-10 macaque (SEQ ID NO: 351); IL-10 chicken (SEQ ID NO: 352); IL-10 guinea pig (SEQ ID NO: 353), and IL-10 zebrafish (SEQ ID NO: 354). [Figure 16] Figure 16 shows a superposition of the crystal structures of IL-10 (1yl6k) bound to receptor R1 and IL-10 (6x93) bound to both receptors R1 and R2. The relatively unstructured region that adopts a different conformation when interacting with R2 is shown in the lower right. [Figure 17]Figure 17. Analysis of P. aeruginosa PAO1 infection of mouse lungs. (A) Bacterial burden (CFU) obtained from mouse lung homogenates 24 or 48 hours post-infection (24 or 48 hpi). (B) Fold change in mRNA expression of different inflammatory markers at 24 hpi with PAO1 at doses of 10 and 10 CFU / mouse. [Figure 18] Figure 18. Generation of controls to validate the protein design loop in foldikine-10. (A) Activation of HEK-blue cells-10 with supernatant of Mycoplasma pneumoniae bacterial cell culture. (B) Measurement of Kdapp for foldikine-10 (MutSC1) and two controls to demonstrate the importance of the designed linker. [Figure 19] Figure 19 is a graph showing the impact of linker composition over foldikine-22. Foldikine-22_3 is the same as foldikine-22_3 (SCIL22_3) used herein, foldikine-22_controlcentre is the same as foldikine-22_3_centrallinkers used herein, and foldikine-22_N-C loop polyG is the same as foldikine-22_3_linkerNCpolygly used herein. It demonstrates that replacement of the engineered loop between the N- and C-termini of one foldikine domain or in the bridge between foldikine domains with polyG causes a significant loss of activity. [Figure 20] FIG. 20. Analysis of the impact of IL-22 hydrophobic mutations compared to IL22 wt. [Figure 21]Figure 21. Analysis of the impact of individual mutations in the hydrophobic core of IL-22. IL-22 is IL-22 WT, C>A at terminus is C to A mutation at position 7; 56 is T56M point mutation in monomeric IL22; 66 is A66M point mutation in monomeric IL22; 95 is V95I point mutation in monomeric IL22; 99 is T99F point mutation in monomeric IL22; 173 is S173L point mutation in monomeric IL22. Bottom: Apparent average Kd determined for WT and mutant constructs. [Figure 22] Figure 22 is a graph showing the response of HEK-Blue™ IL-22 cells to engineered versions of IL-22. HEK-Blue™ IL-22 cells were incubated with bacterial supernatants expressing either IL-22, foldikine-22_1, foldikine-22_3, or a control with a poly-Gly linker at the N- to C-terminus (foldikine-22_3_linkerNCpolygly). After 24 hours of incubation, internal signaling activation was assessed by measuring SEAP levels in the supernatant using Quanti-Blue™. (A) Undiluted supernatant (concentration >30 ng / mL). (B) Eight serial dilutions (each 0.5-fold dilution) of IL-22 starting at 30 ng / L. Concentrations are shown on the x-axis in molar concentrations. [Figure 23] Figure 23 shows the response of HEK-Blue™ IL-10 and IL-22 cells to foldikine-10 / 22 (SCIL10IL22). HEK-Blue™ IL-10 cells were incubated with bacterial supernatants expressing two different chimeras (chimera 3 and chimera 5). After 24 hours of incubation, internal signaling activation was assessed by measuring SEAP levels in the supernatants using Quanti-Blue™. Undiluted supernatants (quantified chimera concentrations >30 ng / mL) were assessed for IL-10 and IL-22 activity, respectively. [Figure 24]Figure 24 shows the response of HEK-Blue™ IL-10 and IL-22 cells to heterodimers fused by a short poly-gly-ser linker. HEK-Blue™ IL-10 cells were incubated with supernatants from bacteria expressing IL-10, IL-22, heterodimer 10-22 (heterodimer 10-22), or heterodimer 2210 (heterodimer 2210). After 24 hours of incubation, internal signaling activation was assessed by measuring SEAP levels in the supernatants using Quanti-Blue™. For panels (A) and (B), undiluted supernatants (quantified chimera concentrations >30 ng / mL) were assessed for IL-10 and IL-22, respectively. [Figure 25] Figure 25 is a schematic diagram of a mutation scheme for forming a foldikine based on a swapped-domain dimeric class I cytokine: (i) a wild-type domain-swapped dimer showing the α-helix D of each monomer unit aligning with the α-helices A, B, and C of the opposing monomer to form an adjacent 3D domain comprising α-helices A, B, and C of a first monomer sequence and α-helix D of a second monomer sequence; (ii) a single-chain foldikine based on fused wild-type monomers in which the monomer sequences are joined to create a single-chain polypeptide by linking the C-terminus of one monomer sequence to the N-terminus of the second monomer sequence using a peptide linker (dashed line). The wild-type linker is shown with a solid line. Boxes indicate the α-helices that form each of the left and right 3D domains. As depicted, there are two peptide linkers that span the first (left) and second (right) 3D tertiary structure domains to provide structural / conformational stability. [Figure 26]Figure 26 is a schematic diagram of a mutation scheme for forming a foldikine based on a dimeric class I cytokine: (i) a wild-type dimer showing the left and right domains of the cytokine dimer, each containing α-helices A, B, C, and D of the first and second monomer sequences, respectively; (ii) a single-chain foldikine based on fused wild-type monomers, in which the monomer sequences have been joined to create a single-chain polypeptide by linking the C-terminus of each monomer to the respective N-terminus of each monomer (dashed lines) and then removing the internal wild-type linker from one of the original monomer sequences to create new N- and C-termini. The wild-type linker is shown with a solid line. Boxes indicate the α-helices that form each of the left and right 3D domains. As depicted, there are two peptide linkers that span the first (left) and second (right) 3D tertiary structure domains to provide structural / conformational stability. [Figure 27] Figure 27 is a schematic diagram of a mutation scheme for forming a foldikine based on a dimeric class II cytokine: (i) a wild-type dimer showing the left and right domains of the cytokine dimer, each containing α-helices A, B, C, D, E, F, and G of the first and second monomer sequences, respectively; (ii) a single-chain foldikine based on fused wild-type monomers in which the monomer sequences have been joined to create a single-chain polypeptide by linking the C-terminus of one monomer to the N-terminus of the same monomer and removing internal wild-type linkers from each of the original monomer sequences to create new N- and C-termini, which are then joined by two new inter-domain peptide linkers (dashed lines) to form the single-chain polypeptide. The wild-type linkers are shown with solid lines. Boxes indicate the α-helices that form each of the left and right 3D domains. As depicted, there are two peptide linkers that span the first (left) and second (right) 3D tertiary structure domains to provide structural / conformational stability. [Figure 28]Figure 28 is a schematic diagram of a mutation scheme for forming a foldikine based on a swapped-domain dimeric class II cytokine: (i) a wild-type domain-swapped dimer showing α-helices D, E, and F of each monomer unit aligning with α-helices A, B, and C of the opposing monomer to form an adjacent 3D domain comprising α-helices A, B, and C of the first monomer sequence and α-helices D, E, and F of the second monomer sequence; (ii) a single-chain foldikine based on fused wild-type monomers in which the monomer sequences are joined to create a single-chain polypeptide by linking the C-terminus of one monomer sequence to the N-terminus of the second monomer sequence using a peptide linker (dashed line). The wild-type linker is shown with a solid line. Boxes indicate the α-helices that form each of the left and right 3D domains. As depicted, there are two peptide linkers that span the first (left) and second (right) 3D tertiary structure domains to provide structural / conformational stability. [Figure 29] Figure 29 is a graph demonstrating the expression of MutSC1 foldikine in Escherichia coli (E. coli) cells. IL-10 polypeptide sequences in wild-type IL-10 and MutSC1 foldikine were quantified by ELISA from both pelleted cells (pel) and supernatants (sup). Colony-forming unit (CFU) counts were normalized to the femtosecond micrograms of IL-10 estimated in each culture. MutSC1 expression was detected in both the pellet and the supernatant. The functionality of E. coli-expressed MutSC1 was assessed in HEK-blue cells. Both pellet and supernatant MutSC1 activated the p-STAT3 cascade in this reporter cell line. MutSC1_His indicates results for His-tagged foldikine. [Figure 30]Figure 30 is a graph demonstrating the expression of MutSC1 foldikine in L. lactis cells. The IL-10 polypeptide sequence in wild-type IL-10 and MutSC1 was quantified by ELISA from both pelleted cells (pel) and supernatant (sp). HEK-Blue10 reporter cells were used to verify the functionality of the protein expressed by L. lactis. On the y-axis, fg was quantified by ELISA and normalized to CFU counted in two different cultures, IL-10 and MutSC1. [Figure 31] Figure 31 is a schematic diagram of the mutation scheme for forming a foldikine according to method A based on a class I cytokine. (Top part) Wild-type cytokine type I monomers, each containing α-helices A, B, C, and D; (bottom part) Single-chain foldikines based on fused wild-type monomers in which the monomer sequences have been joined by opening the loop between α-helices B and C of both cytokine monomers, linking the new N-terminus of one cytokine monomer to the new C-terminus of the other cytokine monomer, and conversely, linking the natural N-terminus and C-terminus of one of the cytokine monomers. The wild-type linker is shown with a solid line. Boxes indicate the α-helices that form each of the left and right 3D domains. As depicted, there are two peptide linkers that span the first (left) and second (right) 3D tertiary structure domains to provide structural / conformational stability. [Figure 32] Figure 32 shows a 3D representation of a foldikine according to method A based on a dimeric class I cytokine with a newly introduced interlinker (linker between two cytokine monomers) in black as well as a new intralinker (connecting the native N- and C-termini of one of the cytokine monomers). Nt and Ct indicate the N- and C-termini of the original cytokine monomer. [Figure 33]Figure 33 is a schematic diagram of a mutation scheme for forming a foldikine according to Method B based on two class II cytokines: (top part) wild-type cytokine type II monomers, each containing α-helices A, B, C, D, E, F, and G of the first and second monomer sequences, respectively; (bottom part) a single-chain foldikine based on fused wild-type monomers in which monomer sequences have been joined to create a single-chain polypeptide by linking the C-terminus of one monomer to the N-terminus of the other monomer, and conversely, removing the internal wild-type linker between α-helices C and D from one original monomer sequence to create new N- and C-termini (Nt and Ct) to form a single-chain polypeptide. The wild-type linker is shown with a solid line. Boxes indicate the α-helices that form each of the left and right 3D domains. As depicted, there are two peptide linkers that span the first (left) and second (right) 3D tertiary structure domains to provide structural / conformational stability. [Figure 34] Figure 34. 3D representation of a foldikine according to method A based on a dimeric class II cytokine: the discontinuous black line is the newly introduced interlinker, and the new Nter and new Cter indicate the newly created N- and C-termini after opening the loop in one monomer. [Figure 35] Figure 35 shows a titration plot of HEK Blue™ cells in response to IL-2 using supernatants from CHO cells transiently expressing IL-2 WT and IL-2 foldikine (SCIL2 with different interlinkers composed of proline (ORK2_6-8) or glycine / serine (ORK_9-11)). Proteins were expressed using CHO as the expression system. Vmax was set to 0.9. [Figure 36] Figure 36. Titration plot of HEK Blue™ cells in response to IL-22 with supernatant of M. pneumoniae cells transiently expressing IL-22 WT or IL-22 foldikine (ORK22 with different interlinkers composed of proline (ORK17-19) or glycine / serine (ORK_24-26)). [Figure 37]Figure 37 shows a titration plot of HEK Blue™ cells in response to IL22 using supernatants from CHO cells transiently expressing IL-22 WT (9,76E-11M) and IL-22 foldikines (ORIK22-12 (4,32E-11M) with a poly-pro interlinker and ORIK22_14 (2,41E-09M) with a poly-Gly interlinker). The proteins were expressed using CHO as the expression system. [Figure 38] FIG. 38. Titration plot of HEK Blue™ cells in response to IL-2 with supernatants from CHO cells transiently expressing IL-2 WT and the chimeric foldikine IL2 / IL-4 (IL2-IL4 polyPro linker). [Figure 39] Figure 39 shows a titration plot of HEK Blue™ cells in response to IL-4 using supernatants from CHO cells transiently expressing IL-2 WT and the chimeric foldikine IL2 / IL-4 (IL2-IL4 polyPro linker). For comparison purposes, the activity of commercially purchased IL-4 (6507-IL-010 / CF, Biotechne) is shown. [Figure 40] FIG. 40. Titration plot of HEK Blue™ cells in response to IL-2 with supernatant of CHO cells transiently expressing IL-2 WT and foldikine ORK2_013. [Figure 41] Figure 41 shows a titration plot of HEK Blue™ cells in response to IL-22. The Y-axis shows the calculated EC-50 in nM. The X-axis indicates the different IL variants expressed in CHO and tested. In all cases, comparative values ​​are shown for IL-22 WT. The different variants represent different circularly permuted versions of the WT cytokine, joining Nt and Ct by protein design (ModelX and FoldX) engineered linkers and opening different loops. LF means that the protein was expressed but lost activity in the HEK reporter. Proteins were expressed using CHO as the expression system. Above each bar, the average EC-50 in nM for each variant is shown. [Figure 42] Figure 42 shows a titration plot of HEK Blue™ cells in response to IFNβ. The X-axis indicates the different IL variants expressed and tested in CHO. In all cases, comparative values ​​are shown for WT IFNβ. The different variants represent different circularly permuted versions of the WT cytokine, joining the Nt and Ct by protein design (ModelX and FoldX) engineered linkers and opening different loops. Proteins were expressed using CHO as the expression system. Above each bar, the average EC-50 in nM for each variant is shown. [Figure 43] Figure 43 shows a titration plot of HEK Blue™ cells in response to IL-2. The X-axis indicates the different IL variants expressed and tested in CHO. In all cases, WT IL-2 values ​​are shown for comparison. The different variants represent different circularly permuted versions of the WT cytokine, conjugated with Nt and Ct by protein design (ModelX and FoldX) engineered linkers and opening different loops. Proteins were expressed using CHO as the expression system. Above each bar, the average EC-50 in nM for each variant is shown. [Figure 44] FIG. 44 shows a titration plot of HEK Dual cells in response to IFNγ with supernatants from CHO cells transiently expressing IFNγ WT and the foldikine ORKIFNg-002. [Figure 45]Figure 45 is a schematic diagram of the mutation scheme for forming a foldikine according to method C based on a dimeric class I cytokine. (Top part) Wild-type monomer type I (cytokine 1 and cytokine 2) each containing α-helices A, B, C, and D; (bottom part) Single-chain foldikine based on fused wild-type monomers in which the monomer sequences are joined by opening the loop between α-helices B and C of both cytokine monomers. The loop between helices B and C of monomer 2 is opened, and helix B of monomer 2 is linked to the native N-terminus of monomer 1, and helix C is linked to the native C-terminus of monomer 1. Boxes indicate the α-helices that form each of the left and right 3D domains. As depicted, there are two peptide linkers spanning the first (left) and second (right) 3D tertiary structure domains to provide structural / conformational stability. [Figure 46] Figure 46 shows a 3D representation of a foldikine according to method C, based on a dimeric class I cytokine with a newly introduced interlinker (linker between two cytokine monomers) indicated by a dashed line. Nt and Ct indicate the N- and C-termini of the original cytokine monomer. [Figure 47]Figure 47 is a schematic diagram of a mutational scheme for forming foldikines according to method C based on class II cytokines. (Top part) Wild-type type II monomers (cytokines 1 and 2), each containing α-helices A, B, C, D, E, F, and G; (bottom part) a single-chain foldikine based on fused wild-type type II cytokine monomers in which the monomer sequences have been joined by opening the loop between α-helices B and C of one cytokine monomer (cytokine 1), linking the novel C-terminus of the opened cytokine monomer (cytokine 1, helix B) to the native N-terminus of the other cytokine monomer (cytokine 2, helix A), and linking the novel N-terminus of the opened cytokine monomer (cytokine 1, helix C) to the native C-terminus of the other cytokine monomer (cytokine 2; helix F). Boxes indicate the α-helices that form each of the left and right 3D domains. As depicted, there are two peptide linkers spanning the first (left) and second (right) 3D tertiary structure domains to provide structural / conformational stability. [Figure 48] Figure 48 shows a 3D representation of a foldikine according to method C, based on a dimeric class II cytokine with a bold newly introduced interlinker (linker between two cytokine monomers). Nt and Ct indicate the N- and C-termini of the original cytokine monomer. DETAILED DESCRIPTION OF THE INVENTION

[0039] All documents cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs (e.g., cell culture, molecular genetics, nucleic acid chemistry, and biochemistry).

[0040] Unless otherwise indicated, the practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, chemical methods, pharmaceutical formulation, and animal delivery and treatment, which are within the capabilities of those of ordinary skill in the art. Such techniques are described in the literature, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice, Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J. Lilley and J. E. Dahlberg, 1992, and Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press, each of which general texts is incorporated herein by reference.

[0041] To facilitate the understanding of the present invention, several terms are defined herein.

[0042] The term "amino acid" in the context of the present invention is used in its broadest sense and is meant to include naturally occurring L-α-amino acids or residues. The commonly used one- and three-letter abbreviations for naturally occurring amino acids are used herein: A=Ala, C=Cys, D=Asp, E=Glu, F=Phe, G=Gly, H=His, I=Ile, K=Lys, L=Leu, M=Met, N=Asn, P=Pro, Q=Gln, R=Arg, S=Ser, T=Thr, V=Val, W=Trp, and Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2nd ed., pp. 71-92, Worth Publishers, New York). The generic term "amino acid" further encompasses chemically modified amino acids, such as D-amino acids, retro-inverso amino acids, and amino acid analogs; naturally occurring amino acids not normally incorporated into proteins, such as norleucine; and chemically synthesized compounds possessing properties known in the art to be characteristic of amino acids, such as β-amino acids. For example, analogs or mimetics of phenylalanine and proline, which can restrict the conformation of peptide compounds in the same way as natural Phe and Pro, are included within the definition of amino acid. Such analogs and mimetics are referred to herein as "functional equivalents" of the respective amino acids. Other examples of amino acids are listed in Roberts and Vellaccio, "The Peptides: Analysis, Synthesis, Biology," Gross and Meiehofer, eds., Vol. 5, p. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference.

[0043] The term "peptide," as used herein (e.g., in the context of a foldikine protein as disclosed herein), refers to multiple amino acids linked together in a linear or cyclic chain. The term oligopeptide is typically used to refer to peptides having from 2 to about 50 or more amino acids. Peptides of more than about 50 amino acids are often referred to as polypeptides or proteins. However, in the present invention, the term "peptide" is not limited to any particular number of amino acids and is used interchangeably with the terms "polypeptide" and "protein," unless otherwise indicated. Given that minor modifications to the primary sequence of the peptides / proteins of the present disclosure can be made without substantially altering the activity of the polypeptides according to the present invention, the present disclosure and various aspects and embodiments of the present invention should further be considered to encompass any polypeptide sequence that is substantially identical to the specific amino acid sequences disclosed herein. For example, the claimed invention encompasses polypeptide sequences having at least 80% identity to a polypeptide disclosed herein (as may be defined by the SEQ ID NOs of the polypeptide sequences disclosed herein), at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, or about 100% identity to a polypeptide sequence of a SEQ ID NO explicitly disclosed herein.

[0044] As used herein, the term "cytokine" refers to any one of a broad and loose category of small proteins important in cell signaling. Cytokines are peptides that cannot cross the lipid bilayer of a cell and enter the cytoplasm. Cytokines have been shown to be involved in autocrine, paracrine, and endocrine signaling as immunomodulators. The term "foldikine," as used herein, refers to a single-chain cytokine derived from two covalently linked wild-type cytokine monomers and folded to form a dimeric 3D domain structure. Generally, the peptide sequence of each 3D domain of the covalent dimer also contains one or more amino acid mutations, substitutions, and / or deletions to (i) dimerize the cytokine monomers and / or (ii) structurally restrict the configuration of the novel 3D dimer unit, and therefore is not naturally occurring.

[0045] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to deoxyribonucleotide (DNA) or ribonucleotide (RNA) polymers, or mixed polymers, in either linear or cyclic conformations and in single- or double-stranded form. In the present invention, such DNA or RNA polymers can contain natural nucleotides, non-natural or synthetic nucleotides, and mixtures thereof. Non-natural nucleotides include analogs of natural nucleotides and nucleotides modified at the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). Examples of modified nucleic acids include PNAs and morpholino nucleic acids. Generally, analogs of a particular nucleotide have the same base-pairing specificity, i.e., an analog of G will base pair with C. In the present invention, these terms should not be considered limiting with respect to the length of the polymer. In certain embodiments, the 5' and / or 3' ends of the polynucleotides of the present disclosure can be modified to improve sequence stability to actively prevent degradation. Suitable modifications in this context include, but are not limited to, biotinylated nucleotides and phosphorothioate nucleotides.

[0046] As used herein, a "gene" is a segment of nucleic acid (typically DNA) involved in producing a polypeptide or ribonucleic acid gene product. It includes regions preceding and following the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons). Advantageously, the term also encompasses the coding and / or transcribed region that encodes a gene product, as well as regulatory sequences required for gene expression (e.g., enhancers, silencers, promoters, terminators, etc.), which may be adjacent to or separate from the associated coding sequence. Preferred genes according to the present invention are those that encode cytokines, particularly those that encode dimeric cytokines or "foldikines" according to the present disclosure. Taking into account codon redundancy, the present disclosure and claimed invention encompasses polynucleotide sequences having at least 70% identity to the polynucleotides disclosed herein, such as the SEQ ID NOs disclosed herein, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity, or about 100% identity to polynucleotide sequences encoding the SEQ ID NOs explicitly disclosed herein.

[0047] In the context of this disclosure, the terms "individual," "subject," or "patient" are used interchangeably to refer to an animal that may be suffering from a medical (pathological) condition and that may respond to a therapeutic molecule or medical treatment of the present invention. The animal to be treated is preferably a mammal, such as a human, non-human primate, cow, sheep, pig, dog, cat, rabbit, mouse, or rat. Preferably, the subject is a human.

[0048] The terms "treat," "treating," "treatment," or "therapy" as used herein in the context of a disease state or condition refer to the reduction in severity and / or frequency of one or more pathogenic symptoms, the elimination of symptoms, and / or the prevention of symptoms and / or the underlying cause or causes of such disease or condition. In some cases, such treatment may involve the reduction or amelioration of physiological damage caused by the disease or condition.

[0049] The concept of phordikine To improve cytokine efficiency and reduce the concentration required for therapeutic effect, it is necessary to improve cytokine foldability or decrease the rate of degradation and serum clearance by genetically engineering mutations that improve binding affinity, reduce aggregation, or fusion to other protein domains such as Fcs (https: / / pubmed.ncbi.nlm.nih.gov / 32994996 / ) or chemical modification (https: / / www.nature.com / articles / s41551-021-00797-8; https: / / www.mdpi.com / 2079-4991 / 9 / 8 / 1074; https: / / link.springer.com / article / 10.1007 / s11912-019-0760-z).

[0050] Reducing toxicity is often due to the fact that a particular cytokine binds to a variety of cells in the body, some of which are desired targets and others that have undesirable effects. Most cytokines bind to two or three different receptors, and some are promiscuous, recognizing multiple cytokines. Receptors for a particular cytokine are not expressed proportionally at the same levels on all cells; therefore, it should be possible to alter the affinity for a receptor to favor binding to specific cell populations and thus reduce toxicity (https: / / doi.org / 10.1111 / all.15132; https: / / pubmed.ncbi.nlm.nih.gov / 33737461 / ).

[0051] In some cases, a combination of two different cytokines, either antagonistic or agonistic, or a mixture of both, may be useful for a particular treatment (https: / / academic.oup.com / abt / article / 4 / 2 / 123 / 6309385). Linking two cytokines by inserting a linker sequence between the N-terminus of one molecule and the C-terminus of the other may be one way to solve some or all of the above problems. However, even when such cytokines act by forming noncovalent dimers, linking two cytokines via their N- or C-termini is not always possible due to incompatible binding modes to the receptor. Furthermore, a flexible linker may be susceptible to proteolysis and may not increase binding affinity due to conformational flexibility between the two monomers.

[0052] Here, the concept of foldikines may offer significant advantages over both wild-type cytokines and the prior art. As noted above, foldikines are molecules composed of two modified cytokine domains, each of which is referred to herein as a "foldikine domain"; in this regard, it should be noted that at least one of the two "foldikine domains" is not structurally or sequence identical to the cytokine monomer domain from which the foldikine domain is based, as described herein. In aspects and embodiments of the foldikines of the present invention, one of the two foldikine domains may be considered to be contiguous. A foldikine domain may have a non-native sequence order compared to the corresponding cytokine monomer / domain, while another foldikine domain is split into two fragments of equal or different size connected to the natural or newly created N- and C-termini of the continuous foldikine domain (see Figure 1). The continuous and split domains of foldikines are sometimes referred to herein as 3D domains.

[0053] Thus, provided is a single-chain dimeric cytokine polypeptide comprising (a) a first cytokine monomer domain or functional portion thereof and (b) a second cytokine monomer domain or functional portion thereof, wherein the sequence of one of the cytokine monomer domains or functional portions thereof is not contiguous but is interrupted by the sequence of the other cytokine monomer domain or functional portion thereof.

[0054] A cytokine monomer domain of a single-chain dimeric cytokine polypeptide that is not contiguous in sequence is a cytokine monomer domain that comprises the free N-terminus of the artificial single-chain dimeric cytokine polypeptide, which cytokine monomer domain consists of or forms part of a so-called "split domain" throughout this application.

[0055] The sequence of the other cytokine monomer domain or functional portion thereof may have its natural sequence order or may have a different sequence order. The second cytokine monomer domain is also referred to throughout this application as the "contiguous domain," which is connected to the split domain by two peptide linkers ("interlinkers").

[0056] Benefits of Foldikine Having two functionally independent domains with fixed orientation within the same molecule offers some interesting possibilities to answer some of the above-mentioned questions, independent of the mode of production and administration (see Figure 2).

[0057] In embodiments, as shown, for example, in Figures 3-6, 25-28, 31-34, and 45-48, foldikines have the advantage of having two linkers between adjacent (left and right) 3D domains (which themselves can be thought of as "continuous" and "separate"), such that the entropic cost of binding is primarily borne by the first 3D domain, and the presence of the two linkers restricts the mobility of the second 3D domain, thereby increasing the structural / conformational stability of the folded dimer and potentially resulting in improved binding to its target receptor. Furthermore, providing two independent 3D domains connected by two linkers allows for the creation of homo- or heterodimeric cytokines with novel properties, e.g., giving more freedom for mutations that can be introduced at the surface of the 3D domain that interacts with the receptor, which may allow for greater selectivity for specific cell types depending on the level of expression of the target receptor.

[0058] In some advantageous embodiments, a heterodimeric foldikine according to the present invention may exhibit improved binding of one domain to its target receptor, while the other domain may exhibit reduced binding to its target receptor. In such embodiments, the first domain may be advantageously used to target specific cells, while the second domain may have reduced affinity and therefore advantageously / predominantly bind only to cells that also express the receptor for the first domain. In embodiments, a foldikine may also increase the binding affinity of both domains, thus significantly reducing the effective physiological concentration that may be required to achieve a particular physiological effect. For example, it may be desirable to construct an antagonist molecule with two domains that have high affinity for one receptor and no affinity for the second receptor (the receptor for the corresponding wild-type cytokine), so that the foldikine can effectively compete with the corresponding monomeric physiological cytokine. Having two 3D domains allows for many possible combinations of homing, affinity, and specificity, allowing for the combination of agonist and antagonist functions in a single molecule.

[0059] Design of foldikines based on the cytokine family Although we have developed the concept of foldikines for helix-bundle cytokines, which include the majority of interleukins, we anticipate that a similar approach may be possible for other proteins composed of helix-bundle structures or cytokines with other folds. Helix-bundle interleukins are classified as follows: (A) Class I helical cytokines, which can be subdivided into long-chain helical (IL6, IL11, IL12A, IL23A, IL27A, IL31, CLCF1, CNTF, CTF1, LIF, OSM, and CSF3) or short-chain helical (IL2, IL3, IL4, IL5, IL7, IL9, IL13, IL15, IL21, TSLP, GM-CSF, CSF1, and CSF2), or (B) Class II cytokines that are type I interferons, such as IL10-like proteins (IL10, IL19, IL20, IL22, IL24, IL26, IL28A, IL28B, and IL29), and IFNα (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21), IFNω (IFNW1), IFNε (IFNE), IFNκ (IFNK), and IFNβ (IFNB1).

[0060] Although the majority of helix-bundle cytokines share a similar monomer structure within each class, with type I cytokines typically having four conserved α-helices and type II cytokines typically having six conserved α-helices, IFNγ, IL-5, and IL-10 are "swapped-domain" dimeric proteins. In swapped-domain dimeric proteins such as IL-10, the monomeric structure of the standard helix-bundle cytokine unfolds and wraps around the other open IL-10 monomer in an antiparallel manner, together generating two functional split cytokine domains and defining two adjacent 3D domains (see, for example, Figures 3 and 25).

[0061] Depending on the class of interleukin and whether the interleukin is a monomer or a swapped dimer, different strategies for generating foldikines can be employed, as described herein.

[0062] As used herein, the α-helices of a cytokine monomer are numbered from N-terminus to C-terminus in alphabetical order A, B, C, and D (for class I cytokines), or A, B, C, D, E, and F (for class II cytokines), or ordinal (1st through 4th, or 6th, depending on the class of cytokine).

[0063] Foldikines containing swapped domain cytokines According to this embodiment, the single-chain dimeric cytokine polypeptide according to the invention comprises a first and / or second cytokine monomer(s) that form a swapped domain dimer.

[0064] According to one embodiment, a single-chain dimeric cytokine polypeptide according to the present invention comprises a first and a second cytokine monomer that form a swapped domain dimer. Specifically, the first and second cytokine monomers that form the swapped domain dimer may be derived from the same cytokine or from two different cytokines (two different class I cytokines or two different class II cytokines).

[0065] In the case of swapped domain cytokines (e.g., IFNγ, IL-5, and IL-10), the scheme for designing a single-chain dimeric cytokine (i.e., a foldikine) of the present disclosure may depend on whether the swapped domain cytokine is fused to a second swapped domain cytokine or to a cytokine that does not form a swapped domain.

[0066] For example, as described with reference to Figures 3, 25, and 28, in various embodiments in which a foldikine is created from a naturally occurring swapped domain dimer, the method can be to link the N-terminus of a first cytokine monomer to the C-terminus of a second cytokine monomer such that a "continuous 3D (foldikine) domain" (the right-hand domain in Figure 3) is formed and a split 3D (foldikine) domain (the left-hand domain in Figure 3) is formed in the swapped domain foldikine dimer. Note, however, that in this case the C-terminal helix of the most N-terminal monomer is linked to the N-terminal helix of the most C-terminal monomer, so that the "continuous" 3D domain has an inverted helix / sequence compared to the wild-type cytokine (Figures 25 and 28). Furthermore, it should be noted that each domain in 3D space is composed of portions of two separate polypeptide monomers.

[0067] In any such aspect and embodiment, the linker sequence bridging the C-terminus of one monomer and the N-terminus of the other monomer can have any suitable sequence, preferentially including a sequence that defines a conformationally restrained structure, i.e., a "structured" linker. In this manner, the artificial linker serves to conformationally restrain the 3D domain structure in an appropriate / desired structural orientation. Such linkers can have about 3 to about 20 amino acid residues, about 3 to about 16 amino acid residues, about 4 to about 12 amino acid residues, about 4 to about 8 amino acid residues, about 3 to 8 amino acid residues, or about 3 to 6 amino acid residues. Advantageously, such linkers do not contain multiple Gly and / or Ser residues adjacent to one another, for example, advantageously, the linker peptide contains no more than 5 adjacent Gly and / or Ser residues, suitably no more than 3 adjacent Gly and / or Ser residues, no more than 2 Gly and / or Ser residues, only isolated Gly and / or Ser residues, or in some embodiments no Gly and / or Ser residues.

[0068] Thus, there is provided a single-chain dimeric cytokine polypeptide comprising a first cytokine monomer and a second cytokine monomer that form a swapped domain dimer, wherein a linker peptide bridges the N-terminus of the first cytokine monomer and the C-terminus of the second cytokine monomer.

[0069] In some embodiments, the cytokine monomers that form the swapped domain dimer are class I cytokines, and the single-chain dimeric cytokine polypeptide comprises, from N- to C-terminus, a first cytokine monomer domain comprising helices A-C of a second cytokine monomer and helix D of the first cytokine monomer (see, e.g., Figure 25, "Split Domain"), and a second cytokine monomer domain comprising helix D of the second cytokine monomer and helices A-C of the first cytokine monomer (see, e.g., Figure 25, "Continuous Domain"), wherein a linker peptide bridges helix D of the second cytokine monomer with helix A of the first cytokine monomer.

[0070] Exemplary homomeric foldikines based on swapped domain class I cytokines include: TEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLCTEEIFQGIGTLESQTVQGGTVERLFK hhhhhhhhhhhhhh hhhh hhhhhhhhhh hhhhhh HA HB NLSLIKKYIDGQKKKCG EERRRVNQFLDYLQEFLGVMNTEWIIES (NtCt) hhhhhhhhhhhhhhhhhhhhhhhhhhhhhh HC HD TEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLCTEEIFQGIGTLESQTVQGGTVERLFKNL hhhhhhhhhhhhh hhhh hhh hhhhhhhhhh hhhhhhhh HA HB HC SLIKKYIDGQ KKKCGEERRRVNQFLDYLQEFLGVMNTEWIES hhhhhhhhhh hhhhhhhhhhhhhhhhh HD comprising or consisting of (NtCt) is a peptide linker (sequence of SEQ ID NO: 101-(NtCt)-SEQ ID NO: 102), or a dimeric IL-5 that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the sequence and retains at least the same stability and / or at least the same level of interaction with the IL-5 receptor. The positions of the helices are represented by a series of "h"s in the above sequences.

[0071] In some embodiments, the cytokine monomers that form the swapped domain dimer are class II cytokines, and the single-chain dimeric cytokine polypeptide comprises, from N- to C-terminus, a first cytokine monomer domain comprising helices A-C of a second cytokine monomer and helices D-F of a first cytokine monomer (see, e.g., Figure 28, "Split Domain"), and a second cytokine monomer domain comprising helices D-F of the second cytokine monomer and helices A-C of the first cytokine monomer (see, e.g., Figure 28, "Continuous Domain"), wherein a linker peptide bridges helix F of the second cytokine monomer with helix A of the first cytokine monomer.

[0072] Exemplary homomeric foldikines based on swapped domain class II cytokines include dimeric IL-10. According to one embodiment, the dimeric IL-10 is CTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ hhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhhhhh HA HB AENQDPDIKAHVNSLGENLKTLRLR LRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINY hhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhhhhh HC HD HE IEAYMTMKIRN (NtCt)PNMLR DLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEV hhhhhhhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhh HF HA HB MPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKS KAVEQVKNAFNKLQEKGIYKAMSEFDIF hhhhhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhh HC HD HE INYIEAYMTM_YY hhhhhhhhhh HF ((NtCt) is a peptide linker) (SEQ ID NO: 11-(NtCt)-SEQ ID NO: 12) or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto, and retaining at least the same stability and / or at least the same level of interaction with the IL-10 receptor. The positions of the helices are represented by a series of "h"s in the above sequence.

[0073] According to one embodiment, the NtCt linker is a peptide linker and comprises or consists of the sequence NGGLDY (SEQ ID NO: 13).

[0074] Specifically, the single-chain dimeric IL-10 comprises or consists of SEQ ID NO: 9 (the so-called MutSC1 polypeptide of Example 1) or SEQ ID NO: 10 (the so-called MutSC2 polypeptide of Example 1), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto, and can retain at least the same stability and / or at least the same level of interaction with the IL-10 receptor.

[0075] Another exemplary homomeric foldikine based on a swapped domain class II cytokine is dimeric IFNγ.

[0076] According to one embodiment, the dimeric IFNγ has the sequence QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQK hhhhhhhhhhhh hh hhhhh hhhhhhhhhhhhhhhhhh hh HA HB SVETIKEDMNVKFFNSN KKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPMLF(NtCt) hhhhhhhhhhhhhh hhhhhhhhhh hhhhhhhh hhhhhh HC HD HE HF MQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDD hhhhhhhhhhhhh hhhhh hhhhhhhhhhhhhhhhhhhh HA HB QSIQKSVETIKEDMNVKFF NSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQ hhhhhhhhhhhhhhhh hhhhhhhhhh hhhhhhh---hhhhhh HC HD HE HF MLFRGRRASQ_YY ((NtCt) is a peptide linker) (sequence SEQ ID NO: 14-(NtCt)-SEQ ID NO: 15), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto, and retains at least the same stability and / or at least the same level of interaction with the IFNγ receptor. The positions of the helices are represented by a series of "h"s in the above sequences.

[0077] According to one embodiment, the NtCt linker comprises or consists of the sequence EGPG (SEQ ID NO: 16) or ASKPHPGQLWY (SEQ ID NO: 17).

[0078] Specifically, the single chain dimeric IFNγ comprises or consists of SEQ ID NO: 18 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical thereto and can retain at least the same stability and / or at least the same level of interaction with the IFNγ receptor.

[0079] Foldikines containing cytokine(s) that do not form swapped domain cytokines According to this aspect of the invention, the single-chain dimeric cytokine polypeptide comprises (a) a first cytokine monomer domain or a functional portion thereof and (b) a second cytokine monomer domain or a functional portion thereof, wherein the sequence of the second cytokine monomer domain or a functional portion thereof is contiguous, while the sequence of the first cytokine monomer domain is such that the first sequence portion of the first cytokine monomer is located N-terminally of the second cytokine monomer or a functional portion thereof, and the second sequence portion of the first cytokine monomer is located C-terminally of the second cytokine monomer or a functional portion thereof.

[0080] There are three different ways to make a foldikine, which can include two of the same or different class I cytokines, two of the same or different class II cytokines, or one class I cytokine and one class II cytokine.

[0081] According to one embodiment, the first and second cytokine monomers are class I cytokine monomers that do not form swapped domain dimers, and the first and second cytokine monomers are the same or different.

[0082] According to another embodiment, the first and second cytokine monomers are class II cytokine monomers that do not form swapped domain dimers, and the first and second cytokine monomers are the same or different.

[0083] According to another embodiment, the first cytokine monomer is a class I cytokine monomer and the second cytokine monomer is a class II cytokine monomer, or the first cytokine monomer is a class II cytokine monomer and the second cytokine monomer is a class I cytokine monomer.

[0084] Foldikine formation method A According to this method of forming a foldikine, a loop is opened in each cytokine monomer, and the new N-terminus of the open loop of cytokine monomer A is joined with the new C-terminus of the open loop of cytokine monomer B, and the new C-terminus of the open loop of monomer A is joined with the new N-terminus of the open loop of cytokine monomer B. The native (or original) N-terminus and native (or original) C-terminus of cytokine monomer A are further joined (or, alternatively, the native / original N-terminus and native / original C-terminus of cytokine monomer B are joined).

[0085] According to this method A of making a foldikine, the single-chain dimeric cytokine polypeptide comprises: (a) a first cytokine monomer domain or a functional portion thereof; and (b) a second cytokine monomer domain or a functional portion thereof, wherein the sequence of the second cytokine monomer domain or a functional portion thereof is contiguous (in particular inserted within the sequence of the first cytokine monomer domain), and wherein a first sequence portion of the first cytokine monomer domain that is located N-terminal to the second cytokine monomer domain or a functional portion thereof corresponds to the N-terminal portion of a naturally occurring cytokine monomer domain, while a second sequence portion of the first cytokine monomer domain that is located C-terminal to the second cytokine monomer domain or a functional portion thereof corresponds to the C-terminal portion of a naturally occurring cytokine monomer domain.

[0086] Structurally, the single-chain dimeric cytokine polypeptide is i) a split domain comprising the free N- and C-termini of a single-chain dimeric cytokine polypeptide and the first and second sequence portions of a first cytokine monomer domain in their natural sequence order (i.e., the first portion of the first cytokine monomer domain is the N-terminal portion of the first cytokine monomer domain and is at the N-terminus of the single-chain dimeric cytokine polypeptide, while the second portion of the first cytokine monomer domain is the C-terminal portion of the first cytokine monomer domain and is at the C-terminus of the single-chain dimeric cytokine polypeptide); and ii) a contiguous domain comprising a second cytokine monomer domain or a functional portion thereof constructed as a first sequence portion and a second sequence portion of the second cytokine monomer domain, wherein the first and second sequence portions are in reverse order compared to their natural sequence order (i.e., the second portion is a C-terminal portion of the second cytokine monomer domain and is N-side of the first portion of the second cytokine monomer domain, which is an N-terminal portion of the second cytokine monomer domain). Including, In a split domain, the sequences of the first and second sequence portions of the first cytokine monomer domain are separated by the sequence of the contiguous domain.

[0087] For class I cytokines that do not form swapped domain dimers, the formation of a foldikine may involve opening the loops of each monomer (particularly between α-helices B and C), closing the N- and C-termini of one of the monomers via a linker, and then linking the two cytokines via two linkers (see, e.g., Figures 31-32).

[0088] According to one embodiment, there is provided a single-chain dimeric class I polypeptide comprising two class I cytokine monomers, wherein the loop between alpha helices C and D is opened in each cytokine monomer, said single-chain dimeric cytokine polypeptide comprising: a) a linker peptide bridging the second α-helix (α-helix B) of a first class I cytokine monomer (cytokine 2 in Figure 31) with the third α-helix (α-helix C) of a second class I cytokine monomer (cytokine 1 in Figure 31); b) a linker peptide bridging the fourth α-helix (C-terminal α-helix, or α-helix D) of the second class I cytokine monomer and the first α-helix (N-terminal α-helix, or α-helix A) of the second class I cytokine monomer; and c) a linker peptide bridging the second α-helix (α-helix B) of the second class I cytokine monomer with the third α-helix (α-helix C) of the first class I cytokine monomer. Includes.

[0089] In said single-chain dimeric cytokine, the natural N- and C-termini of the first class I cytokine monomer are free.

[0090] In total, the single-chain dimeric class I polypeptide comprises, from N-terminus to C-terminus, i) an N-terminal portion of a first class I cytokine monomer comprising α-helices A and B of the first class I cytokine monomer; ii) a linker peptide bridging α-helix B of the first class I cytokine monomer with α-helix C of a second class I cytokine monomer; iii) a C-terminal portion of the second class I cytokine monomer comprising α-helices C and D of the second class I cytokine monomer; and iv) a linker peptide bridging α-helix B of the first class I cytokine monomer with α-helix C of a second class I cytokine monomer. and vii) a C-terminal portion of the first class I cytokine monomer comprising α-helix C and α-helix D of the first class I cytokine monomer (Figures 31 and 32).

[0091] In some embodiments, once a class I foldikine is made according to method A, the native Nt and Ct of a second class I cytokine monomer are joined to create a circular permutant variant in which the loop is opened in the first second cytokine monomer.

[0092] For class II cytokines that do not form swapped domain dimers, the formation of a foldikine may involve linking two cytokines via two linkers after opening a loop in each monomer (specifically between α-helices C and D) and closing the N- and C-termini of one of the monomers via the linker (see, e.g., Figures 5 and 27).

[0093] According to one embodiment, there is provided a single-chain dimeric type II cytokine polypeptide comprising two cytokine monomers derived from class II cytokine(s) that do not form swapped domain dimers, wherein the loop between alpha helices C and D is opened in each cytokine monomer, wherein said single-chain dimeric type II cytokine polypeptide comprises: a) a linker sequence bridges the third α-helix (α-helix C) of a first class II cytokine monomer (cytokine 1 in Figure 27) with the fourth α-helix (α-helix D) of a second class II cytokine monomer (cytokine 2 in Figure 27); b) a linker sequence bridges the fourth alpha helix (alpha helix D) of the first class II cytokine monomer and the third alpha helix (alpha helix C) of the second class II cytokine monomer; c) a linker sequence bridges the first α-helix (the N-terminal α-helix, or α-helix A) of the first class II cytokine monomer and the sixth α-helix (the C-terminal α-helix, or α-helix F) of the first class II cytokine monomer; d) the third and fourth α-helices (α-helices C and D) of each of said first and second class II cytokine monomers are not cross-linked.

[0094] In total, the single-chain dimeric class II polypeptide comprises, from N-terminus to C-terminus, i) an N-terminal portion of a first class II cytokine monomer comprising α-helices A-C of the first class II cytokine monomer; ii) a linker peptide bridging α-helix C of the first class II cytokine monomer with α-helix D of a second class II cytokine monomer; iii) a C-terminal portion of a second class II cytokine monomer comprising α-helices D-F of the second class II cytokine monomer; and iv) a linker peptide bridging α-helix C of the first class II cytokine monomer with α-helix D of a second class II cytokine monomer. and vii) a C-terminal portion of the first Class II cytokine monomer comprising α-helix D of the first Class II cytokine monomer.

[0095] In any such embodiment, the two class II cytokine monomers are derived from one class II cytokine that does not form swapped domain dimers, or from two different class II cytokines that do not form swapped domain dimers.

[0096] Exemplary homomeric foldikines based on class II cytokines include i) Array RLDKSNFQQPYITNRTFMLAKEASLADNNTTDVRLIGEKLFHGVSMSERCYLMKQVLNFT hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhh HA HB LEEVLFPQSDRFQPYMQEVVPFLARLSNRLS (Xn1) HIQRNVQKLKDTVKKLGESGE h---hhhh hhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhh hhh HC HD IKAIGELDLLFMSLRNACI(NtCt)LDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKL hhhh hhhhhhhhhhh hhhhhhhhhhhhhhhhhhh HE HF HA FHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLS (Xn2) HIQR h hhhhhhhhhhhhhhh hhh hhhhhhhhhhhhhhhhhhh HB HC NVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI hhhhhhhhhhh hhhhhhh hhhhhhhhhhh HD HE HF a dimeric IL-22 which may comprise or consist of (SEQ ID NO:325-(Xn1)-SEQ ID NO:326-(NtCt)-SEQ ID NO:327-(Xn2)-SEQ ID NO:328) or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine (IL-22); ii) dimeric IL-19 (which may comprise or consist of the sequence SEQ ID NO:195-(Xn1)-SEQ ID NO:196-(NtCt)-SEQ ID NO:197-(Xn2)-SEQ ID NO:198 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), iii) dimeric IL-20 (which may comprise or consist of the sequence SEQ ID NO:200-(Xn1)-SEQ ID NO:201-(NtCt)-SEQ ID NO:202-(Xn2)-SEQ ID NO:203 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), iv) dimeric IL-26 (which may comprise or consist of the sequence SEQ ID NO:210-(Xn1)-SEQ ID NO:211-(NtCt)-SEQ ID NO:212-(Xn2)-SEQ ID NO:213 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), v) dimeric IFNλ2 (which may comprise or consist of the sequence SEQ ID NO:215-(Xn1)-SEQ ID NO:216-(NtCt)-SEQ ID NO:217-(Xn2)-SEQ ID NO:218 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), vi) dimeric IFNλ3 (which may comprise or consist of the sequence SEQ ID NO:220-(Xn1)-SEQ ID NO:221-(NtCt)-SEQ ID NO:222-(Xn2)-SEQ ID NO:223 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), vii) dimeric IFNλ1 (which may comprise or consist of the sequence SEQ ID NO:225-(Xn1)-SEQ ID NO:226-(NtCt)-SEQ ID NO:227-(Xn2)-SEQ ID NO:228 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine, viii) dimeric IFNα1 / 13 (which may comprise or consist of the sequence SEQ ID NO:230-(Xn1)-SEQ ID NO:231-(NtCt)-SEQ ID NO:232-(Xn2)-SEQ ID NO:233 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), ix) dimeric IFNα2 (which may comprise or consist of the sequence SEQ ID NO:235-(Xn1)-SEQ ID NO:236-(NtCt)-SEQ ID NO:237-(Xn2)-SEQ ID NO:238 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), x) dimeric IFNβ (which may comprise or consist of the sequence SEQ ID NO:240-(Xn1)-SEQ ID NO:241-(NtCt)-SEQ ID NO:242-(Xn2)-SEQ ID NO:243 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), xi) dimeric IFNΩ1 (which may comprise or consist of the sequence SEQ ID NO:245-(Xn1)-SEQ ID NO:246-(NtCt)-SEQ ID NO:247-(Xn2)-SEQ ID NO:248 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), xii) dimeric IFNε (which may comprise or consist of the sequence SEQ ID NO:250-(Xn1)-SEQ ID NO:251-(NtCt)-SEQ ID NO:252-(Xn2)-SEQ ID NO:253 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), xiii) dimeric IFNκ (which may comprise or consist of the sequence SEQ ID NO:255-(Xn1)-SEQ ID NO:256-(NtCt)-SEQ ID NO:257-(Xn2)-SEQ ID NO:258 or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine), are listed, Here, (NtCt), (Xn1) and (Xn2) are peptide linkers.

[0097] In any such aspect and embodiment, the linker sequence (NtCt) bridging the N-terminus and C-terminus of one monomer can have any suitable sequence, preferentially including sequences that define a conformationally constrained structure, i.e., a "structured" linker. Such linkers can have about 3 to about 20 amino acid residues, about 3 to about 16 amino acid residues, about 4 to about 12 amino acid residues, about 4 to about 8 amino acid residues, about 3 to about 8 amino acid residues, or about 3 to about 6 amino acid residues. In some embodiments, such linkers do not contain multiple adjacent Gly and / or Ser residues; for example, the linker peptide contains no more than five adjacent Gly and / or Ser residues, preferably no more than three adjacent Gly and / or Ser residues, no more than two Gly and / or Ser residues, contains only isolated Gly and / or Ser residues, or in some embodiments, contains no Gly and / or Ser residues.

[0098] Linkers can be designed by modeling to interact with receptors, as we found that the designed linkers were superior to poly-Gly-Ser linkers of the same length.

[0099] Exemplary structured (NtCt) linkers comprise or consist of the sequence of ANGT (SEQ ID NO: 329), or ANGV (SEQ ID NO: 330), or TDYDSQTN (SEQ ID NO: 303).

[0100] Another exemplary structured (NtCt) linker comprises or consists of the sequence EGPG (SEQ ID NO: 304), particularly when the foldikine is dimeric IFN-β.

[0101] Similarly, the artificial linkers (Xn1) and (Xn2) bridging two monomer sequences can have any suitable sequence, particularly sequences that define a conformationally constrained structure, i.e., a "structured" linker. Such linkers can have about 3 to about 20 amino acid residues, about 3 to about 16 amino acid residues, about 4 to about 12 amino acid residues, about 4 to about 8 amino acid residues, about 3 to about 8 amino acid residues, or about 3 to about 6 amino acid residues. Advantageously, such linkers do not contain multiple adjacent Gly and / or Ser residues; for example, the linker peptide contains no more than five adjacent Gly and / or Ser residues, preferably no more than three adjacent Gly and / or Ser residues, no more than two Gly and / or Ser residues, only isolated Gly and / or Ser residues, or, in some embodiments, no Gly and / or Ser residues. Exemplary pairs of suitable linkers bridging two monomer sequences are: i) sequences designed using protein design algorithms (i.e., FoldX, ModelX) such that the linker interacts with itself or with either of the two cytokine domains, thereby adopting a preferred conformation (exemplary sequences include Xn1=TCMPGGSKT (SEQ ID NO: 337) and Xn2=RLELLP (SEQ ID NO: 331), or Xn1=NRLKKLMASSD (SEQ ID NO: 332) and Xn2=RLELLP (SEQ ID NO: 331)); ii) flexible sequences composed of varying proportions of Gly and Ser residues (exemplary sequences include Xn1 = Xn2 = GGG, or GGGSGG (SEQ ID NO: 273), or GGGSGGSGG (SEQ ID NO: 333), or GGGSGGSGGSGG (SEQ ID NO: 334)), provided that the linker length remains short (preferably no more than 6, 5, 4, or 3 residues) and prevents the folding of part of the split domain into part of the continuous domain; or iii) Rigid sequences composed of Pro residues that create PolyPro helices of any length (exemplary sequences include Xn1 = Xn2 = GPG, or GPPPPPPPG (SEQ ID NO: 335), or GPPPPPPPPPG (SEQ ID NO: 336)). (particularly when the foldikine is dimeric IL-22).

[0102] In some embodiments, once a class II foldikine is made according to method A, the native Nt and Ct of a second class II cytokine monomer are joined to create a circularly permuted variant in which the loop is opened in the first second class II cytokine monomer.

[0103] According to some embodiments, there is provided a single chain dimeric IL-22 polypeptide comprising or consisting of the sequence of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, or SEQ ID NO:288, or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, which retains at least the same stability and / or at least the same level of interaction with IL-22 receptor 1 and / or IL-22 receptor 2 as an IL-22 polypeptide.

[0104] According to some embodiments, there is provided a single chain chimeric IL-10-IL-22 polypeptide comprising or consisting of the sequence of SEQ ID NO: 76, or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retains at least the same stability and / or at least the same level of interaction with the IL-10 receptor and the IL-22 receptor.

[0105] Foldikines containing both class I and class II cytokines can be designed by the same method A described for foldikines based on class I or class II cytokines.

[0106] According to this embodiment, in the single-chain dimeric cytokine polypeptide of the invention, one of the first and second cytokine monomers is a class I cytokine monomer, and the other of the first and second cytokine monomers is a class II cytokine monomer.

[0107] In the first scheme, two cytokines are linked via two artificial peptide linkers after opening the natural loops of the type I and type II cytokines and closing the natural N- and C-termini of the type II cytokine or type I cytokine via linkers.

[0108] Foldikine formation method B According to this method of forming a foldikine, the native N-terminus of cytokine monomer A is joined to the native C-terminus of cytokine monomer B, and the native C-terminus of cytokine monomer B is joined to the native N-terminus of cytokine monomer A. The loop is opened in either cytokine monomer A or cytokine monomer B.

[0109] Experiments performed by linking the native Nt and Ct, opening the protein at different loops, and creating circular permutations of the monomeric cytokine (see Example 4) show which loops can be opened without compromising the functionality of the cytokine.

[0110] According to this method B of making a foldikine, the single-chain dimeric cytokine polypeptide comprises (a) a first cytokine monomer domain or a functional portion thereof and (b) a second cytokine monomer domain or a functional portion thereof, wherein the sequence of the second cytokine monomer domain or a functional portion thereof is contiguous, while the sequence of the first cytokine monomer domain is such that a first sequence portion of the first cytokine monomer domain located N-terminal to the second cytokine monomer domain or a functional portion thereof corresponds to the C-terminal portion of a naturally occurring cytokine monomer domain, and a second sequence portion of the first cytokine monomer domain located C-terminal to the second cytokine monomer domain or a functional portion thereof corresponds to the N-terminal portion of a naturally occurring cytokine monomer domain.

[0111] Structurally, the single-chain dimeric cytokine polypeptide is i) a split domain comprising the free N- and C-termini of a single-chain dimeric cytokine polypeptide and the first and second sequence portions of a first cytokine monomer domain in reverse order compared to their natural sequence order (i.e., the second portion, which is the C-terminal portion of the first cytokine monomer domain, is N-side of the first portion of the first cytokine monomer domain, which is the N-terminal portion of the first cytokine monomer domain); and ii) a contiguous domain comprising a second cytokine monomer domain or a functional portion thereof in its natural sequence (i.e., from N-terminus to C-terminus); In a split domain, the sequences of the first and second sequence portions of the first cytokine monomer domain are separated by the sequence of the contiguous domain.

[0112] For class I cytokines that do not form swapped-domain dimers, foldikines are formed according to Method B by linking two monomeric class I cytokines via two linkers, bridging the N- and C-termini of each cytokine monomer, respectively, followed by opening a loop in one of the monomers to create a new N- and C-termini in the single-chain polypeptide. The loop opened in one class I cytokine monomer can be the loop between α-helices A and B, between α-helices B and C, or between α-helices C and D.

[0113] According to some embodiments, there is provided a single-chain dimeric class I polypeptide comprising two class I cytokine monomer domains or functional portions thereof, wherein the single-chain dimeric cytokine polypeptide comprises: a) a linker peptide bridges the fourth alpha helix (alpha helix D) of a first class I cytokine monomer (e.g., cytokine 2 in Figure 26) with the first alpha helix (alpha helix A) of a second class I cytokine monomer (e.g., cytokine 1 in Figure 26); b) a linker peptide bridges the fourth α-helix (C-terminal α-helix, or α-helix D) of the second class I cytokine monomer with the first α-helix (N-terminal α-helix, or α-helix A) of the first class I cytokine monomer; c) alpha helices A and B of the first class I cytokine monomer are not cross-linked, or alpha helices B and C of the first class I cytokine monomer are not cross-linked, or alpha helices C and D of the first class I cytokine monomer are not cross-linked.

[0114] In the single-chain dimeric cytokine, the new N- and C-termini created by opening the loops of the first class I cytokine monomer are free.

[0115] According to one embodiment, the loop between alpha helices B and C is opened in one cytokine monomer, and the single-chain dimeric class I polypeptide comprises, from N-terminus to C-terminus, i) a C-terminal portion of a first class I cytokine monomer comprising alpha helices C and D of the first class I cytokine monomer, ii) a linker peptide bridging alpha helix D of the first class I cytokine monomer and alpha helix A of a second class I cytokine monomer, iii) a portion of the second class I cytokine monomer comprising alpha helices A-D of the second class I cytokine monomer, iv) a linker peptide bridging alpha helix D of the second class I cytokine monomer and alpha helix A of the first class I cytokine monomer, and v) an N-terminal portion of the first class I cytokine monomer comprising alpha helices A and B.

[0116] According to one embodiment, the loop between alpha helices A and B is opened in one cytokine monomer, and the single-chain dimeric class I polypeptide comprises, from N-terminus to C-terminus, i) a C-terminal portion of a first class I cytokine monomer comprising alpha helices B-D of the first class I cytokine monomer, ii) a linker peptide bridging alpha helix D of the first class I cytokine monomer and alpha helix A of a second class I cytokine monomer, iii) a portion of the second class I cytokine monomer comprising alpha helices A-D of the second class I cytokine monomer, iv) a linker peptide bridging alpha helix D of the second class I cytokine monomer and alpha helix A of the first class I cytokine monomer, and v) an N-terminal portion of the first class I cytokine monomer comprising alpha helix A.

[0117] According to one embodiment, the loop between alpha helices C and D is opened in one cytokine monomer, and the single-chain dimeric class I polypeptide comprises, from N-terminus to C-terminus, i) a C-terminal portion of a first class I cytokine monomer comprising alpha helix D of the first class I cytokine monomer, ii) a linker peptide bridging alpha helix D of the first class I cytokine monomer and alpha helix A of a second class I cytokine monomer, iii) a portion of the second class I cytokine monomer comprising alpha helices A-D of the second class I cytokine monomer, iv) a linker peptide bridging alpha helix D of the second class I cytokine monomer and alpha helix A of the first class I cytokine monomer, and v) an N-terminal portion of the first class I cytokine monomer comprising alpha helices A-C.

[0118] In the context of the IL-2 cytokine monomer, and with reference to the sequence of SEQ ID NO: 79 (without the signal peptide), it has been specifically shown that the monomer can open at the loop between alpha helices A and B at positions 49-54 (particularly between residues 52-53) or 69-76 (particularly between residues 68-69), between alpha helices A and B, at the loop between positions 91-101 (particularly between residues 96-97), between alpha helices B and C, or at the loop between positions 117-125 (particularly between residues 120-121) between alpha helices C and D (see Figure 43).

[0119] In some embodiments, once a class I foldikine is produced according to method B, the native Nt and Ct of a second class I cytokine monomer are combined to create a circularly permuted variant with an open loop in the first second class I cytokine monomer.

[0120] An example of a single-chain dimeric Class I polypeptide comprising IL-2 and IL-4 cytokine monomers (IL-4 contains consecutive domains) comprises or consists of the sequence of SEQ ID NO: 295, or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, that retains at least the same stability and / or at least the same level of interaction with the IL-2 receptor and the IL-4 receptor.

[0121] Exemplary heterodimeric foldikines based on class I cytokines include: MYRMQLLSCIALSLALVTNS LRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSII hhhhh-hhhhhhhhhh hhhhhhhhhhhhhhhhh IL-2 HC IL-2 HD STLTGPPPPPGHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHH H hhhhhhhhhhhhhh hhh hhhhhhhhhhhhhhhhhh IL-4 HA IL-4 HB EKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh IL-4 HC IL-4 HD GPPPPGTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh---hhhhhh IL2-HA IL-2 HB LNLAQS* Examples include:

[0122] Exemplary homomeric foldikines based on short-chain class I cytokines include: i) dimeric IL-2 (which may comprise or consist of SEQ ID NO:89-(Xn1)-SEQ ID NO:90-(Xn2)-SEQ ID NO:91); ii) dimeric IL-4 (which may comprise or consist of SEQ ID NO: 93-(Xn1)-SEQ ID NO: 94-(Xn2)-SEQ ID NO: 95); iii) dimeric IL-3 (which may comprise or consist of SEQ ID NO: 97-(Xn1)-SEQ ID NO: 98-(Xn2)-SEQ ID NO: 99); iv) dimeric IL-7 (which may comprise or consist of SEQ ID NO: 103-(Xn1)-SEQ ID NO: 104-(Xn2)-SEQ ID NO: 105); v) dimeric IL-9 (which may comprise or consist of SEQ ID NO: 107-(Xn1)-SEQ ID NO: 108-(Xn2)-SEQ ID NO: 109); vi) dimeric IL-15 (which may comprise or consist of SEQ ID NO: 110-(Xn1)-SEQ ID NO: 111-(Xn22)-SEQ ID NO: 112); vii) dimeric IL-21 (which may comprise or consist of SEQ ID NO: 113-(Xn1)-SEQ ID NO: 114-(Xn)-SEQ ID NO: 115); viii) dimeric TSLP (which may comprise or consist of SEQ ID NO: 116-(Xn1)-SEQ ID NO: 117-(Xn2)-SEQ ID NO: 118), and ix) Dimeric GM-CSF (which may comprise or consist of SEQ ID NO: 120-(Xn1)-SEQ ID NO: 121-(Xn2)-SEQ ID NO: 12-SEQ ID NO: 101) Or sequences that are at least 95%, 98% or 99% identical thereto and that retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric short-chain class I cytokine; and Here, (Xn1) and (Xn2) are peptide linkers.

[0123] Exemplary homomeric foldikines based on long-chain class I cytokines include: i) dimeric IL-6 (which may comprise or consist of SEQ ID NO: 135-(Xn1)-SEQ ID NO: 136-(Xn2)-SEQ ID NO: 137); ii) dimeric IL-11 (which may comprise or consist of SEQ ID NO: 139-(Xn1)-SEQ ID NO: 140-(Xn2)-SEQ ID NO: 141); iii) dimeric IL-12α (which may comprise or consist of SEQ ID NO: 143-(Xn1)-SEQ ID NO: 144-(Xn2)-SEQ ID NO: 145); iv) dimeric IL-23α (which may comprise or consist of SEQ ID NO: 147-(Xn1)-SEQ ID NO: 148-(Xn2)-SEQ ID NO: 149); v) dimeric IL-27α (which may comprise or consist of SEQ ID NO: 151-(Xn1)-SEQ ID NO: 153-(Xn2)-SEQ ID NO: 153); vi) dimeric IL-31 (which may comprise or consist of SEQ ID NO: 154-(Xn1)-SEQ ID NO: 155-(Xn2)-SEQ ID NO: 156); vii) dimeric CLCF1 (which may comprise or consist of SEQ ID NO: 158-(Xn1)-SEQ ID NO: 159-(Xn2)-SEQ ID NO: 160); viii) dimeric ONCM (which may comprise or consist of SEQ ID NO: 161-(Xn1)-SEQ ID NO: 162-(Xn2)-SEQ ID NO: 163); ix) dimeric CTF1 (which may comprise or consist of SEQ ID NO: 165-(Xn1)-SEQ ID NO: 166-(Xn2)-SEQ ID NO: 167); x) dimeric CNTF (which may comprise or consist of SEQ ID NO: 169-(Xn1)-SEQ ID NO: 170-(Xn2)-SEQ ID NO: 171); xi) dimeric LIF (which may comprise or consist of SEQ ID NO: 173-(Xn1)-SEQ ID NO: 174-(Xn2)-SEQ ID NO: 175), and xii) dimeric CSF3 (which may comprise or consist of SEQ ID NO: 177-(Xn1)-SEQ ID NO: 178-(Xn2)-SEQ ID NO: 179) Or sequences at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, which retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric long chain class I cytokine; and Here, (Xn1) and (Xn2) are peptide linkers.

[0124] In any such aspect and embodiment, the linker peptides (Xn1) and (Xn2) bridging the α-helices, e.g., between the C-terminus of one monomer and the N-terminus of the other, can have any suitable sequence and preferentially include sequences that define conformationally constrained structures, i.e., "structured" linkers. Such linkers can have about 3 to about 20 amino acid residues, about 3 to about 16 amino acid residues, about 4 to about 12 amino acid residues, about 4 to about 8 amino acid residues, about 3 to about 8 amino acid residues, or about 3 to about 6 amino acid residues. In some embodiments, such linkers do not contain multiple adjacent Gly and / or Ser residues; for example, the linker peptide contains no more than five adjacent Gly and / or Ser residues, preferably no more than three adjacent Gly and / or Ser residues, no more than two Gly and / or Ser residues, contains only isolated Gly and / or Ser residues, or in some embodiments, contains no Gly and / or Ser residues. Preferentially, such linkers contain at least two consecutive Pro residues, preferably 3, 4, 5, or 6 consecutive Pro residues, forming a rigid PolyPro helix. In some embodiments, peptide linkers Xn1 and Xn2 are identical.

[0125] In some embodiments, the dimeric IL-2 comprises or consists of SEQ ID NO:89-(Xn1)-SEQ ID NO:90-(Xn2)-SEQ ID NO:91, where the peptide linkers Xn1 and Xn2 are identical and comprise or consist of the sequence GPPPPG (SEQ ID NO:270), GPPPPPG (SEQ ID NO:271), GPPPPPPG (SEQ ID NO:272), GGGSGG (SEQ ID NO:273), GGGSGGG (SEQ ID NO:274), or GGGSGGGG (SEQ ID NO:275). In these embodiments, the dimeric IL-2 can further comprise a Lys residue immediately N-terminal to SEQ ID NO:89 and a Gln-Ser dipeptide immediately C-terminal to SEQ ID NO:91 (the sequence K-SEQ ID NO:89-(Xn1)-SEQ ID NO:90-(Xn2)-SEQ ID NO:91-QS).

[0126] For class II cytokines that do not form swapped-domain dimers, forming a foldikine according to method B involves linking two monomeric class II cytokines via two linkers, bridging the N- and C-termini of each cytokine monomer, respectively, followed by opening a loop in one of the monomers to create a new N- and C-termini in the single-chain polypeptide. The loop opened in one class II cytokine monomer can be the loop between α-helices A and B, between α-helices B and C, between α-helices C and D, between α-helices D and E, or between α-helices E and F.

[0127] According to some embodiments, there is provided a single-chain dimeric type II cytokine polypeptide comprising two cytokine monomers of class II cytokine(s) that do not form swapped domain dimers, wherein said single-chain dimeric type II cytokine polypeptide comprises: a) a linker sequence bridging the first α-helix (the N-terminal α-helix, or α-helix A) of a first class II cytokine monomer (e.g., cytokine 1 in Figure 33) and the sixth α-helix (the C-terminal α-helix, or α-helix F) of a second class II cytokine monomer (e.g., cytokine 2 in Figure 33); b) a linker sequence bridges the sixth α-helix (C-terminal α-helix, or α-helix F) of the first class II cytokine monomer with the first α-helix (C-terminal α-helix, or α-helix A) of the second class II cytokine monomer; c) the first and second α-helices (α-helices A and B), the second and third α-helices (α-helices B and C), the third and fourth α-helices (α-helices C and D), the fourth and fifth α-helices (α-helices D and E), or the fifth and sixth α-helices (α-helices E and F) of the first class II cytokine monomer are not cross-linked.

[0128] In the single-chain dimeric cytokine, the new N- and C-termini created by opening the loops of the first class II cytokine monomer are free.

[0129] According to one embodiment, the loop between α-helices B and C is opened in a first Class II cytokine monomer, and the single-chain dimeric Class II polypeptide comprises, from N-terminus to C-terminus, i) a C-terminal portion of a first Class II cytokine monomer comprising α-helices C through F of the first Class II cytokine monomer, ii) a linker peptide bridging α-helix F of the first Class II cytokine monomer and α-helix A of a second Class II cytokine monomer, iii) a portion of the second Class II cytokine monomer comprising α-helices A through F of the second Class II cytokine monomer, iv) a linker peptide bridging α-helix F of the second Class II cytokine monomer and α-helix A of the first Class II cytokine monomer, and v) an N-terminal portion of the second Class II cytokine monomer comprising α-helices A and B.

[0130] According to one embodiment, the loop between α-helices C and D is opened in a first Class II cytokine monomer, and the single-chain dimeric Class II polypeptide comprises, from N-terminus to C-terminus, i) a C-terminal portion of the first Class II cytokine monomer comprising α-helices D-F of the first Class II cytokine monomer, ii) a linker peptide bridging α-helix F of the first Class II cytokine monomer and α-helix A of a second Class II cytokine monomer, iii) a portion of the second Class II cytokine monomer comprising α-helices A-F of the second Class II cytokine monomer, iv) a linker peptide bridging α-helix F of the second Class II cytokine monomer and α-helix A of the first Class II cytokine monomer, and v) an N-terminal portion of the first Class II cytokine monomer comprising α-helices A-C.

[0131] According to one embodiment, the loop between alpha helices E and F is opened in a first Class II cytokine monomer, and the single-chain dimeric Class II polypeptide comprises, from N-terminus to C-terminus, i) a C-terminal portion of a first Class II cytokine monomer comprising alpha helix F of the first Class II cytokine monomer, ii) a linker peptide bridging alpha helix F of the first Class II cytokine monomer and alpha helix A of a second Class II cytokine monomer, iii) a portion of the second Class II cytokine monomer comprising alpha helices A-F of the second Class II cytokine monomer, iv) a linker peptide bridging alpha helix F of the second Class II cytokine monomer and alpha helix A of the first Class II cytokine monomer, and v) an N-terminal portion of the first Class II cytokine monomer comprising alpha helices A-E.

[0132] In the context of the IL-22 cytokine monomer, and with reference to the sequence of SEQ ID NO: 291 (without the signal peptide), it has been specifically shown that the monomer can open at the loop at positions 102-113 between alpha helices B and C (particularly between residues at positions 108-109), or at the loop at positions 129-140 between alpha helices C and D (particularly between residues at positions 134-135), or at the loop at positions 165-167 between alpha helices E and F (particularly between residues at positions 164-165) (see Figure 41).

[0133] In the context of the IFNβ cytokine monomer, and with reference to the sequence of SEQ ID NO: 291 (without the signal peptide), it has been specifically shown that the monomer can open at the loop between α-helices A and B at positions 34-51 (particularly between residues 49-50), or at the loop between α-helices B and C at positions 71-81 (particularly between residues 75-76), or at the loop between α-helices C and D at positions 108-121 (particularly between residues 117-118) (see Figure 42).

[0134] In any such aspect and embodiment, the linker peptides (Xn1) and (Xn2) bridging the α-helices, e.g., between the C-terminus of one monomer and the N-terminus of the other, can have any suitable sequence and preferentially include sequences that define conformationally constrained structures, i.e., "structured" linkers. Such linkers can have about 3 to about 20 amino acid residues, about 3 to about 16 amino acid residues, about 4 to about 12 amino acid residues, about 4 to about 8 amino acid residues, about 3 to about 8 amino acid residues, or about 3 to about 6 amino acid residues. In some embodiments, such linkers do not contain multiple adjacent Gly and / or Ser residues; for example, the linker peptide contains no more than five adjacent Gly and / or Ser residues, preferably no more than three adjacent Gly and / or Ser residues, no more than two Gly and / or Ser residues, contains only isolated Gly and / or Ser residues, or in some embodiments, contains no Gly and / or Ser residues. Preferentially, such a linker contains at least two consecutive Pro residues, preferably 3, 4, 5 or 6 consecutive Pro residues, forming a rigid PolyPro helix.

[0135] In some embodiments, the peptide linkers Xn1 and Xn2 are the same.

[0136] Foldikines containing both type I and type II cytokine monomers can be designed by the same method B as described for class I and class II cytokine-based foldikines.

[0137] In this scheme, the two cytokines are linked via two artificial linker peptides that bridge the natural N-terminus of cytokine type I with the natural C-terminus of cytokine type II and the natural C-terminus of cytokine type I with the natural N-terminus of cytokine type II, opening the loop of cytokine type I (see, e.g., Figure 6i) or cytokine type II (not shown).

[0138] In some embodiments, once a class II foldikine is made according to method B, the native Nt and Ct of a second class II cytokine monomer are joined to create a circularly permuted variant in which the loop is opened in the first second class II cytokine monomer.

[0139] In any such aspect and embodiment, the linker sequence bridging the N- and C-termini of the type I and type II cytokines can have any suitable sequence, preferentially including sequences that define a conformationally constrained structure, e.g., a "structured" linker. Such linkers can have about 3 to about 20 amino acid residues, about 3 to about 16 amino acid residues, about 4 to about 12 amino acid residues, about 4 to about 8 amino acid residues, about 3 to about 8 amino acid residues, or about 3 to about 6 amino acid residues. Advantageously, such linkers do not contain multiple adjacent Gly and / or Ser residues; for example, the linker peptide contains no more than five adjacent Gly and / or Ser residues, preferably no more than three adjacent Gly and / or Ser residues, no more than two Gly and / or Ser residues, contains only isolated Gly and / or Ser residues, or, in some embodiments, contains no Gly and / or Ser residues. Similarly, the artificial linker bridging two monomer sequences can have any suitable sequence, particularly sequences that define a conformationally constrained structure, i.e., a "structured" linker. Such linkers can have about 3 to about 20 amino acid residues, about 3 to about 16 amino acid residues, about 4 to about 12 amino acid residues, about 4 to about 8 amino acid residues, about 3 to about 8 amino acid residues, or about 3 to about 6 amino acid residues. Advantageously, such linkers do not contain multiple adjacent Gly and / or Ser residues; for example, the linker peptide contains no more than five adjacent Gly and / or Ser residues, preferably no more than three adjacent Gly and / or Ser residues, no more than two Gly and / or Ser residues, only isolated Gly and / or Ser residues, or, in some embodiments, no Gly and / or Ser residues.

[0140] In any such aspect and embodiment, any such artificial linker peptide sequence (e.g., a linker bridging the N- and C-termini of a type I cytokine with the ends of an open loop of a type II cytokine, and a linker connecting the N- and C-termini of a type II cytokine (if that option is selected)) can have any suitable sequence, preferentially including sequences that define conformationally constrained structures, e.g., "structured" linkers. Such linkers can have from about 3 to about 20 amino acid residues, from about 3 to about 16 amino acid residues, from about 4 to about 12 amino acid residues, from about 4 to about 8 amino acid residues, from about 3 to about 8 amino acid residues, or from about 3 to about 6 amino acid residues. Advantageously, such linkers do not contain multiple Gly and / or Ser residues adjacent to one another, for example the linker peptide contains no more than 5 adjacent Gly and / or Ser residues, preferably no more than 3 adjacent Gly and / or Ser residues, no more than 2 Gly and / or Ser residues, contains only isolated Gly and / or Ser residues, or in some embodiments contains no Gly and / or Ser residues.

[0141] Formation of foldikine method C In the case of class I cytokines, according to method C of forming foldikines, a loop is opened in one class I cytokine monomer B, the new N-terminus of the opened loop of cytokine monomer B is joined to the natural (or original) C-terminus of cytokine monomer A, and the new C-terminus of the open loop of monomer B is joined to the natural (or original) N-terminus of cytokine monomer A.

[0142] According to this method C of making a foldikine, the single-chain dimeric cytokine polypeptide comprises (a) a first class I cytokine monomer domain or functional portion thereof and (b) a second class I cytokine monomer domain or functional portion thereof, wherein the sequence of the first class I cytokine monomer domain or functional portion thereof is contiguous, while the sequence of the second cytokine monomer domain is such that a first sequence portion of the second class I cytokine monomer located N-terminal to the first class I cytokine monomer or functional portion thereof corresponds to the N-terminal portion of a naturally occurring class I cytokine monomer, and a second sequence portion of the second class I cytokine monomer located C-terminal to the first class I cytokine monomer or functional portion thereof corresponds to the C-terminal portion of a naturally occurring class I cytokine monomer.

[0143] The single-chain dimeric class I cytokine polypeptide is b) a split domain comprising the free N- and C-termini of a single-chain dimeric class I cytokine polypeptide and a first and second sequence portion of a second class I cytokine monomer domain; and ii) a contiguous domain comprising the first class I cytokine monomer domain or a functional portion thereof; Including, The sequences of the first and second sequence portions of the second class I cytokine monomer domain are separated by the sequence of the contiguous domain in the split domain.

[0144] In the case of class I cytokines, forming a foldikine according to method C may involve opening a loop in one monomer (eg, between α-helices B and C).

[0145] According to one embodiment, a single-chain dimeric Class I polypeptide is provided comprising two Class I cytokine monomers, wherein the loop between alpha helices B and C is opened in one cytokine monomer, wherein in said single-chain dimeric cytokine polypeptide: a) a linker peptide bridges the fourth alpha helix (alpha helix D) of a first class I cytokine monomer (e.g., cytokine 1 in Figure 45) with the third alpha helix (alpha helix C) of a second class I cytokine monomer (e.g., cytokine 2 in Figure 45); b) a linker peptide bridges a first α-helix (the N-terminal α-helix, or α-helix A) of a first class I cytokine monomer with a second α-helix (α-helix B) of a second class I cytokine monomer; c) α-helices B and C of said second class I cytokine monomer are not cross-linked.

[0146] In said single chain dimeric cytokine, the original N- and C-termini of the second class I cytokine monomer are free.

[0147] Overall, the single-chain dimeric class I polypeptide comprises, from N-terminus to C-terminus, i) the N-terminal portion of the second class I cytokine monomer, comprising α-helices A and B of the second class I cytokine monomer; ii) a linker peptide (Xn1) bridging α-helix B of the second class I cytokine monomer and α-helix A of the first class I cytokine monomer; iii) a contiguous domain of the first class I cytokine monomer, comprising α-helices A through D; iv) a linker peptide (Xn2) bridging α-helix D of the first class I cytokine monomer and α-helix C of the second class I cytokine monomer; and vii) the C-terminal portion of the second class I cytokine monomer, comprising α-helices C and D of the first class I cytokine monomer.

[0148] The artificial linkers (Xn1) and (Xn2) bridging the two monomer sequences can have any suitable sequence, particularly sequences that define a conformationally constrained structure, i.e., a "structured" linker. Such linkers can have about 3 to about 20 amino acid residues, about 3 to about 16 amino acid residues, about 4 to about 12 amino acid residues, about 4 to about 8 amino acid residues, about 3 to about 8 amino acid residues, or about 3 to about 6 amino acid residues. Advantageously, such linkers do not contain multiple adjacent Gly and / or Ser residues; for example, the linker peptide contains no more than five adjacent Gly and / or Ser residues, preferably no more than three adjacent Gly and / or Ser residues, no more than two Gly and / or Ser residues, only isolated Gly and / or Ser residues, or, in some embodiments, no Gly and / or Ser residues.

[0149] According to some embodiments, the sequence of SEQ ID NO: X338: SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhh---hhhhhhhh HA HB QSKGGGSGGSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPL hhhhhhhhhhhhhhhhhhhhhhhhhhhhhh---hhhh HA HB EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGSGG hhhhh-hhhhhhhhhh hhhhhhhhhhhhhhhhs HC HD NFLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTG hhhhh-hhhhhhhhhh hhhhhhhhhhhhhhhhs HC HD or single-chain circularly permuted dimeric IL-2 polypeptides comprising or consisting of a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto and retaining at least the same stability and / or at least the same level of interaction with the IL-2 receptor.

[0150] In some embodiments, once a class II foldikine is made according to method C, the native Nt and Ct of a second class I cytokine monomer are joined to create a circularly permuted variant in which the loop is opened in the first second class I cytokine monomer.

[0151] According to some embodiments, there is provided a single-chain circularly permuted dimeric IL-2 polypeptide comprising or consisting of the sequence of SEQ ID NO: 296, or a sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and which retains at least the same stability and / or at least the same level of interaction with the IL-2 receptor.

[0152] In the case of a class II cytokine, according to this method C of making a foldikine, the single-chain dimeric cytokine polypeptide comprises (a) a first class II cytokine monomer domain or a functional portion thereof and (b) a second class II cytokine monomer domain or a functional portion thereof, wherein the sequence of the second class II cytokine monomer domain or a functional portion thereof is contiguous, while the sequence of the first class II cytokine monomer domain is such that a first sequence portion of the first class II cytokine monomer domain located N-terminal to the second class II cytokine monomer domain or a functional portion thereof corresponds to the N-terminal portion of a naturally occurring class II cytokine monomer domain, and a second sequence portion of the first class II cytokine monomer domain located C-terminal to the second class II cytokine monomer domain or a functional portion thereof corresponds to the C-terminal portion of a naturally occurring class II cytokine monomer domain.

[0153] In the case of a class II cytokine, the inserted sequence of the second class II cytokine monomer is in its natural order.

[0154] According to this method C of making foldikines, a single-chain dimeric class II cytokine polypeptide is i) a split domain comprising the free N- and C-termini of a single-chain dimeric class II cytokine polypeptide and the first and second sequence portions of a first class II cytokine monomer domain in their natural sequence order; and ii) a contiguous domain comprising a second class II cytokine monomer domain or a functional portion thereof (see Figure 47); The sequences of the first and second sequence portions of the first class II cytokine monomer domain are separated in a split domain by the sequence of a contiguous domain of the second class II cytokine monomer domain.

[0155] In the case of class II cytokines, forming a foldikine according to method C may involve opening a loop in one monomer (specifically between alpha helices B and C of one monomer), joining the newly created C-terminus with the native Ct of the other cytokine monomer, and joining the newly created N-terminus with the native N-terminus of the other cytokine monomer.

[0156] According to one embodiment, a single-chain dimeric Class II polypeptide is provided comprising two Class II cytokine monomers, wherein the loop between alpha helices B and C is opened in a first cytokine monomer, wherein in said single-chain dimeric cytokine polypeptide: a) a linker peptide bridges the second α-helix (α-helix B) of a first class II cytokine monomer (cytokine 1 in Figure 47) with the first α-helix (α-helix A) of a second class II cytokine monomer (cytokine 2 in Figure 47); b) A linker peptide bridges the third α-helix (α-helix C) of a first class II cytokine monomer with the first α-helix (N-terminal α-helix, or α-helix A) of a second class II cytokine monomer.

[0157] In the single-chain dimeric cytokine, the natural N- and C-termini of the first class II cytokine monomer are free, and the inserted sequence of the second class II cytokine monomer follows its natural sequence order.

[0158] Overall, the single-chain dimeric Class II polypeptide comprises, from N-terminus to C-terminus, i) the N-terminal portion of a first Class II cytokine monomer comprising alpha helices A and B of the first Class II cytokine monomer; ii) a linker peptide (Xn1) bridging alpha helix B of the first Class II cytokine monomer with alpha helix A of a second Class II cytokine monomer; iii) the native sequence of the second Class II cytokine monomer comprising alpha helices A through F of the second Class II cytokine monomer; iv) a linker peptide (Xn2) bridging alpha helix F of the second Class II cytokine monomer with alpha helix C of the first Class II cytokine monomer; and vii) the N-terminal portion of the first Class II cytokine monomer comprising alpha helices C through F of the first Class II cytokine monomer.

[0159] Similarly, the artificial linkers (Xn1) and (Xn2) bridging the two monomer sequences can have any suitable sequence, as disclosed above in Methods A and B for making foldikines.

[0160] In some embodiments, once a class II foldikine is made according to method C, the native Nt and Ct of a second class II cytokine monomer are joined to create a circularly permuted variant with an open loop in the first second class II cytokine monomer.

[0161] Furthermore, foldikines containing both type I and type II cytokine monomer domains can be designed using the same method C described for class I or class II cytokine-based foldikines.

[0162] In scheme C, two cytokine monomers are linked via two linkers after opening the native loop of the type II cytokine and closing the new N- and C-termini of the open loop via two artificial linkers to the native N- and C-termini of the type I cytokine. Furthermore, the native loop of the type I cytokine can be opened and the native N- and C-termini of the type II cytokine closed via linkers (see Figure 6ii), or the native loop of the type I cytokine can be opened and new N- and C-termini linked to the native N- and C-termini of the type II cytokine, while a circular permutation version thereof can be made in which the native N- and C-termini of the type I cytokine are joined and the loop is opened in the type II cytokine to new N- and C-termini.

[0163] Structural template used to define the angle between the two 3D domains of foldikine When designing a foldikine from a pair of monomeric cytokines, a template / reference based on the swapped domain cytokine can be used. Advantageously, any such template / reference will have a similar structure to the desired resulting foldikine, since in this way any beneficial angles between the two globular domains that may be important in binding the four receptors to the synthetic foldikine can be maintained (see Figures 7 and 8).

[0164] Foldikine-containing polypeptides Further provided is a polypeptide comprising a single-chain dimeric polypeptide according to the invention.

[0165] In these embodiments, the single-chain dimeric polypeptide can be fused or covalently linked to any peptide, such as a protein tag or other polypeptide.

[0166] Recombinant Vectors, Host Cells and Expression Control sequences / regulatory sequences The terms "control sequence" or "regulatory sequence" are used interchangeably herein to refer to any nucleotide sequence capable of increasing or decreasing expression of a particular gene. This regulation can be applied by either affecting transcription or translation rates or by altering the stability of the sequence. In embodiments, polynucleotide sequences of the present disclosure include regulatory elements such as, but not limited to, promoters, enhancers, selectable markers, origins of replication, linker sequences, polyA sequences, terminator sequences, and degradation sequences. In certain embodiments, polynucleotides according to the present disclosure include one or more suitable control sequences. In certain embodiments, the control sequences are identical for all polynucleotides of the present disclosure. In alternative embodiments, different control sequences are used for or within different polynucleotides. In certain embodiments, the control sequences are naturally occurring in the target prokaryotic or eukaryotic cell. In other embodiments, the control sequences are adapted to perform their intended function in the target cell, e.g., bacteria. In some embodiments, polynucleotides of the present disclosure may encode tag sequences to improve purification or localization. Both oligonucleotide motifs and sequences and amino acid motifs or sequences that bind to other oligonucleotides or proteins are contemplated.

[0167] promoter A polynucleotide of the present disclosure may suitably comprise a promoter sequence, or promoter, operably linked to a nucleotide sequence encoding a polypeptide of the present disclosure. As used herein, "promoter" refers to a regulatory region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. A promoter may also contain subregions to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. At least one module in a promoter functions to position the start site for RNA synthesis. The most well-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, additional elements overlapping the start site itself help define the start point.

[0168] Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the start site, although many promoters have been shown to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, and as a result, promoter function is maintained even when elements are inverted or moved relative to one another. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription.

[0169] A promoter can be constitutive, inducible, activatable, repressible, tissue-specific, or any combination thereof. A promoter promotes expression, or promotes transcription, of a nucleic acid sequence that it regulates. As used herein, a promoter is considered "operably linked" when it is in the correct functional location and orientation with respect to the nucleic acid sequence that it regulates, and regulates ("promotes") transcription initiation and / or expression of that sequence. The term "operably linked" is used interchangeably herein with the terms "operably positioned," "under control," and "under transcriptional control."

[0170] Promoters can be classified as strong or weak based on their affinity for RNA polymerase (and / or σ factor), which relates to how closely the promoter sequence resembles the ideal consensus sequence for the polymerase. The strength of a promoter depends on whether transcription initiation occurs frequently or infrequently at that promoter. Different promoters with different strengths can be used as desired.

[0171] Suitable promoter sequences may be natural or synthetic. For example, they may be those naturally associated with a gene or sequence obtained by isolating the 5' non-coding sequence located upstream of the coding segment of a given gene or sequence. Such promoters may be referred to as "endogenous." Similarly, activators / enhancers may be those naturally associated with a nucleic acid sequence, located either within, downstream, or upstream of the nucleic acid sequence.

[0172] In some embodiments, the encoding nucleic acid segment can be under the control of a recombinant or heterologous promoter, where recombinant or heterologous promoter refers to a promoter that is not normally associated with the encoded nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from any suitable eukaryotic or prokaryotic cell, and "non-naturally occurring" synthetic promoters or enhancers, such as those containing different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering. In addition to synthetically producing promoter and enhancer nucleic acid sequences, sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including polymerase chain reaction (PCR).

[0173] In certain embodiments, the promoter can be a constitutive promoter, which is understood by those skilled in the art to be a promoter whose expression is constant under standard culture conditions, i.e., a promoter that expresses a gene product at a substantially constant expression level.

[0174] In alternative embodiments, the promoter can be an inducible (conditional) promoter. As used herein, an "inducible promoter" is characterized by initiating or enhancing transcriptional activity when in the presence, influence, or contact with an inducer or inducing agent. An "inducer" or "inducing agent" can be an endogenous or usually exogenous condition, compound, or protein that contacts a promoter or transcription machinery in such a way that it is active in inducing transcriptional activity from an inducible promoter. Inducible promoters, and more specifically bacterial inducible promoter systems, have been described in great detail in the art (e.g., Brautaset et al., Positively regulated bacterial expression systems, Microbial biotechnology, 2009).

[0175] Inducible promoters for use in accordance with the present disclosure function in microbial cells, such as bacterial cells. Examples of inducible promoters for use herein include, but are not limited to, bacteriophage promoters (e.g., PlsIcon, T3, T7, SP6, PL) and bacterial promoters (e.g., Pbad, PmgrB, Ptrc2, Plac / ara, Ptac, Pm), or hybrids thereof (e.g., PLlacO, PLtetO).Examples of bacterial promoters for use in accordance with the present disclosure include positively regulated E. coli promoters such as the positively regulated σ promoter (e.g., inducible pBad / araC promoter, Lux cassette right promoter, modified lambda Prm promoter, plac Or2-62(positive), pBad / AraC with additional REN sites, pBad, P(Las)TetO, P(Las)CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), as promoter (e.g., Pdps), σ promoter (e.g., heat shock), σ promoter (e.g., glnAp2); negatively regulated σ promoter (e.g., promoter(PRM+), modified lambda Prm promoter, TetR-TetR-4C P(Las)TetO, P(Las)CIO, P(Lac)IQ, RecA_DlexO_DLac01, dapAp, FecA, Pspac-hy, pel, plux-cl, plux-lac, CinR, CinL, glucose regulation, modified Pr, modified Prm+, FecA, Pcya, recA(SOS), RecA(SOS), EmrR regulation, Betl regulation, pLac_lux, pTet_Lac, pLac / Mnt, pTet / Mnt, LsrA / cI, pLux / cI, Lacl, LacIQ, pLacIQl, pLas / cI, pLas / Lux, pLux / Las, pRecA with LexA binding site, reverse BBa_R0011, pLacI / ara-1, pLacIq, rrnB Negatively regulated E. coli promoters include, but are not limited to, negatively regulated E. coli promoters such as PI, cadC, hns, PfhuA, pBad / araC, nhaA, OmpF, RcnR), the σ promoter (e.g., Lutz-Bujard LacO with alternative sigma factor σ), the σ promoter (e.g., Lutz-Bujard LacO with alternative sigma factor σ), and the σ promoter (e.g., glnAp2); and negatively regulated B. subtilis promoters, such as the repressible B. subtilis σA promoter (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank) and the σ promoter.Other inducible microbial and / or bacterial promoters can be used in accordance with the present disclosure.

[0176] Inducible promoters for use in accordance with the present disclosure can be induced (or repressed) by one or more physiological condition(s), such as changes in pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more exogenous or endogenous inducer(s). Exogenous inducers or inducers can include, but are not limited to, amino acids and amino acid analogs, sugars and polysaccharides, nucleic acids, protein transcription activators and repressors, cytokines, toxins, petroleum-based compounds, metal-containing compounds, salts, ions, enzyme substrate analogs, hormones, or combinations thereof.

[0177] Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to those of skill in the art. Examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems including the tetracycline repressor protein (tetR) and the tetracycline operator sequence (tetO) and the tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, and gaecdysone receptor, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., α-glucosamine- and α-glucosamine-responsive promoters), and the like. Examples of promoters include, but are not limited to, chemically / biochemically regulated promoters and physically regulated promoters, such as promoters derived from yeast, mouse, and human metallothionein (a protein that binds and sequesters metal ions) genes, pathogen-regulated promoters (e.g., induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells). In certain embodiments, the promoter is the TetR promoter, which is part of the Tet-On or Tet-Off system (Krueger et al., Tetracycline derivatives: alternative effectors for Tet transregulators, Biotechniques, 2004 and Loew et al., Improved Tet-responsive promoters with minimized background expression, BioMedCentral Biotechnology, 2010).

[0178] Promoter sequences suitable for expression in Mycoplasma bacteria include, but are not limited to, the P3 promoter (48) and the synthetic promoter pSynL. Other promoters have also been used successfully in the Mycoplasma field, such as the SynMyco promoter (https: / / doi.org / 10.1093 / dnares / dsz012) or other promoters used in intermediate versions derived from the Tn4001 transposon (10.1007 / BF00382099).

[0179] Other promoters for use in accordance with the present disclosure can include any suitable promoter derived from bacterial, viral, or eukaryotic sources (particularly for expression in Lactococcus lactis or E. coli).

[0180] In various other embodiments in which the target cell is a mammalian cell, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat may be used to obtain high levels of expression of the transgene. The use of other viral or mammalian cell or bacterial phage promoters known in the art is also contemplated to achieve expression of the transgene, provided that the expression level is sufficient for the given purpose.

[0181] Enhancer In some embodiments of the present disclosure, promoters may or may not be used in conjunction with "enhancers," which are also involved in transcriptional activation. Enhancers are one or more regions of DNA that can bind proteins (i.e., trans-acting factors) to increase the level of transcription of a gene. Enhancers can be located in functional regions upstream or downstream of a promoter. They are typically located at the 5' end of a coding region, but can also be located away from the promoter sequence, for example, within an intron region of a gene or within 3' of the coding region of a gene.

[0182] Terminator In some embodiments, the polynucleotides of the present disclosure can contain a terminator sequence or terminator. As used herein, a "terminator" is a nucleic acid sequence that stops transcription. A terminator can be unidirectional or bidirectional. A terminator is composed of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. A terminator sequence prevents transcriptional activation of a downstream nucleic acid sequence by an upstream promoter. Thus, in certain embodiments, a terminator that terminates the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve a desired gene / protein expression level.

[0183] The most commonly used type of terminator is the forward terminator. When placed downstream of a nucleic acid sequence that is normally transcribed, a forward transcription terminator will terminate transcription. In some embodiments, a bidirectional transcription terminator is provided, which typically terminates transcription on both the forward and reverse strands. In some embodiments, a reverse transcription terminator is included, which typically terminates transcription only on the reverse strand. In prokaryotic systems, terminators typically fall into two categories: (i) rho-independent terminators and (ii) rho-dependent terminators. Rho-independent terminators generally consist of a palindromic sequence that forms a stem-loop rich in GC base pairs followed by a series of uracil bases.

[0184] Terminators for use in accordance with the present disclosure include any transcription terminator described herein or known to those of skill in the art. Examples of terminators include, but are not limited to, gene termination sequences, such as the bovine growth hormone terminator, and viral termination sequences, such as the TO terminator, the TE terminator, Lambda T1, and the T1T2 terminators found in bacterial systems. In some embodiments, the termination signal can be a sequence that cannot be transcribed or translated, such as a sequence resulting from sequence cleavage.

[0185] Other genetic elements are known in the art and can be used in accordance with the present disclosure.

[0186] Expression vector Any suitable polynucleotide of the present disclosure can be part of an expression vector, such as a plasmid, optionally a non-replicating plasmid, a phagemid, a bacteriophage, a bacteriophage-derived vector, an artificial chromosome, a minicircle, a lentiviral vector, a retroviral vector, an adenoviral or adeno-associated viral vector, a piggyBac vector, or a tol2 vector. Those skilled in the art are aware of these various types of constructs, and their generation and manipulation are detailed in numerous examples (Sambrook et al., Molecular cloning: a laboratory manual, ISBN 0879693096, 1989 and the corresponding latest 4th edition, Cold Spring Harbor Laboratory Press, 2012). Furthermore, it is clear to those skilled in the art that plasmid DNA or (circular) recombinant DNA is commonly referred to as copy DNA, complement DNA, or the abbreviation "cDNA," which can be used interchangeably.

[0187] The term "expression vector" or "expression construct" refers to a genetic construct containing a nucleic acid encoding a gene product, in which some or all of the nucleic acid coding sequence is transcribable. The transcript may, but need not, be translated into a protein. Thus, in certain embodiments, expression includes both transcription of a gene and translation of RNA into a gene product.

[0188] Particularly useful vectors are contemplated to be those in which the coding portion of the DNA segment, whether encoding a full-length protein or a smaller polypeptide, is placed under the transcriptional control of a promoter, as described above.

[0189] Naturally, it will be important to use a promoter that effectively directs expression of the polypeptide of the present disclosure in the cell type or organism selected for expression. The use of promoter and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology; see, for example, Sambrook et al. (1989), which is incorporated herein by reference. Any suitable promoter that directs the desired level of expression of the encoded polypeptide can be used in the vector or plasmid of the present disclosure.

[0190] The particular promoter used to control the expression of a nucleic acid is not believed to be important, so long as it is capable of expressing the desired polypeptide at the desired level in the target cell or organism. Thus, when mycoplasma is used, it may be preferable to place the polynucleotide coding region adjacent to and under the control of a promoter that is expressible in mycoplasma. Similarly, if a polypeptide is to be expressed in E. coli or L. lactis, an appropriate promoter suitable for expression in E. coli or L. lactis should be used.

[0191] When generating recombinant bacteria or other cells, it can be useful to include one or more "reporter genes." Generally, a reporter gene encodes a polypeptide not otherwise produced by the host cell, or a protein or factor produced by the host cell but at a much lower level, or a mutant form of a polypeptide not otherwise produced by the host cell. A reporter gene can encode an enzyme that produces a calorimetric or fluorometric change in the host cell that is detectable by in situ analysis and is a quantitative or semi-quantitative function of transcriptional activation. Exemplary reporter genes encode esterases, phosphatases, proteases, and other proteins that are detected by their chromophore- or fluorophore-producing activity, as would be known to one of skill in the art. Well-known examples of such reporter genes are E. coli β-galactosidase, luciferase, and chloramphenicol-acetyltransferase (CAT). Alternatively, a reporter gene may encode a selectable marker, e.g., by conferring resistance to a selection agent on the host cell. For example, the gene neo confers resistance to the antibiotic neomycin on cells. It is contemplated that virtually any host cell system compatible with the reporter gene cassette can be used to determine the regulatory units. Thus, mammalian cells, or other eukaryotic cells, insect, bacterial, or plant cells can be used.

[0192] Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG initiation code and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would be able to readily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in-frame" with the reading frame of the desired coding sequence. Furthermore, it is well known that certain bacteria can utilize alternative (non-ATG) initiation sites; therefore, initiation signals containing such alternative sites are also contemplated. Exogenous translational control signals and initiation codons can be natural or synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements.

[0193] As used herein, the terms "artificial" and "recombinant" cells are intended to mean cells into which an exogenous DNA segment or gene, such as a polynucleotide encoding a polypeptide (foldikine) of the present disclosure, has been introduced. Artificial cells are therefore distinguishable from naturally occurring cells that do not contain recombinantly introduced exogenous DNA segments or genes. Recombinant cells include those that have introduced cDNA or genomic genes, and also include genes located adjacent to a promoter not naturally associated with the particular introduced gene.

[0194] To express the foldikine polypeptide of the present invention, an expression vector can be prepared containing a polynucleic acid encoding the foldikine under the control of one or more promoters. To bring the coding sequence under the control of a promoter, the 5' end of the transcription initiation site of the transcriptional open reading frame is generally located about 1 to about 50 nucleotides downstream (i.e., 3') of the selected promoter. The upstream promoter stimulates transcription of the DNA and promotes expression of the encoded polypeptide. One or more enhancer elements can also be associated with the expression construct.

[0195] Many standard techniques are available for constructing expression vectors containing appropriate nucleic acids and transcriptional / translational control sequences to achieve expression of proteins or polypeptides in a variety of host expression systems. Cell types available for expression include, but are not limited to, M. pneumoniae, E. coli, and bacteria such as L. lactis, S. aureus, and B. subtilis transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors.

[0196] Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are generally used in connection with these hosts. The vector usually carries a replication site, as well as marking sequences which allow phenotypic selection of transformed cells. For example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin resistance and tetracycline resistance and thus provides an easy means of identifying transformed cells. pBR plasmids or other microbial plasmids or phages must also contain, or be modified to contain, promoters that can be used by the microorganism for expression of its own proteins.

[0197] Mycoplasma tends to have difficulty maintaining replicating vectors. However, transposons derived from Tn4001 vectors (with different resistance markers) can be used. The transposon contains a transposase and is located outside a pair of inverted repeats. Foreign DNA sequences that may be inserted into the chromosome are located within the inverted repeats. When the vector is inserted into a cell, the transposase catalyzes the excision of the inverted repeats, randomly introducing internal sequences into the chromosome. Therefore, foreign DNA fragments of different lengths can be introduced into the M. pneumoniae chromosome.

[0198] Additionally, phage vectors containing replicon and control sequences compatible with host microorganisms can be used as transforming vectors in connection with these hosts. For example, phage lambda GEM™-11 can be utilized to generate recombinant phage vectors that can be used to transform host cells such as M. pneumoniae and E. coli.

[0199] Additional useful vectors include the pIN vectors (Inouye and Inouye, 1985), pQE (His-tagged) vectors (Qiagen), and pGEX vectors, which are used to generate glutathione-S-transferase (GST) soluble fusion proteins for subsequent purification, isolation, or cleavage. Other suitable fusion proteins are with β-galactosidase, ubiquitin, etc.

[0200] It is contemplated that the polypeptides of the present disclosure can be expressed in heterologous systems, such as bacteria, where cytokines are not normally expressed, or, in some cases, can be expressed in eukaryotic cells, such as human cells.

[0201] In certain embodiments, the polynucleotide of the present disclosure can comprise a bicistronic expression construct. In further embodiments, the polynucleotide is integrated, i.e., inserted, into a cellular genome, preferably a bacterial genomic sequence, such as M. pneumoniae, L. lactis, or E. coli. In some embodiments, the polynucleotide can be part of a cellular genome, for example, a de novo designed cellular genome or a mutagenized or synthesized bacterial genome. In further embodiments, the nucleotide arrangement is comprised in a bacterial artificial chromosome or a yeast artificial chromosome.

[0202] In certain embodiments, the polynucleotides of the present disclosure can be incorporated into genetically engineered Mycoplasma strains, such as the attenuated CV2 strain (Δmpn133, Δmpn372) (58) or CV8, which are based on CV2 and in which the mpn051 gene has been replaced with the gpsA gene from Mycoplasma penetrans. These polynucleotides can also be evaluated in other Mycoplasma species of interest.

[0203] In certain embodiments, polynucleotides of the present disclosure may be comprised in a bacteriophage. As used herein, the term "bacteriophage" refers to a virus that can infect and optionally replicate within bacteria and archaea, and can be modified for therapeutic purposes as described in the art (e.g., Principi et al., Advantages and Limitations of Bacteriophages for the Treatment of Bacterial Infections, Frontiers in Pharmacology, 2019).

[0204] In further embodiments, a concatenation of various sequence elements (e.g., polynucleotides of the present disclosure) can be considered an "operon." As used herein, "operon" refers to a functional unit of DNA containing a cluster of genes, all of which are controlled by a single promoter. Thus, genes from an operon are generally co-transcribed. Genes transcribed from an operon can be transcribed into a single mRNA strand to generate a monocistronic mRNA that can be translated together in the cytoplasm or can be spliced ​​and translated separately.

[0205] Vectors and plasmids of the present disclosure also typically contain an "origin of replication" or "ORI," which refers to a sequence from which replication is initiated in either prokaryotes or eukaryotes. DNA replication can proceed bidirectionally or unidirectionally from this point. Commonly used prokaryotic origins of replication include, but are not limited to, pMB1, modified pMB1, pBR322, ColEl, ColEl derivatives, FI, R6K, p15A, pSClO1, and pUC.

[0206] To allow a polynucleotide to be inserted and cloned, i.e., propagated, a vector typically contains one or more unique restriction sites and is capable of autonomous replication in a given cell or vehicle organism so that the cloned sequence is reproducible. As noted above, such vectors may also contain a selection marker, such as an antibiotic resistance gene, to allow for the selection of recipient cells containing the vector, as described above.

[0207] Expressed Polypeptides In embodiments, a polypeptide of the present disclosure—encoded by a polynucleotide of the present disclosure—can include a "display signal" or "exposure signal" and / or a "secretion signal." Thus, a polynucleotide of the present disclosure can include a nucleic acid sequence encoding one or more such sequences.

[0208] The term "display signal" or "exposure signal" refers to a peptide sequence that directs a fusion / linked peptide (e.g., a foldikine of the present disclosure) to be exposed on the cell membrane. The cell may be a bacterial cell or a eukaryotic cell, as desired. "Exposed" or "displayed" means that the polypeptide of interest is permanently physically attached to the cell in which it is produced, preferably to the cell surface on the outside of the cell. Exposure signal sequences are known to those skilled in the art. In embodiments, the exposure signal sequence is a sequence that naturally occurs in bacteria such as E. coli, L. lactis, or Mycoplasma (e.g., M. pneumoniae). In some embodiments, the exposure signal sequence is a non-naturally occurring bacterial sequence.

[0209] As used herein, the term "secretion signal" refers to a peptide sequence that induces or mediates secretion of a polypeptide from the cell in which it is expressed. That is, a secreted polypeptide containing a secretion signal is no longer physically attached to the cell in which it is produced and can be secreted into the extracellular space, as opposed to when the polypeptide is exposed or displayed.

[0210] In embodiments, the secretion signal sequence can be a sequence that occurs naturally in bacteria, such as E. coli, L. lactis, or Mycoplasma. In alternative embodiments, the secretion signal sequence is a non-naturally occurring bacterial sequence. Mycoplasma secretion signals are described, for example, in International Patent Application WO 2016 / 135281. In embodiments, concatenated secretion signals can be used to enhance secretion of a polypeptide, for example, different secretion signals can be incorporated at different positions in a polypeptide according to the present disclosure.

[0211] In various embodiments, the signal sequence may be removed from the protein to which it is linked by proteolytic cleavage after the protein is exposed (i.e., displayed) on the cell surface or after the protein is secreted. In certain embodiments, the exposed or secretory signal sequence is located at the N-terminus of the polypeptide.

[0212] Diseases and Disorders The medicaments, compositions, methods, and uses of the present invention may be particularly suitable for treating a wide range of diseases and disorders, including, for example, any disease or disorder that would benefit from a reduction or increase in the rate of inflammatory responses or cell proliferation. Specifically, diseases and disorders that can be treated according to the present invention include cancer and / or proliferative or neoplastic diseases (especially solid tumors) and inflammatory diseases or disorders. The present invention may also be suitable for treating infectious diseases or promoting tissue regeneration after tissue-damaging events or diseases.

[0213] Those skilled in the art will appreciate that proliferative disorders can be associated with: (1) pathological proliferation of normally quiescent or normally proliferating cells, (2) pathological migration of cells from their normal location (e.g., tumor cell metastasis), (3) pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenase, gelatinase, and elastase), which can result in unwanted turnover of cell matrix, and / or (4) pathological angiogenesis, such as occurs in proliferative retinopathies and tumor metastasis. Exemplary proliferative disorders include cancer, benign neoplasms, and progressive angiogenesis associated with disease states (defined above as pathological angiogenesis).

[0214] The compositions, medicaments, methods and uses of the present invention may have beneficial effects in treating and / or alleviating the symptoms of a wide range of proliferative diseases and disorders, for example by preventing cell proliferation and promoting cell death, particularly of pathogenic cells.

[0215] The present invention may have utility in multiple cancer types and / or have a beneficial effect on tumor progression in vivo and / or in vitro (e.g., reversing tumor progression). Specifically, the present invention may be useful in the treatment of lung cancer (especially lung adenocarcinoma), cervical cancer, breast cancer, heart cancer, colon cancer, prostate cancer, brain glioblastoma, pancreatic cancer, leukemia (e.g., acute monocytic leukemia), lymphoma, kidney cancer, colorectal cancer, bladder cancer, testicular cancer, gastrointestinal cancer, liver cancer (e.g., hepatocellular carcinoma), and / or glioblastoma. The present invention may also be useful in the treatment of one or more of skin cancer (e.g., melanoma), head and / or neck cancer, gallbladder cancer, uterine cancer, stomach cancer, thyroid cancer, laryngeal cancer, lip and / or oral cavity cancer, throat cancer, eye cancer, and bone cancer.

[0216] In some particular embodiments, the cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, extrahepatic cancer, Ewing's sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumor, Burkitt's lymphoma, cystic leukemia ... Lactinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasm, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, osteofibrous histiocytoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brainstem glioma, hairy cell leukemia, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, pancreatic islet cell tumor, pancreatic neuroendocrine tumor, Films tumor and other childhood renal tumors, Langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck, midline carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low-grade ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus cancer, and nasal cavity cancer. The cancer may be selected from any one or more of the group consisting of: thyroid cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi's sarcoma, rhabdomyosarcoma, Sézary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia.

[0217] Furthermore, the present disclosure also encompasses therapeutic uses of the therapeutic agents and compositions of the present invention, as well as methods for suppressing or preventing local invasion or metastasis, or both, of any type of primary cancer. For example, the primary cancer may be melanoma, non-small cell lung, small cell lung, lung, hepatocellular carcinoma, retinoblastoma, astrocytoma, glioblastoma, gum, tongue, leukemia, neuroblastoma, head, neck, breast, pancreas, prostate, kidney, bone, testis, ovary, mesothelioma, cervix, gastrointestinal tract, lymphoma, brain, colon, or bladder. In certain embodiments, the primary cancer may be liver cancer. For example, the liver cancer may be hepatocellular carcinoma (HCC) and / or liver metastasis.

[0218] Additionally, the present disclosure can be used to prevent cancer or treat pre-cancerous or pre-malignant cells, including metaplasia, dysplasia, and hyperplasia. It can also be used to inhibit unwanted benign cells, such as squamous cell metaplasia, dysplasia, benign prostatic hyperplasia, hyperplastic lesions, etc. In some embodiments, the progression to cancer or to more severe forms of cancer can be stopped, halted, or delayed by the uses and methods of the present disclosure, including the therapeutic agents disclosed herein.

[0219] Thus, the present invention provides agents and compositions for use in medicine, in particular for use in the treatment of cancer selected from lung cancer (particularly lung adenocarcinoma or lung squamous cell carcinoma), bladder cancer, cervical cancer, breast cancer, colon cancer, glioblastoma, pancreatic cancer, acute monocytic leukemia, kidney cancer, colorectal cancer, skin cancer (e.g., melanoma), stomach cancer, thyroid cancer, bone cancer, and liver cancer. Methods for the treatment of such diseases are also provided. The uses and methods may comprise administering an agent according to the present invention to a patient in need thereof.

[0220] The compositions and medicaments of the present disclosure may also be useful in cancer immunotherapy and / or immuno-oncology treatment.

[0221] inflammation In parallel, the inflammatory response is an adaptive response to various physical, chemical, or biological insults. There are various types of inflammation: classical inflammation, homeostatic inflammation, low-grade inflammation, an adaptive response to stress known as parainflammation, or metabolically induced inflammation. The "foldikines" of the present disclosure can be used to treat inflammatory diseases, for example, in scenarios where the immune system mistakenly attacks the body's own cells or tissues. Associated symptoms of such immunological diseases or disorders include pain, redness, swelling, stiffness, or damage to other / adjacent healthy tissues, among many others. The present invention provides agents and compositions for use in medicine, particularly for use in the treatment of allergies, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury, transplant rejection, diabetes, Alzheimer's disease, osteoarthritis, fibromyalgia, muscular low back pain, arthritis, muscular neck pain, myocarditis, ankylosing spondylitis (AS), antiphospholipid syndrome (APS), myositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, and fibrosis.

[0222] Viral, bacterial and fungal infections The "foldikines" of the present disclosure may be useful in the treatment of viral, bacterial, or fungal infections. Any of these microorganisms may induce an inflammatory response and the release of physiological agents that alter the immune system. In other cases, such infectious agents may produce and release toxins. To counterbalance the immune system response, foldikines may also be applied in the setting of rubella, roseola, smallpox, chikunguya virus infection, measles, norovirus infection, coronavirus infection, shingles, hepatitis, herpes, dengue fever, Ebola, Lassa fever, Marburg hemorrhagic fever, polio, meningitis, encephalitis, or rabies.

[0223] Because cytokines also play an important role in orchestrating immune-mediated cellular repair and regeneration responses, the present invention may also provide additional benefits in promoting tissue repair following damage associated with such infections and infectious agents.

[0224] tissue regeneration The immune system plays a central role in tissue repair and regeneration. The immune response to tissue injury is crucial in determining the rate and outcome of the healing process and the restoration of organ function. Therefore, controlling immune components via cytokines is an attractive approach in regenerative medicine. Thus, the foldikines of the present disclosure may also be useful in methods and therapeutic uses for tissue regeneration, for example, by promoting proper cell proliferation and differentiation and preventing fibrosis. Examples of such uses are in the treatment of conditions such as colitis, wound healing, hair growth, liver regeneration, and matrix synthesis.

[0225] The compositions, medicaments, methods and uses of the present invention may be beneficial in the treatment of any or all of such diseases and disorders.

[0226] therapeutic composition The single-chain dimeric cytokines, polynucleotides, or artificial bacteriophages, recombinant bacteria, or cells (e.g., "therapeutics") of the present invention can be incorporated into pharmaceutical compositions for use in treating animals, preferably humans. The therapeutic agents of the present invention (or their derivatives) can be used to treat one or more diseases or infectious diseases depending on the activity / properties of the single-chain dimeric cytokines of the present invention. In various embodiments, nucleic acids encoding therapeutic peptides can be inserted into expression constructs / vectors, particularly bacteriophage vectors, viral or bacterial genomes, and incorporated into pharmaceutical formulations / medicines for the same purpose. Similarly, the foldikines of the present disclosure can be delivered via conventional delivery methods such as aerosol, intratracheal administration, injection, suppository, etc., or can be administered via recombinant means.

[0227] As one skilled in the art will appreciate, potential therapeutic agents, such as those disclosed herein, can be tested in animal models such as rabbits or mice before being approved for use in human subjects. Thus, the polypeptides, bacteriophages, bacteria, or cells of the present disclosure can be expressed or delivered in vivo in rabbits or mice, or ex vivo in rabbit or mouse cells, as well as in humans / human cells. In the present invention, appropriate expression cassettes and expression constructs / vectors can be designed specifically for each animal system.

[0228] The therapeutic peptides and nucleic acids of the invention may be particularly suitable for treating diseases, conditions, and / or infections that can be targeted (and treated) intracellularly, for example, by directing bacteria or bacteriophages to the desired area of ​​an animal (cells / tissues) where they are administered by conventional methods. They may also be suitable for in vitro and extracellular applications. As used herein, the terms "therapeutic agent" and "active agent" encompass peptides and nucleic acids encoding the polypeptides of the invention, as well as vectors, bacteriophages, viruses, bacteria, and other cells that contain the peptides and / or nucleic acids described herein. Therapeutic nucleic acids of the invention include modified / artificial bacteriophage, viral, or bacterial genomes.

[0229] Therapeutic uses and applications of the therapeutic agents of the present invention include any disease, disorder or other medical condition that may be treatable by expressing or delivering the single-chain dimeric cytokine of the present invention in a subject treated by any delivery methodology applied in the clinic.

[0230] In aspects and embodiments of the invention, particularly preferred diseases include cancer and other proliferative diseases or disorders, inflammatory diseases or disorders, viral, bacterial and fungal infections, and tissue regeneration, as disclosed elsewhere herein.

[0231] One or more additional pharmaceutically acceptable "carriers" (such as diluents, adjuvants, excipients, or vehicles) can be combined with the therapeutic agent(s) of the present invention in a pharmaceutical composition. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The pharmaceutical formulations and compositions of the present invention can be formulated to meet regulatory standards and administered orally, intravenously, topically, or via other standard routes. As used herein, the term "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as otherwise described, conventional media or agents are contemplated for use in therapeutic compositions. Supplementary active ingredients can also be incorporated into the compositions. The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to humans.

[0232] In the present invention, the therapeutic agent(s) can be manufactured into a pharmaceutical product or formulated into a pharmaceutical composition. When administered to a subject, the therapeutic agent is suitably administered as a component of a composition comprising a pharmaceutically acceptable vehicle. The molecules, compounds, and compositions of the present invention can be administered by any convenient route, for example, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, intranasally, epidurally, orally, sublingually, intranasally, intravaginally, transdermally, rectally, by inhalation, or topically to the skin. Administration can be systemic or local. Known delivery systems also include encapsulation in, for example, microgels, liposomes, microparticles, microcapsules, capsules, etc., any of which can be used to administer the agents of the present invention in some embodiments. Any other suitable delivery system known in the art is also contemplated for use in the present invention.

[0233] Acceptable pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical vehicles can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Additionally, auxiliary substances, stabilizers, thickeners, lubricants, and coloring agents can also be used. When administered to a subject, pharmaceutically acceptable vehicles are preferably sterile. Water is a preferred vehicle, particularly when the compounds of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions of the present invention, if desired, can also contain minor amounts of wetting or emulsifying agents, or buffers.

[0234] The medicaments and pharmaceutical compositions of the present invention can take the form of liquids, solutions, suspensions, lotions, gels, tablets, pills, pellets, powders, modified-release formulations (capsules containing liquids or powders), suppositories, emulsions, aerosols, sprays, capsules, liposomes, microparticles, or any other suitable formulation known in the art. Other examples of suitable pharmaceutical vehicles are described in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, see for example pages 1447-1676.

[0235] In some embodiments, the therapeutic compositions or medicaments of the present invention are formulated, according to commonly used procedures, as pharmaceutical compositions adapted for oral administration (more preferably for humans). Compositions for oral delivery can be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Thus, in various embodiments, the pharmaceutically acceptable vehicle can be a capsule, tablet, or pill.

[0236] Orally administered compositions can contain one or more drugs, sweeteners such as fructose, aspartame, or saccharin, flavorings such as peppermint, wintergreen oil, or cherry, coloring agents, and preservatives to provide a pharmaceutically palatable formulation. When the composition is in tablet or pill form, the composition can be coated to delay disintegration and absorption in the gastrointestinal tract, providing sustained release of the active agent over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compositions. In these dosage forms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells and displaces the drug or drug composition through an opening. These dosage forms can provide an essentially zero-order delivery profile, as opposed to the spiked profiles of immediate-release formulations. A time-delay material, such as glycerol monostearate or glycerol stearate, can also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade. For oral formulations, the location of release can be the stomach, the small intestine (duodenum, jejunum, ileum), or the large intestine. Those skilled in the art can prepare formulations that will not dissolve in the stomach but will release the substance in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the gastric environment by protecting the peptide (or derivative) or by releasing the peptide (or derivative) beyond the gastric environment, such as the intestine. To ensure full gastric resistance, a coating impermeable to at least pH 5.0 may be essential.Examples of more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac, which can be used as blend films.

[0237] Surfactants may be added as wetting agents to aid in dissolving the therapeutic agent(s) into the aqueous environment. Surfactants may include anionic surfactants such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic surfactants may also be used, such as benzalkonium chloride or benzethonium chloride. Nonionic detergents that may be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glyceryl monostearate, polysorbates 20, 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. When used, these surfactants may be present in the peptide, nucleic acid, or derivative formulations either alone or as a mixture in different ratios.

[0238] Typically, compositions for intravenous administration comprise sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent.

[0239] Mixtures of the polypeptides, polynucleotides, vectors, bacteriophages, bacteria, and / or other cells described herein can be prepared in water, suitably mixed with one or more excipients, carriers, or diluents. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form will be sterile and will have sufficient fluidity to allow infusion with a suitable syringe. Preferably, the composition will be stable under the conditions of manufacture and storage and will be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0240] For example, for parenteral administration in an aqueous solution, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art.

[0241] Another suitable route of administration for the therapeutic compositions of the present invention is via pulmonary or nasal delivery.

[0242] To enhance cellular uptake of the therapeutic agents of the invention, additives such as fatty acids, oleic acid, linoleic acid, linolenic acid, etc. may be included.

[0243] The therapeutic agents of the present invention may also, in some embodiments, be formulated into compositions for topical application to the skin of a subject.

[0244] In embodiments of the present invention, a therapeutic composition may contain only one therapeutic agent of the present invention, or may contain two or more, e.g., two complementary therapeutic agents of the present invention. For example, inhibition of different inflammatory response pathways in a target animal can be achieved by multiple single-chain dimeric cytokines of the present disclosure, or by single-chain dimeric cytokines capable of targeting two or more different receptors. Specifically, a single-chain dimeric cytokine / foldikine of the present invention may comprise a cytokine monomer of one type (e.g., IL-10) linked to a cytokine monomer of a different type (e.g., IL-22) such that each domain preferentially targets a different receptor and potentially a different inflammatory pathway. When two (or more) therapeutic agents are contemplated, different peptides or encoding nucleic acid constructs, such as bacteriophages, viruses, or bacteria, may be incorporated into the same pharmaceutical composition or may be produced separately. It will be understood that when two (or more) pharmaceutical compositions are manufactured for administration to the same individual, the compositions may be administered simultaneously, sequentially, or separately, as indicated / needed.

[0245] Methods for delivery of therapeutic agents and compositions Any suitable delivery method for administering the therapeutic agents / compositions of the present invention may be used in accordance with the present disclosure. The preferred route of administration is determined by a skilled medical practitioner and may depend on one or more factors.

[0246] Delivery of the therapeutic agent or composition of the present invention can be systemic or local. For example, the route of administration can be determined by the location and / or nature of the disease, e.g., cancer or inflammatory condition, being treated, and can include intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, topical (e.g., near the tumor / target tissue), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration, as described, for example, in U.S. Pat. Nos. 5,543,158, 5,641,515, and 5,399,363. Typically, convenient forms of systemic administration include oral, parenteral, intranasal, sublingual, rectal, transdermal, or any combination thereof.

[0247] In various embodiments, the therapeutic agent / composition can be administered directly to the target tissue, organ, or tumor. In some particularly beneficial embodiments, the therapeutic compositions of the present disclosure can be delivered by injection into the vascular system of a subject.

[0248] In some embodiments, (continuous) administration / administration over a long period of time may be preferred and may be achieved by any suitable mechanism, for example, by implanting a catheter into the tumor, vasculature or tissue. Suitably, the dose of therapeutic composition administered by continuous perfusion may be equivalent to the dose given by single or multiple injections adjusted over the period during which perfusion occurs.

[0249] Injection of nucleic acid constructs, vectors, and / or cells can be delivered by syringe or any other method used for injecting solutions, so long as the expression construct (e.g., bacteria) can pass through a needle of the specific gauge required for injection. In some embodiments, needle-free injection systems known to those skilled in the art can be used, for example, as described in U.S. Patent No. 5,846,233.

[0250] Therapeutic Treatment Regimes and Administration Treatment regimens vary and may often depend on the type and / or location of the disease, such as a tumor, the stage / progression of the disease, and / or the health and age of the patient. For example, some tumors may respond well to treatment with localized and / or high-concentration administration of the therapeutic compositions of the present disclosure, while other diseases (and individuals) may benefit from more diffuse and / or lower doses and / or longer-term administration of the therapeutic agent. A skilled clinician will be able to determine the appropriate therapeutic treatment regime in each situation.

[0251] In various aspects and embodiments, the present disclosure provides therapeutic uses and methods for treating a subject by administering one or more therapeutic agents disclosed herein. Accordingly, the present disclosure provides uses and methods for reducing tumor size and / or growth by promoting the death of targeted tumor cells. The uses and methods comprise administering a therapeutically effective amount of a therapeutic composition or agent of the present disclosure, such as a single-chain cytokine of the present invention, or an agent that expresses a single-chain cytokine, to an individual or a first population of cancer cells or target cells of the individual. Thus, advantageously, by using a vector or delivery agent (e.g., a bacterium, virus, cell, or bacteriophage) that can replicate and express and / or release multiple copies of the single-chain cytokine at the target site, it is not necessary to directly administer the therapeutic agent to all target cells or regions (e.g., within a tumor) of a subject with an initial administration of the therapeutic agent or vehicle. Rather, an enhanced therapeutic effect can be achieved by allowing the therapeutic agent to propagate or produce multiple copies of the single-chain cytokine of the present disclosure.

[0252] An effective amount of a single chain cytokine or other therapeutic agent of the present disclosure or pharmaceutical composition thereof may include an amount sufficient to induce oncolysis / killing of a target cancer cell, and / or lysis or disruption of the growth of a target cancer cell, or inhibition or reduction in the growth or size of a target cancer cell or tumor. Reduction in tumor or target cancer cell growth may be manifested, for example, by cell death, a slowed replication rate or reduced growth rate of a tumor containing the cells, or prolonged survival of a subject containing the cancer cells. In alternative aspects and embodiments, an effective amount of a single chain cytokine or other therapeutic agent of the present disclosure or pharmaceutical composition thereof may include an amount sufficient to reduce an inflammatory response in a target individual, tissue, or organ, such as the lung.

[0253] Thus, in some embodiments, there are provided therapeutic uses and methods for treating a subject having cancer or a tumor or an inflammatory disease or disorder (such as a lung disease, e.g., asthma) comprising administering to the subject an effective amount of a therapeutic agent of the present disclosure. In some embodiments, the tumor can be completely or substantially eliminated. In some embodiments, the inflammatory response can be completely or substantially eliminated.

[0254] The uses and methods of the present disclosure can include administering a therapeutic agent to a subject or individual. Administration can be by any suitable means, for example, injection or ingestion. Administration can be systemic or local, for example, directly to a tumor site. In one embodiment, systemic delivery is preferred.

[0255] Therapeutic compositions and / or therapies can include various "unit doses," which can be defined as containing a predetermined amount of a therapeutic agent or composition. It is within the skill of one of ordinary skill in the clinical arts to determine the appropriate amount to be administered, as well as the particular route and formulation of administration. The unit dose can be administered, for example, as a single injection or tablet, but can also be administered over an extended period of time, such as via continuous infusion over a set period of time.

[0256] In certain embodiments, the therapeutic agent may be administered in one or more doses over a series of time periods (e.g., over a 12-hour or 24-hour period), which together constitute a "unit dose." In other embodiments, each individual dose may be considered a "unit dose."

[0257] Therapeutic compositions and / or medicaments of the present disclosure can be administered in multiple doses (e.g., 2, 3, 4, 5, 6 or more doses) over a suitable treatment period, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, weeks, months, or years, as deemed appropriate. The frequency of administration of the therapeutic agents and / or pharmaceutical compositions described herein can be determined by one of skill in the art, but can be, for example, once daily, twice daily, once weekly, once every two weeks, once monthly, once every two months, once every three months, once every six months, etc.

[0258] Furthermore, according to the therapeutic treatment regime that can be devised, the unit dosage can vary depending on the stage or type of treatment, as well as taking into account the size and / or age of the subject, or the type of disease intended to be treated. For example, in some embodiments, the first dosage of a therapeutic agent or pharmaceutical composition of the present disclosure administered to a subject can be lower or higher than any second, third, or subsequent dosage that may be administered. In other embodiments, during a first administration period, the dosage can be lower or higher than the dosage administered during a second, third, or subsequent administration period. One skilled in the art can determine the duration of any such administration period, for example, about one day, about one week, or about one month, or any intermediate or longer period.

[0259] In some examples, the subject being treated may be administered one or more additional therapeutic agents or may undergo one or more additional conventional therapies (such as radiation therapy or chemotherapy) or complementary therapies.

[0260] Combination therapy In various embodiments, the uses and methods of the present disclosure involve administering a therapeutic agent of the present disclosure (e.g., a polypeptide, polynucleotide, vector, or cell (e.g., a recombinant bacterial cell disclosed herein)), or a pharmaceutical composition containing a therapeutic agent of the present disclosure, in combination with one or more additional therapies or therapeutic agents. The one or more additional therapies or therapeutic agents can be administered simultaneously, sequentially, or separately (before or after) a therapeutic agent according to the present disclosure.

[0261] The additional therapeutic agent may be another therapeutic agent according to the present disclosure, such that two therapeutic agents of the present invention are administered in combination. Other examples of additional therapies or therapeutic agents may include, but are not limited to, chemotherapy, radiation, oncolytic virotherapy with an additional virus, treatment with an immunomodulatory protein, an anti-cancer agent, an anti-inflammatory agent, or any combination thereof.

[0262] In certain embodiments, the additional therapeutic agent is an anti-cancer agent or cancer therapy. Anti-cancer agents may include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, cytokines, immune checkpoint inhibitors, anti-angiogenic agents, apoptosis inducers, anti-cancer antibodies, and / or anti-cyclin-dependent kinase agents. Cancer therapies may include chemotherapy, biological therapy, radiation therapy, immunotherapy, hormonal therapy, anti-vascular therapy, cryotherapy, toxin therapy, and / or surgery, or a combination thereof.

[0263] In various embodiments, cancer treatment may include surgery. Such surgery may include resection, in which all or part of the cancerous tissue is physically removed (e.g., by resection) and / or otherwise destroyed from the subject. Tumor resection is the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery). In some embodiments, it may be determined that a tumor (or a portion thereof) should be removed by resection, or alternatively, the tumor may be determined to be unsuitable for resection. In such situations, embodiments of the present invention may include treating a subject with a therapeutic agent or composition of the present disclosure to improve the results of the resection or to enable the tumor or a portion thereof to be resected. Thus, therapeutic treatment of the present disclosure may increase the resectability of the tumor, for example, due to a reduction in tumor margins or by removal of invaded areas. Additional treatment following resection may serve to remove minimal residual disease at the tumor site.

[0264] kit In embodiments, the present disclosure provides kits for administering the single-chain dimeric cytokines, encoding polynucleotides, vectors, cells, or other agents (such as bacteria or bacteriophages) described herein. In certain embodiments, the kits of the present disclosure can include a "therapeutic agent" such as a single-chain dimeric cytokine-encoding polynucleotide, vector, cell (e.g., bacteria), or bacteriophage, or a (pharmaceutical) composition comprising such a therapeutic agent, as described above. In certain embodiments, the kits of the present disclosure can further include one or more components, such as instructions for use, devices, additional reagents, and components such as tubing, containers, syringes, etc., for carrying out the methods and therapies disclosed herein. In various embodiments, the kits of the present disclosure can further include one or more active agents that can be administered in combination (separately, sequentially, or simultaneously) with the therapeutic agent of the present disclosure, such as at least one selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, an anti-inflammatory agent, and / or any combination thereof.

[0265] In some embodiments, kits according to the present disclosure can include one or more containers containing a therapeutic agent, one or more additional active agents, and / or any of the reagents described herein.

[0266] In some embodiments, the kit may further include an apparatus or device for administering a therapeutic agent (such as a recombinant bacterium) of the present disclosure, and / or any additional active agents, to a subject. Suitable apparatus or devices may include one or more of a hypodermic needle, an intravenous needle, a catheter, a needle-free injector, an inhaler, and / or a liquid dispenser.

[0267] The kit instructions may suitably include instructions as to what should be included in the kit and how it should be properly used, for example, how the various components of the kit should be administered to an individual, including timing, concentrations and amounts, appropriate methods of administration and how / if an individual should be monitored during use / treatment. [Example]

[0268] Example 1: Generation of IL-10 variants and single-chain dimeric IL-10 Unless otherwise indicated, commercially available reagents and standard techniques in molecular biology and biochemistry were used. Materials and Methods The following procedures used by the applicants are described in Sambrook, J. et al., 1989, supra: analysis of restriction enzyme digestion products on agarose gels and preparation of phosphate buffered saline. General purpose reagents, oligonucleotides, antibodies, chemicals, and solvents were purchased from Merck. Enzymes and polymerases were obtained from New England Biolabs (NEB) unless otherwise indicated.

[0269] method Bacterial strains and culture conditions When used, the WT Mycoplasma pneumoniae M129-B7 strain (ATTC 29342) and all M. pneumoniae strains generated in this study are listed in Table 1. Mycoplasma strains were grown at 37 °C under 5% CO in tissue culture flasks (Corning) containing Hayflick liquid medium. Hayflick contains 800 ml of incomplete medium A (20 g PPLO broth [Difco, #255420], 30 g HEPES [final 100 mM], 25 ml 0.5% phenol red solution [Sigma, #P3532]), 200 ml heat-inactivated horse serum (Life Technologies, #26050088), 20 ml sterile-filtered 50% glucose, and 100 mg ampicillin ml -1 Stock 1 ml (final concentration 100 μg ml -1Hayflick broth was prepared by mixing 0.8% bacto-agar (Difco) with 100% ethanol (100%) of ... -1 Cm: 20 μg ml -1 Pseudomonas aeruginosa PAO1 was grown in Trypticase Soy Broth (TSB, T8907) agar plates at 37°C. For cloning, Escherichia coli NEB® 5-alpha High-Efficiency strain (New England Biolabs) was used; it was grown at 37°C in LB broth or supplemented with ampicillin (100 μg ml -1 ) and grown on LB agar plates supplemented with .

[0270] cell line HAFTL cells are a Ha-ras oncogene-transformed murine pre-B cell line derived from fetal liver (47). BLaER1 is a human B cell precursor leukemia cell line (48); both cell lines were kindly provided by Professor Thomas Graf. The THP-1 cell line is a monocytic cell line derived from peripheral blood purchased from the American College of Cardiology (ATCC). Cells were grown in RPMI (12633012, GIBCO) supplemented with 2 mM L-glutamine (25030081, GIBCO), 100 U / mL penicillin + 100 ng / mL streptomycin (15140122, GIBCO), and 20% FBS (10270-106, GIBCO).

[0271] Plasmid generation All plasmids generated in this study were analyzed by the Gibson method. 47Where necessary, gene synthesis (gBlock double-stranded fragments) and oligonucleotide synthesis were performed by Integrated DNA Technologies (IDT) Corporation. Gene amplification was performed using Phusion DNA polymerase (Thermo Fisher Scientific). In some examples, promoter sequences for IL-10 and foldikine_10 variants were generated using the P3 synthetic promoter. 48 and used a secretion signal designated S142 (U.S. Patent Application No. 15 / 553,552, Garrido et al., 2021). The promoter used for the IL-22 variants and IFN-lambda was pSynL (GTTAACGATTAAGATCAAAAAGTGCCTGGTATCGTAAATAATATTGTATAATTAAAAAAGAAT; SEQ ID NO: 339). A detailed list of the plasmids, strains, and their gene portions is also provided in Table 1. All plasmids were verified by PCR and Sanger sequencing (GATC biotech).

[0272] Generation of M. pneumoniae strain CV8 CV8 is a derivative of the attenuated CV2 strain (Δmpn133, Δmpn372) in which mpn051 has been replaced by the gpsA gene from Mycoplasma penetrans. 58 The enzymes encoded by these two genes are involved in the oxidation of glycerol-3-phosphate, producing the metabolic by-product NADH in the case of the GpsA enzyme, or H2O2 in the case of the GlpD enzyme encoded by mpn051; this H2O2 production is crucial for the cytotoxic effect of M. pneumoniae. The CV8 strain also carries a second gene deletion in which the puromycin resistance gene (introduced to select for the insertion of the GP35-encoding gene) has been removed from its position in the genome (mpn560). Genome editing tools adapted for mycoplasmas based on the ssDNA recombinase GP35 49As substrates for recombination, we used long stretches of ssDNA, prepared as previously described. 50 .

[0273] The sequences of the primers and plasmids used to generate long stretches of ssDNA are shown in Table 4.

[0274] KK For the expression and secretion of different versions of human cytokines, such as IL-10 (hIL-10), and various foldikine constructs in bacteria, e.g., M. pneumoniae, transposon-based vectors were generated by fusing appropriate promoter sequences to cytokine and foldikine constructs containing the appropriate signal peptides. For the expression and secretion of IL-10 variants and foldikines with N-terminal IL10 sequences, the synthetic promoter P3 was used with the N-terminal signal peptide 142 (s142) for secretion (see Table 1 for gene information for each construct). For the expression and secretion of IL-22 and IFN-lambda variants and the corresponding foldikine constructs, the synthetic promoter pSynL was used, fused to the signal peptide (s142). A tetracycline resistance marker was also used (see Methods herein).

[0275] To generate M. pneumoniae-expressing cells, different plasmids were transformed into either WT or CV8 M. pneumoniae. 2Cells were grown in tissue flasks (Corning) and incubated at 37°C under 5% CO2 until late logarithmic growth phase. Cells were washed twice with pre-chilled electroporation buffer (272 mM sucrose, 8 mM HEPES, pH 7.4), resuspended, scraped, and passed 10 times through a 25-gauge (G25) needle. A 50 μl aliquot of cells in a 0.1 cm cuvette containing 2 μg of the desired plasmid was kept on ice for 20 minutes. Electroporation settings were set to 1250 V / 25 μF / 100 Ω in a BIO-RAD Gene Pulser Xcell instrument. After pulsing, 420 μl of fresh Hayflick was added to the cells. From this culture, 80 μl was aliquoted into a 0.1 cm cuvette containing 2 μg of the desired plasmid. -1 25cm containing 5ml of fresh Hayflick (containing chloramphenicol or tetracycline) 2 From the different M. pneumoniae expressing cells, 50 μl of stock inoculum was added to a 25 cm 2 flask containing 5 ml of fresh Hayflick. 2 The cells were added to tissue flasks (Corning) and grown for 48 hours. The culture medium was then collected, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was stored in 1 ml aliquots at -80°C.

[0276] Disulfide identification by mass spectrometry From the aliquots originally prepared and stored at -80°C, 25 μL of the initial inoculum of hIL-10, MutSC1, or MutSC2 was added to a T25cm plate containing 5 mL Hayflick medium supplemented with Cm. 2 Flasks were inoculated in duplicate. After 24 hours, the medium was discarded from one of the duplicates and replaced with version 13 defined medium (see, e.g., EP 20382261). After 36 hours, the supernatants from both replicates were collected and processed for proteomic analysis. No differences in protein abundance were observed under either set of conditions. For further characterization, samples were grown in version 13 defined medium.

[0277] The supernatant was collected, concentrated using a 3K MWCO column, and centrifuged with 700 μL of supernatant at 12,000 rpm for 15 minutes. Next, 200 μL of the supernatant was added to the same MWCO column, and the mixture was centrifuged at 12,000 rpm for 60 minutes at 4°C. The final concentrated volume of approximately 100 μL was divided into two portions: one for BCA quantification and the other for MS analysis. Each of the newly concentrated aliquots was resuspended in 100 μL of 4% SDS, 0.1 M HEPES. The supernatant lysate was quantified by BCA using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) and sent to the UPF MS Facility for analysis. For sample preparation, cysteines were labeled by alkylation with N-ethylmaleimide (30 nmol, 37°C, 60 minutes), and the samples were then precipitated with 6 volumes of cold acetone. The pellet was dissolved in 6 M urea / 200 mM ammonium bicarbonate, and the sample was reduced with dithiothreitol (30 nmol, 37°C, 60 min) and alkylated with iodoacetamide (60 nmol, 25°C, 30 min) in the dark.

[0278] Samples were then digested in the following manner, using 200 mM ammonium bicarbonate for all dilutions: for hIL-10 and MutSC2, the resulting protein extracts were first diluted in 2 M urea and then incubated with endoproteinase Lys-C (endo-LysC) (1:100 w:w, 37°C, 6 h, Wako, cat# 129-02541); samples were then diluted 2-fold (1:100 w:w) and digested with i) trypsin (37°C, overnight; Promega cat# V5113); ii) chymotrypsin (25°C, overnight; Roche diagnostics cat# 11418467001); or iii) GluC (25°C, overnight; Sigma Aldrich cat# P6181). Samples were first diluted in 6M urea for trypsin digestion (1:100 w:w, 37°C, overnight, Promega cat# V5113), followed by GluC digestion (1:100 w:w, 25°C, overnight, Sigma Aldrich cat# P6181).For MutSC1, the combinatorial digestion mix was: i) endo-LysC + trypsin: diluted in 2M urea for endo-LysC (1:100 w:w, 37°C, 6 hours, Wako, cat# 129-02541), then diluted 2-fold for trypsin (1:100 w:w, 37°C, overnight, Promega cat# V5113); ii) endo-LysC + chymotrypsin: diluted in 2M urea for endo-LysC (1:100 w:w, 37°C, 6 hours, Wako, cat# 129-02541), then diluted 2-fold for trypsin digestion (1:100 w:w, 25°C, overnight, Roche diagnostics cat# 11418467001); iii) LysC + GluC: diluted in 2 M urea for endo-LysC (1:100 w:w, 37°C, 6 h, Wako, cat# 129-02541), then diluted 2-fold for GluC (1:100 w:w, 25°C, overnight, Sigma-Aldrich cat# P6181); and iv) trypsin + GluC: diluted in 6 M urea for trypsin digestion (1:100 w:w, 37°C, overnight, Promega cat# V5113), then digested with GluC (1:100 w:w, 25°C, overnight, Sigma-Aldrich cat# P6181). In all cases, digested peptide samples were acidified with formic acid and desalted on a MicroSpin C18 column (The Nest Group, Inc.) prior to LC-MS / MS analysis.

[0279] The following protocol was used for chromatography and MS analysis: Samples were analyzed using an LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled to an EASY-nLC 1000 (Thermo Fisher Scientific (Proxeon), Odense, Denmark). C with a particle size of 5 μm was used. 18Peptides were loaded onto a 2 cm NanoTrap column with an internal diameter of 100 μm packed with 1.9 μm C18 particles (Thermo Fisher Scientific), and separated by reversed-phase chromatography using a 25 cm column with an internal diameter of 75 μm packed with 1.9 μm C18 particles (Nikkyo Technos Co., Ltd. Japan). The chromatographic gradient was 250 nl min -1The flow rate was 0.01, starting with 93% Buffer A and 7% Buffer B for 5 min and gradually increasing to 65% Buffer A and 35% Buffer B at 120 min. After each run, the column was washed with 10% Buffer A and 90% Buffer B for 15 min. Buffer A, 0.1% formic acid in water; Buffer B, 0.1% formic acid in acetonitrile. The mass spectrometer was operated in positive ionization mode with the nanospray voltage set at 2.1 kV and a source temperature of 300 °C. An Ultramark 1621 was used for external calibration of the FT mass spectrometer prior to analysis, and internal calibration was performed using the background polysiloxane ion signal at m / z 445.1200. Measurements were acquired in data-dependent acquisition (DDA) mode, using a full MS scan with one microscan at a resolution of 60,000 over a mass range of m / z 350–2000, with detection in the Orbitrap. The autogain control (AGC) was set to 1E6, dynamic exclusion (60 s), and charge state filtering was enabled to disqualify singly charged peptides. During each cycle of DDA analysis and after each survey scan, the top 20 most intense ions with multiple charged ions above a threshold ion count of 5,000 were selected for fragmentation. Fragment ion spectra were generated via collision-induced dissociation (CID) at a normalized collision energy of 35% and acquired on an ion trap mass analyzer. The AGC was set to 1E4, with an isolation window of 2.0 m / z, a 10 ms activation time, and a maximum injection time of 100 ms. All data were acquired with Xcalibur software v2.2. Digested bovine serum albumin (New England Biolabs cat# P8108S) was analyzed between each sample to avoid sample carryover and ensure instrument stability; QCloud (53) was used to control the longitudinal performance of the instrument during the project.

[0280] Data were analyzed using the Proteome Discoverer software suite (v2.0, Thermo Fisher Scientific) and the Mascot search engine (v2.6, Matrix Science (54)). Data were searched against M. pneumoniae (87,071 entries) as well as hIL-10, MutSC1, MutSC2, and the entire list of common contaminants (55) and corresponding decoy entries. For peptide identification, a precursor ion mass tolerance of 7 ppm was used for the MS1 level, trypsin was selected as the cleavage enzyme, and a maximum of three failed cleavages was allowed. The fragment ion mass tolerance was set to 0.5 Da for MS2 spectra. Methionine oxidation, N-terminal protein acetylation, carbamidomethylation of cysteine, and N-ethylmaleimide were used as variable modifications. The false discovery rate (FDR) for peptide identification was set to a maximum of 5%. Peptide quantification data were obtained from the "Precursor ion area detector" node from Proteome Discoverer (v2.0) using a mass tolerance of 2 ppm for peptide extracted ion current (XIC). The resulting values ​​were used to calculate the top three compartments of proteins with unique peptides for unclassified proteins.

[0281] MutSC1 was analyzed by parallel reaction monitoring (PRM) chromatography and MS analysis. To confirm that the first cysteine ​​was carbamidomethylated, the sample was digested with endo-LysC + trypsin and analyzed by PRM using an Orbitrap Eclipse (Thermo Fisher Scientific) coupled to an Easy-nanoLC 1200 UPLC system (Thermo Fisher Scientific). Peptides were directly loaded onto an analytical column and analyzed using a 2 μm C 18Separation was performed by reversed-phase chromatography using a particle-packed 75 μm internal diameter 50 cm column spectrometer (Thermo Scientific, San Jose, CA, USA).

[0282] The chromatographic gradient started with 95% Buffer A and 5% Buffer B for 5 min at a flow rate of 300 nL / min, gradually increasing to 25% Buffer B and 75% A over 79 min, and then to 40% Buffer B and 60% A over 11 min. After each run, the column was washed for 10 min with 10% Buffer A (0.1% formic acid in water) and 90% Buffer B (0.1% formic acid in 80% acetonitrile).

[0283] The mass spectrometer was operated in positive ionization mode with a 2.4 kV EASY-Spray nanosource at a source temperature of 305 °C. A full MS scan with one microscan at a resolution of 30,000 was used over the mass range of m / z 350-1400 with detection in an Orbitrap mass analyzer. A PRM method was used for data acquisition with detection in an Orbitrap at a resolution of 60,000, with the quadrupole isolation window set at 1.4 m / z and MSMS over the mass range of m / z 300-2,000. MSMS fragmentation was performed using an HCD at 30 NCE and an automatic gain control (AGC) of 1 × 10. 5 The injection time was set to 502 ms, and the maximum injection time was set to 502 ms. Peptide masses (m / z) were defined in the mass list and shown in Table S2. For PRM data analysis, Skyline-Daily software (4) (v21.1.9.353) was used to generate libraries, which were observed as the output of MaxQuant (1.6.10) searches and predicted using Prosit (5), and the fragment compartments of each peptide were extracted.

[0284] Quantification of interleukin expression in bacterial strains To test the expression potential of each of the candidate mutants, cells were harvested in 1 ml of fresh Hayflick medium (e.g., cells were grown adherently in a flask, the medium was aspirated, and 1 ml of fresh medium was added). Cells were plated in 10 μl dots in solid Hayflick plates and grown for 10 days at 37°C and 5% CO2. Undiluted to 1 × 10 -8 Colony forming units (CFU) were counted over a range of different dilutions up to a dilution of 10. For each condition analyzed, the concentrations of the different expressed IL-10 variants were quantified by ELISA.

[0285] For IL-10 quantification, ELISA MAX Deluxe Set Human IL-10 (Biolegend; #430604) was performed according to the manufacturer's instructions. Supernatants were diluted up to 100,000-fold for this assay to fall within the detection range. As positive controls for each assay, commercial recombinant IL-10 (#200-10, Peprotech) (hIL-10r) or recombinant IL-22 (#200-22, Peprotech) was added at 10 μg ml in PBS. -1 The mutants were reconstituted in PBS and stored in 20 μl aliquots at -80°C. A commercial IL-10 Patron curve, used to estimate mutant concentrations, was fitted to a grade 2 polynomial. The same procedure was followed for IL-22 quantification by ELISA (ELISA MAX™ Deluxe Set Human IL-22, 434504, BioLegend) or interferon lambda (LEGEND MAX™ Human IL-29 (IFN-λ1) ELISA Kit, BioLegend). The IL-22 ELISA kit was used to quantify the SCIL10IL22 chimera of the foldikine-22 molecule (described below).

[0286] Isolation of blood monocytes Blood samples from four healthy donors were provided by Banc de Sang i Teixits (Barcelona, ​​Spain) under contract number 160002 approved by the Spanish Ministry of Science and Technology. Written informed consent was obtained from the donors before sample collection.

[0287] Peripheral blood mononuclear cells (PMBCs) were isolated from buffy coats using Leucosep® tubes according to the manufacturer's instructions. Briefly, 10 mL of blood sample was diluted 1:5 with phosphate-buffered saline (PBS), poured into Leucosep® tubes, and centrifuged at 800 g for 15 minutes at room temperature. PMBCs were collected using a Pasteur pipette, washed twice with PBS, and resuspended in 3 ml PBS. Monocytes were identified as CD14 + were isolated by magnetic labeling and differentiated into macrophages as previously described 54 Briefly, cells were purified using CD14 microbead positive selection and MACS separation columns (Miltenyi Biotec) according to the manufacturer's instructions.

[0288] To differentiate monocyte-derived macrophages, monocytes were plated onto glass coverslips (VWR international) in 6-well plates (Thermo Scientific) at 1.5 × 10 cells / well in warm RPMI 1640 medium (GIBCO) at 37°C for 1 h. 6 Cells / well were allowed to adhere. The medium was then diluted to 20 ng / ml -1 The medium was supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and human recombinant macrophage colony-stimulating factor (M-CSF, Peprotech) to a final concentration of 10. The cells were allowed to differentiate for 6–8 days.

[0289] Cultivation of HEK-blue™ cells The HEK-blue™ IL-10, IL-22, IFN-lambda cell line carrying the SEAP reporter construct was purchased from InvivoGen (InvivoGen, San Diego, CA, USA). Cells were grown in DMEM (Lonza, BE12-604F) supplemented with 10% FBS and 2 mM L-glutamine. HEK-IL-10 cells were supplemented with 100 μg ml-1 normocin and selection antibiotics. HEK-blue IL-22 and HEK-blue-IFN-lambda cells were supplemented with blasticidin, puromycin, and Zeocin™. Cells were passaged when they reached 70% confluence according to the manufacturer's recommendations.

[0290] Testing the activity of engineered candidates in HEK-blue™ cells After supernatant quantification by ELISA (see Methods), 200 μl aliquots of the different supernatants were stored at −80°C. Fresh Hayflick medium was used to dilute the samples to the following concentrations (30, 15, 7.5, 3.75, 1.88, 0.94, 0.47, and 0.23 ng ml -1 ) were prepared for each mutant candidate and also for the recombinant protein. The volume of supernatant required was determined by ELISA quantification and the volume was adjusted according to fresh Hayflick medium to reach the concentrations mentioned above in 20 μl supplement.

[0291] In parallel, HEK-blue™ IL-10, IL-22, or IFN-lambda cell suspensions were cultured at 280,000 cells ml in pre-warmed DMEM (no antibiotics) supplemented with 10% FBS, 2 mM L-glutamine. -1The solution was prepared in a 96-well plate (Nunc Microwell, ThermoFisher Scientific, #167008) at 37°C. Then, 180 μl per well was seeded into a 96-well plate (Nunc Microwell, ThermoFisher Scientific, #167008). From this, 20 μl of each "candidate aliquot" or recombinant protein at a fixed concentration prepared in Hayflick medium was added, and the cells were maintained at 37°C and 5% CO2 for 24 hours (induced HEK cells). After 24 hours, 180 μl of QUANTI-Blue Solution (Alkaline phosphatase detection medium, #repqbs, InvivoGen) was mixed with 20 μl of induced HEK cells in a new 96-well plate. The cells were then incubated for 60 minutes at 37°C, and the absorbance (630 nm) was measured in a Tecan i-control 2.0.10.0 spectrophotometer.

[0292] Generation of single-chain (SC) molecules using ModelX software The proposed SC-IL10 variant was generated by rewiring the X-ray IL-10 dimer structure (PDB: 1y6k, 2ilk). Novel connectivity was intelligently designed with the aid of the ModelX tool suite. The ModelX Bridging command (Cross-Linking mode) was used; it connects all geometrically compatible fragments from a custom protein fragment library (PepXDB_5k) to pairs of residues selected as anchors. The Bridging command allows users to select different peptide lengths; the output is an ensemble of bridged models in which linkers / connections with forbidden phi and psi dihedral angles in the Ramachandran plot are discarded.

[0293] If the peptide is longer than the numerical positions between the anchors, Bridging renumbers the excess residues with residue (res) codes that are not recognized by FoldX in further modeling steps. For this reason, the SC-IL10 design required a numerical rearrangement of the monomer protein residues in the dimer to allow for a numerical 18-amino acid-long gap between the regions to be connected. This is crucial for the extreme case where anchoring residues are adjacent to the numerical gap. To cover that situation, renumbering was performed to create a gap of 18 numerical positions around the regions to be connected, which was sufficient to allow length fragments ranging from 6 to 20 residues without using residue codes. The difference between 18 and 20 is that anchoring points are required for the algorithm to position the bridge, but they are replaced with the terminal residues of the peptide found. Bridging also replaces all edge residues between the anchors with residues from the peptide. For clarity, templates generated before using bridging are referred to as "sewing patterns" or "fusion" patterns (e.g., Figures 3-7, 12, and 25-28). Because numerical gaps are not spatial gaps, bridging can also allow for smaller peptide fragments to create non-contiguous numbering.

[0294] Once the sawing / fusion patterns were generated, large-scale linker screening was performed by running the model through the Bridging algorithm using exhaustive combinations of anchoring points in overlapping sliding windows around the sides of the regions to be joined. Every window was queried for different peptide lengths (6-20 aa). The bridging model was generated using FoldX. 30 The side chains were repaired using the RepairPDB command, and the resulting models were ranked by global energy (FoldX Stability command).

[0295] The generation of MutSC1 was accomplished in several steps: residues 12'-17' (note that residues and atoms of monomer 2 are depicted by ') were deleted because they point away from the C' terminus and do not contact receptor R1 in the x-ray crystal structure; and residues 18'-156' were renumbered as 169-307 and joined with the first monomer sequence to create a single-chain molecule, creating sewing / fusion pattern 1. We selected residues 147 (Ile) and 169 (Asn) as the best anchoring points, which we then connected using a 17-amino acid fragment (residues 73-89) from PDB 2wyu. The original sequence of the selected bridge segment in 2wyu was LDALFAGVKEA-FGGLD-Y (SEQ ID NO: 340). Residues LDALFAGVKEA (SEQ ID NO: 341) and the final Y structurally overlapped with the wild-type IL-10 structure. Therefore, we decided to revert all of these mutations to the original IL-10 sequence, except in cases where the new residue was predicted to improve protein stability. In this case, stability was predicted to be improved by the Asn169 to Tyr mutation, so this mutation was retained.

[0296] Estimating values ​​of dynamic data Bacterial cell culture supernatants were diluted in Hayflick medium to adjust IL-10, IL-22, IFN-lambda, or SCIL10IL22 concentrations to 0.0243 ng ml, encompassing the dynamic range described for HEK-blue™ (see Methods). -1 ~30ng ml -1 To calculate the molar concentration for each IL-10 variant, all IL-10 variants (containing the IL-10 ORF) were assumed to have a molecular weight of 36 KDa, with the exception of MutM, which is a synthetic monomer described in (Josephson et al., 2001) and therefore has a molecular weight of 18 KDa.

[0297] The molecular weight of the foldikine-22 variant is also 36 KDa, but two active domains are considered. For SCIL10IL22, the molecular weight is 36 KDa, but one active site is considered for each interleukin. The change in absorbance at 630 nm due to the different interleukin test concentrations was fitted to a saturation binding model using the following equation: Y=B Max×Xh / (K D h+Xh);

[0298] In this equation, K D is the apparent dissociation constant (because the number of active receptors per cell is unknown), h is the Hill slope, and B Max is the saturating signal. This equation assumes specific binding only; all nonspecific signal was subtracted. If a saturating signal was not reached, B Max was set accordingly for each experiment.

[0299] At least three biological replicates (and a maximum of six biological replicates) were used, with two technical replicates for each condition. D Bmax was fixed to estimate the parameters. Different experiments were analyzed independently and fitted using GraphPad Prism 9 software, Excel or Kaleidagraph.

[0300] Characterization of IL-10-expressing CV8 and WT M. pneumoniae cells For IL-10-expressing WT and CV8 cells, 50 μl of stock inoculum was added to a 25 cm well containing 5 ml of fresh Hayflick. 2Cells were added to tissue flasks (Corning) and grown for 48 hours. The culture medium was then collected and centrifuged at 10,000 rpm for 10 minutes, and the supernatant was stored at -80°C for further quantification. Cells were harvested by scraping in 1 ml PBS and centrifuged at 10,000 rpm for 5 minutes at 4°C; the pellet was resuspended in 1 ml PBS and centrifuged at 10,000 rpm for 5 minutes at 4°C, and the supernatant was discarded. The pellet was then disaggregated in 200 μl of lysis buffer (4% SDS, 0.1 M HEPES). Protein lysates were quantified using the Pierce BCA Protein Assay Kit (23227, Thermo Fisher Scientific) according to the manufacturer's protocol. In parallel, the levels of IL-10 variants in the supernatant were quantified by ELISA as previously described. Secretion capacity was normalized to biomass (protein content of the whole culture) for each strain. Two biological replicates and two technical replicates were performed for each assay.

[0301] Mouse experiments / In vivo experiments Six- to eight-week-old C57Bl / 6 female or male mice (18–20 g) were purchased from Charles River Laboratories (France) and housed under pathogen-free conditions in the PRBB animal facility (registration number B9900073). Animal handling and procedures were in accordance with current European (Directive 86 / 609 / EEC) and national (Real Decreto 53 / 2013) legislation, as well as FELASA and ARRIVE guidelines, and were approved by the Animal Experimentation Ethic Committee (Comite de Etica, Experimentacion Animal y Bioseguridad) of the Barcelona Biomedical Research Park (PRBB) and local government approval.

[0302] M. pneumoniae pulmonary infection in mice T75cm containing 25ml Hayflick liquid medium 2M. pneumoniae WT and CV8 strains were cultured in flasks at 37°C and 5% CO2. After 3-4 days of incubation (when the medium changed color), the supernatant was removed, and the bacterial cultures were washed three times, scraped, and collected in 10 ml of sterile PBS. Approximately 0.5-1 × 10 8 CFU ml -1 (about 0.5~1×10 7 100 μl of bacterial suspension (WT or CV8) containing 100 μL of WT or CV8 bacteria (CFU / mouse) was used for intratracheal (IT) infection in mice previously anesthetized with 2% isoflurane (ISOFLO, Covegan). Two or four days after inoculation (2 or 4 dpi), animals were sacrificed by cervical dislocation, and the lungs were removed and processed. The left lung was weighed and previously 55 The cells were homogenized at 1:10 (wt / vol) in PBS in sterile individual bags (Stomacher 80, Seward Medical) for bacterial load quantification as described (detection limit = 0.7–0.9 Log 10 CFU / lung). (i) The right lung was insufflated with 50 μl of 10% methanol-stabilized formalin (Panreac) for histopathological analysis (inferior aortic lobe); and (ii) the right lung was frozen in N2 for RNA extraction and stored at -80°C until use (inferior lobe). Control animals were inoculated with 100 μl of vehicle solution and treated in parallel. Infections were performed in groups of at least five mice per strain (n≧5).

[0303] Acute pulmonary infection of P. aeruginosa PAO1 in a C57Bl / 6 mouse model P. aeruginosa strain PAO1 was grown in TSB agar plates (#BD211825, Fisher Scientific) at 37°C in 5% CO for 14-16 hours. Bacteria were collected in PBS and the suspension was analyzed at an optical density of 600 nm (OD 600 ) = 0.5. For dose-response assays, approximately 1 × 10 5 or 1 x 10 6 CFU ml -1 (approximately 1×10 4 or approximately 1 x 10 5100 μl of the pulmonary sputum containing 100 μL of PBS ... 5 Lungs infected with 10 CFU were infected with 10 CFU at both time points analyzed. 4 The lungs infected with 100% IFN-γ-glucan (100%) showed a higher bacterial burden than those infected with 100% IFN-γ-glucan (100%) CFU (Fig. 17A). The inflammatory response was predominantly localized at 24 hpi (Fig. 17B). 5 Lungs infected with 10 CFU were either uninfected (PBS) or 10 4 Compared with the CFU-infected group, there was a significant increase in the expression of tnf-a, kc, mip-1a, mcp-1 and il-1b (all p<0.05).

[0304] Immunomodulatory effects of hIL-10-expressing CV8 in vivo To evaluate the immunomodulatory effects of hIL-10-encoding mycoplasma strains, approximately 2 × 10 6 CFU ml -1 (about 1×10 5 C57Bl / 6 mice were infected intratracheally (IT) with 50 μl of P. aeruginosa PAO1 bacterial solution containing approximately 2 × 10 CFU / mouse; 2 hours later, approximately 2 × 10 8 CFU ml -1 (about 1×10 7Mice were infected IT with 50 μl of mycoplasma strain containing 1000 ng / mouse (Figure 19A). Control animals were treated in parallel with either (i) 2 μg of human IL-10 recombinant protein (hIL-10r) (#200-10, Peprotech) or (ii) sterile PBS (vehicle solution). 24 hours after PAO1 infection, animals were sacrificed, and lungs were aseptically removed for experiments as described above.

[0305] Total lung RNA extraction and real-time quantitative PCR (RT-qPCR) analysis Right lungs stored at -80°C were homogenized using an Ultra-Turrax (IKA) and total RNA was isolated using the RNeasy® Mini Kit (Qiagen) according to the manufacturer's instructions. RNA concentration was measured spectrophotometrically using a Nanodrop One (Thermo-Scientific), and sample RNA integrity was confirmed by 1% agarose gel electrophoresis. RNA samples with absorbance readings at 260 nm / 280 nm of 1.8 to 2.1 were used.

[0306] Complementary DNA (cDNA) from whole lung cells was synthesized from total RNA (1 μg) using SuperScript II Reverse Transcriptase Reagent (Invitrogen). PCR amplification was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara), and fluorescence was analyzed using the AriaMx Real-Time PCR System (Agilent Technologies). Primer pairs for detecting gapdH, tnf-a, kc, mip-1a, mcp-1, inf-y, il-1b, and nf-kb2 are shown in Table 4. -ΔΔCt The relative abundance of mRNA in each experimental condition was determined using the α method, using gapdH as an endogenous control, and normalized to the value obtained in the PBS group as follows: ΔCt = mean Ct of analyzed genes − mean Ct of gapdH; ΔΔCt = ΔCt of treated group − ΔCt of PBS group; fold change in mRNA expression = 2 -ΔΔCt .

[0307] Histological and immunohistochemical (IHC) analysis of lung samples After necropsy, the inferior aortic lobes were fixed in formalin (Panreac), trimmed, and automatically processed in an ethanol series and xylene (Leica TP1020 Tissue Processor).Tissues were then embedded in paraffin (Tec2900 Histo-Line Workstation), sectioned at 4 μm (Leica RM2235 Microtome), and stained with hematoxylin and eosin (H&E) according to standard procedures (Leica Autostainer XL).

[0308] Preparations were manually mounted on glass slides and examined by light microscopy. Tissues were evaluated by a trained veterinary pathologist (Histopathology Facility Manager of the Institute for Research in Biomedicine-IRB Barcelona). Alveolar cell infiltrates and perivascular-peribronchial / peribronchiolar infiltrates were used as histopathological parameters to establish a quantitative score (0-5: 0, absent; 1, minimal; 2, mild; 3, moderate; 4, severe; 5, very severe); the final score for each sample was the sum of both parameters. For IHC, a Leica Bond RX was used to detect rabbit monoclonal anti-F4 / 80 (D2S9R) XP (#70076S, Cell Signaling) as a marker for macrophages. 56 , or rabbit polyclonal anti-neutrophil elastase (#Ab68672, Abcam) as a marker for neutrophils. 57Paraffin-embedded tissue sections were stained with . The specificity of the staining was confirmed by staining with a rabbit IgG isotype control (#Ab27478, Abcam). Bright-field images were acquired with a NanoZoomer-2.0 HT C9600 digital scanner (Hamamatsu) equipped with a 20x objective. All images were visualized using NDP.view 2 U123888-01 software (Hamamatsu, Photonics, France) with gamma correction set to 1.8 in the image control panel. The output results from image analysis were expressed as % positive cells [% = 100 × (positive cells / positive cells + negative cells)].

[0309] statistical analysis In all cases, statistical significance was set at p<0.05. Analyses were performed using the Prism software (GraphPad Software) statistical package and are detailed in the legends of each figure.

[0310] material List of IL-10 variants tested and described in the following sections: hIL-10r (commercial IL-10 protein, a naturally occurring swapped domain dimer) WT-IL10 / IL-10 ORF (wt swapped domain dimeric IL-10 expressed and secreted by bacteria, e.g., mycoplasmas) Mut1 to Mut3 point mutation in the wt swapped domain dimer IL-10 MutSC1 foldikine_10, a single-chain dimeric IL-10 in which an engineered peptide linker covalently joins the N- and C-termini of two molecules of the swapped dimer in one of the two 3D domains MutSC2, which is identical to MutSC1 but has the point mutations of Mut3 in each half of the foldikine sequence (with the exception of a mutation at residue 18 in the second half of the polypeptide). MutM, a monomeric version of IL10 previously reported by another group (Josephson et al., 19). MutSC1_linkercontrol19 is a foldikine in which the engineered peptide linker of MutSC1 is replaced by a poly-Gly linker of the same length (5 aa) to assess the impact of the linker composition on the functional activity of the engineered molecule. MutSC1_controlcenter21 is a control construct based on MutSC1 in which new N- and C-termini were generated by truncating one of the linkers (position Asn166) bridging the two foldikine domains. To maintain a single chain, the linker sequence used to bridge the original N- and C-termini in one of MutSC1's foldikine domains was used to bridge the two original N- and C-termini present in the original swapped domain structure. This construct is used to evaluate the impact of having only one of the two central linkers of MutSC1 foldikine, such that the 3D domain pair is bridged by only one linker sequence instead of two. · MutIL10_polygly represents a single-chain version of dimeric IL-10 in which the N-terminus of one IL-10 monomer sequence is linked to the C-terminus of the other IL-10 monomer sequence with a 13-residue polyGly linker, as reported ( Minshawi et al., 2020 ; https: / / doi.org / 10.3389 / fimmu.2020.01794 ).

[0311] M. pneumoniae secretes functional IL-10 dimers with disulfide bridges We first tested whether M. pneumoniae can actively express complex molecules, such as human IL-10 swapped dimers with two disulfide bridges. To this end, we used previously described methods for M. pneumoniae. 58 Human IL-10 fused to the mpn142 secretion signal as described in was cloned.

[0312] We demonstrated by ELISA that the M. pneumoniae WT strain expressed IL-10 in the supernatant at a concentration of approximately 2 fg / cell after 48 h of culture (1 × 10 9 (equivalent to 2 μg from a culture of cells; Table 5). We confirmed by mass spectrometry (MS) that both disulfide bridges were generated in the expressed hIL-10 (Table 2), indicating that the dimer was correctly folded. We next examined primary blood, CD14 isolated from four independent healthy blood donors. + Using monocytes, we (i) modulate the human primary macrophage anti-inflammatory response activation program, and (ii) activate STAT3 by phosphorylation of Tyr705. 35 The functionality of the produced IL-10 was tested for potency (Methods).

[0313] M. pneumoniae expressing wild-type (WT) IL-10 was grown for 2 days, and the supernatant was added to cell cultures containing approximately 1 million circulating monocytes (1:5 ratio). As a positive control, human IL-10 recombinant protein (hIL-10r) was added at 20 ng ml -1 As expected, commercial hIL-10r treatment of monocytes enhanced the expression of anti-inflammatory markers (i.e., CD16, CD163, and MerTK) as well as essential pro-inflammatory receptors (i.e., CD86 and MHC2) compared to untreated cells (Figure 9A). 36,37 IL-10 expression was reduced. Notably, the results using IL-10 (WT_IL-10 ORF) produced by M. pneumoniae were comparable to those using hIL-10r (Fig. 9B). By Western blot, we observed phosphorylation of Tyr705 in STAT3 after 24 hours of exposure of freshly isolated monocytes to the supernatant of M. pneumoniae expressing IL-10 or hIL-10r (Fig. 9C). Together, these results demonstrated that M. pneumoniae can express functional human IL-10.

[0314] Expression of MutSC2 in and secretion of MutSC2 from eukaryotic cells For expression of MutSC2 in eukaryotic cells, Expi293F™ cells were selected.

[0315] Expi293F™ cells are human cells derived from the 293F cell line. They are maintained in suspension culture and grown to high density in Expi293™ Expression Medium. Expi293F™ cells are highly transfectable and produce superior protein yields in transient protein expression compared to standard 293 cell lines.

[0316] For expression, purified DNA and polyethyleneimine (PEI) were used to transfect Expi293F human cells (Thermo Fisher Scientific).

[0317] To identify secreted MutSC2 protein, cells were harvested 3 days after transfection, and MutSC2-containing supernatants were collected by centrifugation at 13,000 rpm for 15 minutes. MutSC2 protein was purified on a Hitrap-Ni column in an automated fast protein liquid chromatography (FPLC; Äkta avant), concentrated through a 10 kDa Amicon centrifugal filter unit (EMD Millipore), resuspended in phosphate-buffered saline (PBS), and stored at -80°C.

[0318] Expression of MutSC1 in and secretion of MutSC1 from Escherichia coli Three pCoofy vector variants (KanR), encoding IL-10 WT, MutSC1, or MutSC1-his tag, were transformed into E. coli BL21. Single colonies transformed with each vector variant were picked and grown overnight at 37°C in LB medium supplemented with kanamycin. When the culture density reached approximately 0.5 OD, the culture was cooled to room temperature.

[0319] From the culture, 1 ml was used for CFU counting in LB plates; 4 ml of the culture was induced with 0.5 mM IPTG and incubated overnight at 20°C. The culture was then centrifuged at 3,500 g for 20 minutes, and the supernatant was collected for ELISA quantification. The cell pellet was resuspended in 300 μl of M-PER protein extraction reagent (Thermo Scientific). IL-10 quantification was performed using a human deluxe IL-10 ELISA kit (Biolegend), and the functionality of the IL-10 variants was checked using HEK-Blue10 reporter cells (InvivoGen).

[0320] Expression of MutSC1 in and secretion of MutSC1 from Lactococcus lactis The Lactococcus lactis strain (NZ9000 - pepN::nisRK;) and transformation vector (pNZ8124) were purchased from MobiTec (Germany). Cells were grown as directed by the manufacturer.

[0321] For electroporation, 40 μl of cells were transfected with 200 ng of PNZ8124 vector encoding IL-10 WT or MutSC1. Electroporation settings were set at 2,000 V, 25 μF, and 200 Ω in a Biorad Genepulser. Next, 1 ml of G / L-M17B + 20 mM MgCl2 + 2 mM CaCl2 was added. The cuvette was kept on ice for 5 minutes and incubated at 30°C for 1–1.5 hours. 100 μl of the culture was plated on M17 agar containing glucose and 10 ng / ml chloramphenicol for 2 days.

[0322] For expression of different proteins, 5 ml cultures were grown overnight at 30°C and the OD of the cultures was adjusted to 0.4. Induction of 9 ml cultures was performed with 1 ng / ml nisin (stock: 1 mg / ml nisin in 0.05% acetic acid, Thermo Fisher Scientific) and incubated for 3 hours. Cells were collected by centrifugation, and expressed cytokines / foldikines in the supernatant were directly quantified by ELISA. Cell pellets were lysed with 300 μl of M-PER extraction reagent (Thermo Scientific). Serial dilutions of cells were plated on bacterial growth medium plates for CFU counting.

[0323] Manipulation of IL-10 for increased receptor affinity The potential problem of lower protein production capacity in M. pneumoniae compared to other bacteria 22 To overcome this limitation and allow beneficial reductions in the number of M. pneumoniae administered to patients, we proposed to engineer mutations in IL-10 with increased binding affinity for the high- and low-affinity receptors R1 and R2, respectively. To generate an IL-10 version with increased affinity for R1, we studied the x-ray crystal structure of hIL-10 crystallized in complex with R1 (PDB 1y6k; 2.5 Å) with good crystal resolution.

[0324] We next performed in silico mutagenesis scanning of the complex interface residues using the PositionScan FoldX command, which individually mutated all R1-contacting IL-10 amino acids to each of the other 19 natural amino acids. 31Based on this analysis of sequence conservation across different species (Figure 15), we identified a set of non-conserved residues (see, e.g., Table 1) that, when mutated and modeled using the FoldX software program, improved binding to R1 without degrading protein stability (Table 6). Using selected mutations, we modeled two multiple mutants (Mut1 (SEQ ID NO: 6) and Mut2 (SEQ ID NO: 7)) in which position 31 of IL-10 was changed from Ser to Lys or Arg, respectively (Figure 10). We further checked that the mutations were independent of the structure used by modeling them in other structures of the IL-10 apo form (PDB 2ilk; 1.6 Å) or the IL-10 holo form with R1 at lower resolution (PDB 1j7v, 2.9 Å) (Table 6).

[0325] At the initiation of this study, no structure of IL-10 bound to R2 was available. Therefore, we investigated additional mutations in IL-10 (e.g., N18I, N92I, and K99N) that have previously been reported to potentially enhance the affinity of the interaction between IL-10 and R2. 9 The Mut1 and Mut2 mutations were combined to yield Mut3 (SEQ ID NO: 8). When these three mutations were modeled in all IL-10 structures used here, we found that they reduced the stability of IL-10 (Table 6). Quite recently, a low-resolution structure (6x93; 3.5 Å) of the complex between IL-10 and R1 / R2 became available. 10 Using this structure to model these mutations, we indeed observed an increase in the stability of IL-10 and an increase in the predicted binding energy to R2 (Table 6). This increase can be explained by a local conformational change in the region of IL-10 that interacts with R2 (Figure 16).

[0326] We then investigated the mutants Mut1-3 and MutM (previously described monomeric forms of IL-10). 25) (Table 1); these were cloned into M. pneumoniae (see Figure 10 for an overview of mutation positions and interactions in the structure).

[0327] Apparent K for multiple designed mutants D Measurement of values To assess the relative affinities of different IL-10 variants, we used a reporter cell line (HEK-blue™) containing a secreted embryonic alkaline phosphatase (SEAP) reporter, which triggers a JAK1 / STAT3-mediated response when IL-10 binds to R1 and R2, resulting in SEAP expression that can be measured by absorbance at 630 nm. The intensity of the signal before saturation is proportional to the activation of STAT3, and a kinetic model 38 is the apparent K D The data can be fitted to obtain values ​​(see Methods).

[0328] Mutants Mut1 and Mut2 (designed to bind better to R1) have similar apparent K D values ​​(1.45e- 10 M and 6.45e- 11 M), but had IL-10 ORF (1.98e- 10 Mut3, which is based on Mut2 and incorporates mutations that promote R2 binding, exhibited an 8-fold increase in K compared to the IL-10-ORF. D (2.61e- 11 This resulted in an apparent increase in M ​​(Fig. 11A). 25 , the apparent K of monomeric IL-10 (MutM) D was confirmed to be lower than that of the IL-10 ORF (Fig. 11A and Table 7). The expression levels of the best mutant (Mut3) and the IL-10 ORF were similar (Fig. 11B).

[0329] Rational design of single-chain IL-10 To obtain the highest possible amount / percentage of active forms of IL-10, we needed to minimize potential sequestration of the expressed protein due to degradation, misfolding, and / or extensive multimerization (27). To achieve this, we linked the N-terminal region of one monomer to the C-terminal region of the other monomer in a swapped dimer (MutSC1) (Figure 12A). After a ModelX Bridging search using Asn18 and Lys157 as anchoring points, we obtained 541 side-chain-repaired models; we ranked these by energy using FoldXv5 (30). The best linker sequence was then determined to be N157-FGGLD-Y18 (SEQ ID NO: 316), in which Asn18 was replaced by Tyr and Lys157 by Asn, and the FGGLD sequence (SEQ ID NO: 342) was inserted. By introducing this linker, we deleted the N-terminal residues (residues 12-17; CTHFPG) in one of the two monomers in the crystal structure. We then mutated the Phe residue after N157 to Asn to improve binding to R1 (resulting in the inserted sequence NGGLD; Figure 12A) (SEQ ID NO: 318); this improved its binding to R1 as well as the overall stability of the SC molecule (Table 6). We then renumbered the sequences. Because the N-terminal sequence of one of the monomers containing Cys12 was deleted (which forms a disulfide bridge with Cys108 in the original IL-10 ORF sequence (in which the new residue 259 is deleted)), we mutated Cys108 to Asn to prevent the formation of a potential spurious disulfide bridge. Cys12 and Cys108 remained structurally equivalent to the other monomers in the swapped dimer. MutSC2 was designed by grafting the Mut3 mutations onto MutSC1 (Figure 12B).

[0330] The resulting polypeptide exhibited improved binding to R1 as well as overall stability of the single-chain molecule (Table 6). This resulted in MutSC1 (Table 1). MutSC2 was designed by grafting the Mut3 mutation into MutSC1 (Figure 12B).

[0331] Characterization of engineered IL-10 mutants in vitro Quite importantly, MutSC2 has a better apparent K than the best mutant (Mut3) in the swapped dimer. D To determine the importance of the linker sequence for the activity of MutSC1 and MutSC2, we replaced the linker sequence N-NGGLD-Y with the poly-Gly linker sequence N-GGGGG-Y (MutSC1_controlGly). The novel control mutant MutSC1_controlGly had a very significant decrease in activity, indicating the importance of short linkers having specific sequences (Figure 11C) and, potentially, sequences that create particularly beneficial structural conformations, such as those that may confer conformational stability to the folded dimeric structure.

[0332] Apparent K of MutSC1 and MutSC2 D To address whether this improvement in activation was specific to the cell type used in the assay, we evaluated IL-10 signaling (as shown by Western blot) through its activation in two different immune cell lines: the human monocytic cell line, THP1, and the murine B cell line, HAFTL. In both cases, significantly higher activation was observed for the two SC mutants compared to the WT IL-10 ORF when similar molar concentrations of the IL-10 ORFs, MutSC1 and MutSC2, were used (Figure 11D).

[0333] Apparent K DTo obtain a quantitative estimate of p-STAT3 activation, we assessed ligand-stimulated STAT3 phosphorylation by FACS in two different cell lines, mouse HAFTL and human BLaER1 (B cell precursor leukemia cell lines). Compared with the IL-10 ORF, both MutSC1 and MutSC2 mutants were significantly better at activating p-STAT3 (Figures 11E and 11F). Taken together, the engineered mutants MutSC1 and MutSC2 resulted in enhanced affinity and maximal activity in different cell types of human and mouse origin. As in HEKblue cells, MutSC2 was approximately twofold better than MutSC1.

[0334] Overall, these results supported the notion that the IL-10 swapped homodimer produced by mycoplasmas is a mixture of unfolded monomeric and active dimeric forms, and therefore converting the swapped dimer to a single-chain molecule (e.g., Figure 3) is an effective strategy to increase the amount of not only secreted protein but (more importantly) secreted active protein in bacteria.

[0335] Characterization of the CV8 chassis strain in vivo M. pneumoniae has been shown to induce an inflammatory response in the lungs of mice 17,39 To investigate this response, we investigated the effect of the nuclease MPN133 (mpn133 40 ) and CARDS toxin (mpn372) 41 is deleted, as well as glyceraldehyde-3-phosphate dehydrogenase GlpD (mpn051) 42 We engineered an attenuated strain of M. pneumoniae, referred to herein as chassis CV8 (EP 20382207.7), in which GpsA is replaced by GpsA, an enzyme with similar metabolic activity but which produces H2O rather than H2O2 (see Methods).

[0336] The pathogenicity of CV8 was tested in a mouse model of intratracheal pulmonary infection (Fig. 13). Similar bacterial burdens were observed for the WT and CV8 strains at both time points analyzed (Fig. 13A), suggesting that deletion of virulence factors does not reduce the colonization capacity of the mycoplasma strain. The pulmonary inflammatory profile was analyzed by RT-qPCR (Fig. 13B) 2 days post-inoculation (2 dpi); notably, at this time point, we have previously shown that the bacterial burden is still significant, but there is sufficient time for an inflammatory response. 58 Compared with the uninfected control group (PBS), the WT strain increased the expression of TNF-A, KC, mip-1a, mcp-1, and il-1b (all p<0.05) (Fig. 13B and Table 8). In the case of CV8, only mip-1a expression changed significantly compared with the PBS control (p<0.05), but it remained at a lower level than in the WT strain.

[0337] To confirm chassis attenuation, we analyzed histological findings in the lungs (Fig. 13C). The WT strain promoted the presence of alveolar and peribronchial / peribronchiolar infiltrates at both 2 and 4 dpi compared with the PBS control (p<0.05 for both). In CV8-infected lungs, we also observed peribronchial / peribronchiolar infiltrates, but at lower levels than those observed for the WT strain at both analyzed time points (p<0.05 for both). These results were consistent with the attenuated phenotype of CV8. We therefore generated CV8_IL-10 ORF, CV8_MutSC1, and CV8_MutSC2, and tested their immunomodulatory effects in vivo. Crucially, we observed no significant differences in the production of functional IL-10 ORF by the WT or CV8 strains (Fig. 13C), nor in the activity of any of the secreted mutants compared with the WT strain (Fig. 13D).

[0338] Engineered IL-10 variants exhibit potent immunomodulatory effects in vivo We developed and used a P. aeruginosa PAO1 infection model as an in vivo tool to study the downregulatory properties of the IL-10 variants generated in this study (see Methods) (Figure 17).

[0339] To test the anti-inflammatory properties of the engineered IL-10 mutants expressed by the CV8 chassis, we administered 1 × 10 5 CFU of PAO1 strain, and 2 hours later, 1 × 10 7 Mice were infected with CFU of mycoplasma strains (CV8, CV8_IL-10 ORF, CV8_MutSC1, or CV8_MutSC2). Control mice received PBS (as a negative control) or 2 μg of hIL-10r (as a positive control) (FIG. 14A).

[0340] First, we analyzed lung bacterial counts of PAO1 and Mycoplasma (Figure 14B). No significant differences were observed at 24 hpi between the analyzed groups, excluding that differences in inflammatory gene expression were biased by the bacterial load present in the lungs.

[0341] We next analyzed the inflammatory profile (Figures 14C and 14D). Indeed, infection with PAO1 resulted in a significant increase in the expression of inflammatory markers. Regarding the CV8 strain, we observed a mild inflammatory response when used alone (Figure 14C), which was additive when PAO1 and CV8 were administered jointly (Figures 14C and 14D). Administration of 2 μg of hIL10r protein to mice infected with PAO1 (PAO1+hIL-10r) significantly reduced the expression of TNF-α and mip-1a (both p<0.01), with similar trends for mcp-1 (p=0.1), inf-y (p=0.08), and IL-1β (p=0.06) (Figures 14C and 14D). In contrast to the results observed for the hIL-10r-treated group, CV8 encoding IL-10 WT (CV8_IL-10 ORF) did not significantly modulate the expression of the analyzed inflammatory markers (Figures 14C and 14D). Inoculation with CV8_MutSC1 or CV8_MutSC2 reduced the expression of inflammatory markers (Figure 14C). In contrast to what we observed in vitro, the properties of MutSC1 and MutSC2 differed in vivo, with MutSC1 proving to be a more effective candidate (Figures 14C and 14D). The effect of MutSC1 expressed by the CV8 strain was comparable to or even better than administration of 2 μg of hIL-10r.

[0342] Remarkably, in histopathological analysis of infected lungs, we did not observe any major differences in the analyzed tissue parameters (Table 9). 43 In all PAO1-infected groups, we observed significant cellular infiltrates with a predominance of neutrophils (PMNs) (Figures 14E and 14F). We found a significant decrease in the number of PMNs in the cellular infiltrates of animals treated with MutSC1 compared with the PAO1 + PBS group, and a non-significant decrease with MutSC2 (Figures 14E and 14F).

[0343] In summary, the combination of the M. pneumoniae CV8 chassis with engineered IL-10 variants showed immunomodulatory potential in vivo, with MutSC1 being the most promising candidate.

[0344] Consideration Systemic administration of IL-10 in humans for the treatment of different pathologies, such as inflammatory bowel disease, allergic asthma, and solid tumors, has been tested in different clinical trials, but has failed in clinical outcomes for these patients due to different issues. 4 Limitations include the low concentration of IL-10 in targeted tissues, or the precise tissue location required for IL-10 to have the desired anti-inflammatory effect but avoid systemic toxicity.

[0345] The use of live biopharmaceutical products encoding IL-10 may be an effective strategy for increasing local concentrations of IL-10 in targeted tissues, as it essentially provides a tiny, self-replicating minimal machine as a drug factory that can be easily removed with antibiotics once its action is complete. Strategies relying on local delivery of IL-10 in situ have been explored for some bacteria, such as L. lactis or Bifidobacterium bifidum in inflammatory bowel disease (IBD), with some preliminary promising results in clinical trials. 44,45 However, bacterial delivery of human proteins can also be hindered by the folding potential of the secreted molecule, the need to create disulfide bridges and / or the need to form dimers (or higher oligomers) to become functional proteins. This is the case for IL-10, which is a swapped homodimeric protein with two disulfide bridges.

[0346] In this study, we engineered human IL-10 to optimize its expression and efficacy when expressed by bacteria in vivo. The ultimate goal was to reduce the bacterial dose administered to patients while preserving the anti-inflammatory properties of IL-10, thereby reducing potential bacterial pathogenicity. To demonstrate the improved properties of our engineered IL-10 molecule ("foldikine"), we chose the lung as the target organ. This choice was based on reports showing that exogenous delivery of IL-10 ameliorates different lung diseases. 14,15,46 Based on this, the lung is a more challenging organ than the intestine due to its reduced microbiome size. 28 This was done for the following reasons.

[0347] In this study, we demonstrated that the human lung bacterium M. pneumoniae can secrete functional IL-10 with disulfide bridges that exhibits activity comparable to that of human IL-10 recombinant protein (hIL-10r).

[0348] Since lung infections, even with non-pathogenic bacteria, can trigger inflammatory responses, the fewer bacteria used, the better. Because M. pneumoniae has limited protein synthesis capacity, one way to reduce the dose applied to the lung for therapy is to increase the activity of the secreted molecule. This can be done by increasing the affinity of IL-10 for its receptor and / or improving its stability and folding ability. 9,29 .

[0349] In this study, we demonstrate a combination of intelligent design, software algorithms (ModelX, FoldX) and data from the literature (e.g., for the designed mutations present in Mut3) as well as significantly better apparent K D and even better apparent K when combining separately beneficial sets of mutations (e.g., foldikine MutSC2) DAn N- and C-terminally linked single-chain mutant (foldikine MutSC1) exhibiting the same phenotype was used to engineer multiple mutant cytokines, such as IL-10 and IL-22 variants (as described herein). Furthermore, MutSC1 and MutSC2 foldikines were expressed at approximately four-fold higher levels than the IL-10 ORF or Mut3. Higher expression and better apparent K D In combination, the WT mycoplasma strain expressing MutSC2 has approximately 60-fold higher relative activity in vitro than the IL-10 ORF strain.

[0350] Consistent with this, CV8 strains expressing IL-10 MutSC1 and MutSC2 in vivo showed a significant downregulatory effect on inflammation in P. aeruginosa infection compared with CV8 expressing the IL-10 ORF or administration of 2 μg of hIL-10r protein. However, we observed that MutSC2 performed better than MutSC1 in some assays in vitro, but the opposite trend was observed in vivo. This may be due to the apparent K difference between the two mutants. D The small difference in (approximately 2-fold) could be due to the fact that the mutations engineered in this study were designed using the human R1 receptor, which differs in some IL-10 interaction positions compared to the mouse R1 receptor on which the in vitro assay was performed.

[0351] Single-chain molecules with dimeric 3D structures not only result in increased functional protein when secreted by bacteria, but they also allow asymmetric molecules to be designed for specific cell type targeting, which can be taken into consideration when designing novel single-chain variants by structurally engineering each of the two interacting surfaces of a single molecule separately.

[0352] Together, we have generated a live biopharmaceutical chassis for bacterial cargo delivery of optimized cytokines (e.g., IL-10) in lung tissue that downregulates inflammatory responses in the context of infection, thereby removing previous constraints on effective cytokine delivery in vivo and paving the way for successful clinical trials.

[0353] With appropriate sequence and structural modifications, the foldikine concept demonstrated above for IL-10 and IL-22 foldikines can be applied to many other cytokines as described elsewhere herein, such as (A) class I helical cytokines that can be subclassified into long-helical types (IL6, IL11, IL12A, IL23A, IL27A, IL31, CLCF1, CNTF, CTF1, LIF, OSM, and CSF3) or short-helical types (IL2, IL3, IL4, IL5, IL7, IL9, IL13, IL15, and IL21, TSLP, CSF1, and CSF2); and (B) class I helical cytokines that can be subclassified into long-helical types (IL6, IL11, IL12A, IL23A, IL27A, IL31, CLCF1, CNTF, CTF1, LIF, OSM, and CSF3) or short-helical types (IL2, IL3, IL4, IL5, IL7, IL9, IL13, IL15, and IL21, TSLP, CSF1, and CSF2); It is contemplated that the present invention is applicable to class II cytokines, including IL10-like cytokines (IL10, IL19, IL20, IL22, IL24, IL26, IL28A, IL28B, and IL29), type I interferons (IFNα (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21), IFNω (IFNW1), IFNε (IFNE), IFNκ (IFNK), and IFNβ (IFNB1).

[0354] [Table 1]

[0355] [Table 2]

[0356] Table 1. This table lists the various constructs generated in this disclosure, as well as the strains in which the different constructs were performed. For each gene construct, we included the amino acid or nucleotide sequence. A list of IL-10 variants was analyzed in this study. The reference molecule is unengineered WT IL-10 as the starting background scaffold. For mutants 1 (Mut1), 2 (Mut2), and 3 (Mut3), the listed positions and chains of the mutated residues correspond to PDB numbering. The light chain retains IL-10. Variants MutSC1 and MutSC2 are single-chain molecules ("foldikines") generated in this study. MutSC2 is based on MutSC1 and incorporates the point mutations shown in Mut3. The mutations in MutSC2 overlap because two identical domains are merged, except for position 18, where the mutation at that position was not compatible with the loop engineered to create a single molecule.

[0357] [Table 3]

[0358] [Table 4]

[0359] Tables 2A and 2B. Proteomic analysis of supernatants of selected mycoplasma strains (WT IL-10 and MutSC2 expressed by M. pneumoniae WT for identification of disulfide bridges). Details of the protocols followed in the proteomics facility using three different digestion procedures are described in the Methods section: Table 2A - Sulfide WT IL-10, and Table 2B - Sulfide MutSC2. In each of them, we showed the different peptides found after digestion and their possible chemical modifications, if any. The presence of carbamidomethyl implies that the peptide cysteine ​​is part of a disulfide bridge. The peptide sequences in Table 2A are SEQ ID NOS: 19-28, respectively, from top to bottom; the peptide sequences in Table 2B are SEQ ID NOS: 27, 27, 28, 28, 20, 21, 29-32, and 22, from top to bottom. The amino acid residues indicated in square brackets form part of the peptide sequence. LysC digested with LysC protease. Chym digested with chymotrypsin. GluC digested with GluC protease. Tryp digested with trypsin. When two names appear together, it means that both proteases were used. A comparable proteomic analysis was performed for sulfide MutSC1, and potential disulfide-bridge cysteine ​​residues were identified at positions Cys12 and Cys62 (data not shown).

[0360] [Table 5]

[0361] Table 3. List of antibodies used in this study. For each of them, we include the clone description and supplier.

[0362] [Table 6]

[0363] Table 4. List of primers used in this study. For each of them, we include the directionality, sequence, source reference, and indicate any chemical modifications present (*: phosphorothioated base, BtnTg: biotinylated 5' end).

[0364] [Table 7]

[0365] Table 5. IL-10 protein secretion capacity per cell (M. pneumoniae WT) of WT and variants (IL-10 ORF, Mut3, and MutSC1) across different biological replicates (R2, R3, and R4). For each of them, we integrated the colony-forming units (CFU) counted for each flask with the IL-10 concentration estimated for each of the supernatants by ELISA (see Methods). We selected MutSC1 expression per cell (fg / CFU) in R2 as a reference to correct the expression values.

[0366] [Table 8]

[0367] Table 6. Energy calculations using FoldXv5 (see Methods). Values ​​are provided in Kcal / mol. For each mutant (Mut1, Mut2, and Mut3), the interaction energies with R1 and R2 and the stability energies in the apo form and complex were calculated independently (Summary Analysis Sheet). Energies were calculated for three independent PDB models (2ilk, 1y6k, and 6x93).

[0368] [Table 9]

[0369] Table 7. Ex vivo characterization of kinetic parameters of IL-10 variants generated in this study expressed and secreted by M. pneumoniae. Above, we show the mean and standard deviation of the different kinetic parameters from several biological replicates (below).

[0370] [Table 10]

[0371] Table 8. Gene expression of inflammatory markers in lungs infected with M. pneumoniae WT or CV8. C57Bl / 6 mice were infected with 100 μl containing 10 CFU per mouse and sacrificed 2 or 4 days post-infection (2 or 4 dpi). Control groups (PBS) were treated with 100 μl of vehicle solution. The readout of these reporter cells was measured as absorbance at 630 nm (see Methods) and is proportional to the ability of each IL-10 variant to activate the IL-10 receptors (R1 and R2). ELISA quantification and HEK-blue-derived absorbance values ​​are displayed on separate sheets for each replicate. All kinetics derived from these results (Bmax, Hill constant (h), and KD) were calculated using a kinetic model using GraphPad Prism software (see Methods). Each calculated parameter is shown in the "Integrated Table" sheet. In experiments where saturation was not reached, Bmax was set to the maximum Bmax in the same biological replicate and is indicated by an asterisk.

[0372] [Table 11]

[0373] Table 9. Histological findings observed in lungs infected with PAO1 and treated with CV8 encoding an IL-10 variant. Control mice were inoculated with PBS or hIL-10r protein. Hematoxylin and eosin (H&E) staining was used to evaluate cellular infiltration (score: 0-5), infiltrated perivascular / peribronchial (score: 0-5), and PMNs in the bronchial / bronchiolar lumen (score: 0-5). A total score (score: 0-15) was calculated by summing all parameters. Immunohistochemistry was used for quantitative determination of neutrophils (neutrophil elastase) or macrophages (FA). A lower score indicates a less inflammatory or pathogenic phenotype.

[0374] Assessment of the impact of engineered linkers in foldikine-10 Construction and Design Above, we described a novel molecule called MutSC1, which has the following amino acid sequence: MutSC1 (SEQ ID NO: 9) MTQENSCTHFPGNLPNMRLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ hhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhhhhh HA HB AENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINY hhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhhhhh HC HD HE IEAYMTMNGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEV hhhhhhhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhh HF HA HB MPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIF hhhhhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhh HC HD HE INYIEAYMTMKIRN Hhhhhhhhhh HF

[0375] The sequences highlighted in bold correspond to the peptide linkers designed to connect the C- and N-termini of the two monomer domains. In bold italics, we indicate the sequences that are part of the two wild-type structured linkers (CENKSKA; SEQ ID NO: 168) between the 3D domains. In the case of the native "swapped dimer" of IL-10, these linker sequences are the native, structured linkers that confer the characteristic bending angle (V-form) between the two domains of the foldikine (or between the left and right domains of the wild-type IL-10 dimer). To assess the impact of the linker composition on the functional activity of the engineered molecule, we generated a control in which the NGGLD sequence (SEQ ID NO: 318) was replaced by a poly-Gly linker of the same length (5 aa) (underlined). This resulted in a molecule designated MutSC1_polyglycontrol_19. > MutSC1_polyglycontrol_19 (SEQ ID NO: 63) MTQENSCTHFPGNLPNMRLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ hhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhhhhh HA HB AENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINY hhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhhhhh HC HD HE IEAYMTMGGGGGYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEV hhhhhhhh hhhhhhhhhhh hhhhhh hhhhhhhhhhhhhhhh HF HA HB MPQAENQDPDIKAHVNSLGENLKTLRLRLRRNHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIF hhhhhh hhhhhhhhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhh hhh HC HD HE INYIEAYMTMKIRN Hhhhhhhhhh HF

[0376] To assess the importance of having two linkers between each 3D domain compared to a single linker and the importance of the rigidity of the V-form at the center of the molecule, the following control polypeptides were synthesized and assayed: A) We duplicated the N-terminal linker of one 3D domain into the other foldikine domain so that each domain was formed from a continuous primary sequence, and to create new N- and C-termini for the polypeptide, we truncated one of the two linkers between the 3D domains at position Asn166 (see underlined sequences and bold italics in MutSC1 and MutSC1_control_center21 indicating the new termini and two non-natural N→C-terminal linkers (bold)). Thus, in contrast to the foldikines of the present invention (as described elsewhere herein), the foldikine "MutSC1_control_center21" has only one structural linker spanning between the left and right 3D domains in the folded protein. MutSC1_controlcenter_21 KSKA VEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNFGLDYLPNMLRDLRDAFSR hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh HD HE HF HA VKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNS hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh HB HC LGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYM hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh HD HE HF TMNFGGLDYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQ hhhhhhhhhhhhhhhhhhhhhhhhhhh HA HB FYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHFLP CEN hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh HC

[0377] result Three different constructs were inserted into the Mycoplasma pneumoniae genome via a transposon (Tn4001, CmR) under the control of a synthetic promoter (p3) (https: / / doi.org / 10.1038 / s41467-017-00239-7) and a secretion signal (s142_OPT) (https: / / doi.org / 10.15252 / msb.202010145). M. pneumoniae was grown in Hayflick medium for 48 h, after which time the supernatants were collected and the concentrations of the different engineered molecules were calculated by ELISA. In all three cases, the supernatant concentrations were higher than 30 ng / ml, the maximum concentration within the dynamic range of HEK-BLUE-IL10 cells.

[0378] A first estimate of the functionality of the molecules was made by assessing the activity of undiluted supernatants in reporter HEK-blue cells. All three constructs activated the STAT3 signaling pathway (see Figure 18).

[0379] Next, we performed eight serial dilutions starting from 30 ng / mL (and diluted each 0.5-fold) to assess HEK blue cell activation (see Figure 18B). We fitted a Michaelis-Menten model to the experimental data (saturation binding, specific binding with a Hill slope, Bmax fixed at 2.3) to obtain the apparent KD. When the engineered loop was replaced with a poly-Gly linker of the same size, this mutant had a very significant decrease in activity (MutSC1_linkercontrol19 / MutSC1_polyglycontrol_19). When N- and C-terminal linkers were introduced into both 3D domains (new N- and C-termini formed between the foldikine domains), there was an approximately 2.5-fold decrease in activity (MutSC1_linkercontrol21 / MutSC1_controlcenter_21).

[0380] The above results suggest that both the precise amino acid composition of the introduced N- and C-terminal linkers and the presence of two linker peptides bridging between adjacent 3D domains are crucial for optimal functionality of foldikines.

[0381] Example 2: Generation of a foldikine from a class II monomeric cytokine with three engineered linkers List of IL-22 variants tested and described in the following sections. The IL-22 fold-in scaffold is based on the scheme depicted in Figure 5 and Figure 27. IL22 WT (PDB=3dlq), a monomeric IL22 based on Il22 xtal expressed in bacteria, e.g., M. pneumoniae Hydrogen_core-1, a monomeric IL22 with mutations T56L, A66M, V95I, T99F, and S173L Hydrogen_core-2, a monomeric IL22 with mutations T56M, A66M, V95I, T99F, S173L, and N68Q CtoA, which is IL22 WT with Cys at position 7 replaced by Ala T56M point mutation in monomeric IL22 A66M point mutation in monomeric IL22 V95I point mutation in monomeric IL22 T99F point mutation in monomeric IL22 S173L point mutation in monomeric IL22 Foldikine-22_1 (SCIL22_1) (SEQ ID NO: 65): A single-chain IL-22 dimer in which a linker has been engineered to connect the N- and C-termini of one of the IL-22 monomer sequences, and the dimeric unit has then been created (e.g., as illustrated in Figures 5 and 27) by fusing that monomer to another IL-22 monomer sequence by breaking the loop sequence in each monomer sequence and connecting the respective new N- and C-termini to form an inter-domain bridging linker, as further described below. Foldikine-22_3 (SCIL22_3) (SEQ ID NO: 66): A single-chain foldikine similar to foldikine-22_1 but with a different engineered linker sequence between one of the novel N- and C-termini, and in which the Cys residue at position 2 of SEQ ID NO: 65 is replaced with a Val residue. Foldikine-22_3_linkerNCpolygly / Foldikine-22_3_NCpolygly contains two engineered linkers bridging between the left and right 3D domains, but replaces the designed / engineered NC-terminal linker with polyGly-Ser. The exact length of the engineered linkers was maintained in the novel polyGly-Ser linker. The foldikine-22_3_centrallinker retains the NC-terminal linker of foldikine-22_3 (SCIL22_3), but changes the two central linkers bridging the left and right 3D domains to polyGly-Ser. We maintained the exact length of the engineered linkers when changing them to polyGly-Ser.

[0382] Construct and Design: Generation of Single-Chain IL-22 Human IL-22 is a naturally monomeric interleukin. We converted IL-22 into a foldikine (referred to as "foldikine-22") using a combination of intelligent design and ModelX and FoldX software, as described when designing the IL-10-based foldikine (referred to herein as "foldikine-10"). Because IL-22 is not naturally a swapped dimer, to generate foldikine-22 it was necessary to create two non-native bridges between separate monomers and also link the N- and C-termini of one of the monomers. The two bridges between the separate monomers are created by opening a loop in each monomer and cross-linking the monomers at each of the open sequences (between their respective novel N- and C-termini), as described and illustrated with respect to Figures 5 and 27. The two novel bridges between the monomers are indicated by underlining and double underlining in the sequence below. Because these bridges are not located at the same position and do not fuse identical sequences, each linker sequence may preferably be different, as illustrated in the following embodiments. Two different mutant designs were generated, one differing in one of the linker sequences (single underlined), and one in which Cys2 was mutated to Val (bold, underlined). In bold, we have labeled the designed sequence ANGT (SEQ ID NO: 329) that connects the N- and C-termini of one of the 3D domains. The bridging linkers between the left and right foldikine domains are TCMPGGSKT (SEQ ID NO: 337) and RLELLP (SEQ ID NO: 331) in foldikine-22_1 and NRLKKLMASSD (SEQ ID NO: 332) and RLELLP (SEQ ID NO: 331) in foldikine-22_3. For ease of reference, structurally conserved helices in foldikine 22 are labeled by the sequential order in which they are found in the original IL-22 WT sequence (i.e., HA, HB, ...), and helical segments are labeled with h.If a helical segment is a slightly distorted helix, such that some residues are not assigned as h in swisspdb, but are surrounded by typical helical segments, this is designated as h-Nres-h (where Nres can be any number of residues indicated as -). Helical segments are defined using the swisspdb software and can vary slightly in length and in the crystal structure depending on the software used to define them. >SCIL22_1 (foldikine-22_1) (SEQ ID NO: 65) G C RLDKSNFQQPYITNRTFMLAKEASLADNNTTDVRLIGEKLFHGVSMSERCYLMKQVLNFT hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhh HA HB LEEVLFPQSDRFQPYMQEVVPFLARLSNRLS TCMPGGSKT HIQRNVQKLKDTVKKLGESGE h---hhhh hhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhh hhh HC HD IKAIGELDLLFMSLRNACIANGTLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKL hhhh hhhhhhhhhhh hhhhhhhhhhhhhhhhhhh HE HF HA FHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLS RLELLP HIQR h hhhhhhhhhhhhhhh hhh hhhhhhhhhhhhhhhhhhh HB HC NVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI hhhhhhhhhhh hhhhhhh hhhhhhhhhhh HD HE HF >SCIL22_3(フォールディカイン-22_3) G V RLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFT hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh HA HB LEEVLFPQSDRFQPYMQEVVPFLARLS NRLKKLMASSD HIQRNVQKLKDTVKKLGESGEIK h---hhhh hhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhhh HC HD AIGELDLLFMSLRNACIANGTLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFH hhhh hhhhhhhhhh hhhhhhhhhhhhhhhh hh HE HF HA GVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLS RLELLP HIQRNV h hhhhhhhhhhhhh hhhhhhhhhhhhhhhhhhh hhhh HB HC QKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI hhhhhhhhhhhhhhhhhhhhhhhhhhhhhh HD HE HF

[0383] To further investigate the importance of the engineered linker sequence, we generated a number of additional mutant foldikine constructs based on the foldikine-22_3 framework: The first construct, foldikine-22_3_linkerNCpolygly, retains the sequence of the two engineered linker bridges of foldikine-22_3 but changes the NC-terminal linker to polyGly. The other construct (foldikine-22_3_centrallinkers) retains the NC-terminal linker (bold) but changes the two central linker bridges between the 3D domains to polyGly-Ser (gray). In both cases, we maintained the exact length of the engineered linkers when changing them to polyGly or polyGly-Ser, respectively.

[0384] > Foldikine-22_3_linkerNCpolygly (SEQ ID NO: 67) GVRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLS NRLKKLMASSD HIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACIGGGGLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLS RLELLP HIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI

[0385] > Foldikine-22_3_centrallinkers (SEQ ID NO: 68) GVRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLS GGSGGSGGSGGHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI-ANGTLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLS GGSGGS HIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI

[0386] Stabilization of the hydrophobic core of IL-22 in the bound state In parallel, we engineered some mutations into the IL-22 framework that were predicted to stabilize the hydrophobic core in the bound conformation based on modeling of the hydrophobic core. Amino acid substitutions that were predicted to provide enhanced stability and that were not 100% conserved by evolution across different species were particularly desirable. Each potential mutation was evaluated individually and in combination.

[0387] The crystallographic structure and sequence chosen as reference for calculating the stability changes upon mutation of the hydrophobic core was 3dlq.PDB. We identified residues T56, A66, V95, T99, and S193 as residues that could potentially stabilize the IL22 protein upon mutation. These residues are marked in grey on the WT Pdb sequence (SEQ ID NO: 69). 3dlq. Pdb sequence (SEQ ID NO: 69) ARLDKSNFQQPYITNRTFMLAKEASLADNNTTDVRLIGEKLFHGVSMSERCYLMKQVLNFTL hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhhh HA HB EEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEI ---hhhh hhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhh HC HD HE KAIGELDLLFMSLRNACI hhh hhhhhhhhhhhh HF

[0388] In Table 10 below, we list five mutations that FoldX predicted to stabilize the hydrophobic core of IL22.

[0389] [Table 12]

[0390] We then introduced hydrophobic mutations individually and collectively into the foldikine-22 construct described above. 3dlq. Pdb sequence - Hydrophobic core stabilizing protein (56) (SEQ ID NO: 343) ARLDKSNFQQPYITNRMFMLAKEASLADNNTTDVRLIGEKLFHGVSMSERCYLMKQVLNFTL hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhhh HA HB EEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEI ---hhhh hhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhh HC HD HE KAIGELDLLFMSLRNACI hhh hhhhhhhhhhhh HF 3dlq. Pdb sequence - Hydrophobic core stabilizing protein (66) (SEQ ID NO: 344) ARLDKSNFQQPYITNRTFMLAKEMSLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTL hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhhh HA HB EEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEI ---hhhh hhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhh HC HD HE KAIGELDLLFMSLRNACI hhh hhhhhhhhhhhh HF 3dlq. Pdb sequence - Hydrophobic core stabilizing protein (95) (SEQ ID NO: 345) ARLDKSNFQQPYITNRTFMLAKEASLADNNTTDVRLIGEKLFHGVSMSERCYLMKQILNFTL hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhhh HA HB EEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEI ---hhhh hhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhh HC HD HE KAIGELDLLFMSLRNACI hhh hhhhhhhhhhhh HF 3dlq. Pdb sequence - Hydrophobic core stabilizing protein (99) (SEQ ID NO: 346) ARLDKSNFQQPYITNRTFMLAKEASLADNNTTDVRLIGEKLFHGVSMSERCYLMKQVLNFML hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhhh HA HB EEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEI ---hhhh hhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhh HC HD HE KAIGELDLLFMSLRNACI hhh hhhhhhhhhhhh HF 3dlq. Pdb sequence - Hydrophobic core stabilizing protein (173) (SEQ ID NO: 347) ARLDKSNFQQPYITNRTFMLAKEASLADNNTTDVRLIGEKLFHGVSMSERCYLMKQVLNFTL hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhhh HA HB EEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGEMGEI ---hhhh hhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhh HC HD HE KAIGELDLLFMSLRNACI hhh hhhhhhhhhhhh HF 3dlq. Pdb sequence - Hydrophobic core stabilizing protein (IL-22hydC) (SEQ ID NO: 70) ARLDKSNFQQPYITNRMFMLAKEMSLADNNTDVRLIGEKLFHGVSMSERCYLMKQILNFML hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhhh HA HB EEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGELGEI ---hhhh hhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhh HC HD HE KAIGELDLLFMSLRNACI hhh hhhhhhhhhhhh HF

[0391] Engineering IL-22 interface mutations A crystallographic structure of mouse IL-22 bound to receptors R1 and R2 is available (6we0.pdb). We generated a humanized model of this crystallographic structure using FoldX software to mutate residues that differ between mouse and human, and, where necessary, ModelX software to model small backbone movements to better accommodate amino acid differences. After obtaining the human and mouse IL-22 structures with the R1 and R2 receptors, we performed a positional scan analysis with FoldX for each of the positions in contact with the receptors. We then analyzed the interaction energy with both receptors and the stability of the complex. We selected positions that could facilitate binding to both the human and mouse receptors.

[0392] In Table 11 below, we list the different positions analyzed in the human structure, their DDGs in the stability of the complex, and their DDGs in the interactions with R1 and R2, respectively. Values ​​provided are in kcal / mol.

[0393] [Table 13]

[0394] In Table 12 below, we list the different positions analyzed in the mouse structure, their DDGs in the stability of the complex, and their DDGs in the interactions with R1 and R2, respectively. Values ​​provided are in kcal / mol.

[0395] [Table 14]

[0396] With these factors, we selected a series of mutation combinations to be explored initially in IL-22 WT. Below, we can find the sequences of the interface-engineered mutants.

[0397] Table 13 (below) indicates mutations made to the wild-type IL-22 sequence that increase both the stability of IL-22 and its interaction with its receptor. Mutations were performed on IL-22 WT and also included in the foldikine-22 scaffold (partial sequence alignments of wild-type and mutants C through E are shown below).

[0398] [Table 15]

[0399] WT22 (SEQ ID NO: 317) APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI Mutant C_IL22 (SEQ ID NO: 71) APISSHCRLEKRNFQQPYITNRTFMLAKEASLADNNIDVRLIGYKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLYRLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI Mutant D_IL22 (SEQ ID NO: 72) APISSHCRLDKSNFQQPYITNRTFMLAWEAMLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI Mutant E_IL22 (SEQ ID NO: 73) APISSHCREDRSNFQQPYITNRTFMLAWEAMLADNNIDVRLIGYKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLYRLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI

[0400] Experimental results Analysis of the impact of linker protein composition on IL-22 protein functionality Replacement of the NC-terminal linker of IL-22 foldikine with a poly-gly linker (foldikine-22_3_linkerNCpolygly) or a central linker (foldikine-22_3_centrallinkers) resulted in a complete loss of function in the protein, supporting the notion that engineered linker composition profoundly influences protein affinity (see Figure 19).

[0401] Analysis of mutations that stabilize the hydrophobic core of IL-22 For mutations introduced onto the IL-22 scaffold (Kd=1.7e-11), we observed an enhancement in the measured KD, which is postulated to be due to stabilization of the protein in the bound state when core stabilizing mutations are introduced into the hydrophobic scaffold (Kd=5.4e-12) (Figure 20).

[0402] We also analyzed the impact of each mutation separately and compared it with IL-22 WT. When the Cys2 amino acid at the N-terminus of the foldikine-22 sequence was mutated to prevent disulfide bridge formation, we observed a complete loss of function. Nevertheless, when different single mutations in the hydrophobic core were evaluated, we observed an enhancement in protein affinity for three of five mutations at positions 66, 95, and 99 (Figure 21). We observed that the mutation at position 173 was deleterious, and the mutation at position 56 appeared to be neutral. This may explain why only a two-fold increase in affinity was achieved for several hydrophobic mutants. Therefore, future versions of IL-22 and foldikine-22 may beneficially include the three mutations at positions 66, 95, and 99. Our results show that stabilizing the hydrophobic core of a foldikine (or monomeric) protein in the bound state can be a successful strategy to enhance protein affinity (Figure 21).

[0403] Analysis of mutations that improve interaction with R1 and R2 receptors The impact of mutations designed to improve the interaction between IL-22 and R1 (mutant C_IL22, SEQ ID NO: 71), R2 (mutant D_IL22, SEQ ID NO: 72) and both (mutant E_IL22, SEQ ID NO: 73) was analyzed. A 10-fold increase in affinity for mutant E_IL22 relative to wt (apparent KD 3.37E-11 vs 1.28E-10) and a 2-fold increase in affinity for mutant D_IL22 were observed.

[0404] Analysis of foldikine-22 variants We inserted the different constructs into the bacterial genome via transposons (Tn4001, TcR) as described in the methods. Bacterial cells (e.g., M. pneumoniae, L. lactis, or E. coli) were grown in Hayflick medium for 48 hours, after which the supernatants were collected and the concentrations of the different IL-22 manipulating molecules were calculated by ELISA. In all three cases, the supernatant concentrations were higher than 30 ng / ml, the maximum concentration falling within the dynamic range of HEK-BLUE-22 cells.

[0405] A first estimate of the functionality of the molecules was made by assessing the activity of undiluted supernatants in reporter HEK-blue cells (see Figure 22A). From these molecules, IL-22, foldikine-22-1, and foldikine-22_3 demonstrated the ability to activate the IL-22-mediated signaling pathway via STAT3. A construct in which the NC terminus of foldikine-22_linker was replaced with a polyGly linker (foldikine-22_3_linkerNCpolygly) did not activate the signaling pathway, suggesting that it was not active (Figures 19 and 22B).

[0406] Next, we performed eight serial dilutions starting from 30 ng / mL (and diluted 0.5-fold at each dilution) to assess the activation of HEK blue cells (see Figure 22B). We fitted a Michaelis-Menten model to the experimental data (saturation binding, specific binding with Hill slope, fixed Bmax at 2.2) to obtain the apparent Kd. The apparent Kd is shown in panel B. Both foldikines showed an approximately 4-fold increase in Kd compared to IL22 WT, verifying the improvement in activity of foldikine-22 compared to wild-type IL-22.

[0407] Chimeric IL-10 / IL-22 A list of chimeric IL-10 / IL-22 chimeric foldikine variants tested and described in the following section. Foldikine 10-22: A chimeric foldikine joining IL-10 and IL-22, where IL-10 corresponds to the closed / continuous foldikine 3D domain and IL-22 corresponds to the open / split domain (SEQ ID NO: 74). Chimera 3 / Foldikine 10-22_3 is based on Foldikine 10-22 in which the Cys residue at position 1 has been replaced with an Ala residue. (SEQ ID NO: 75) Chimera 5 / Foldikine 10-22_5 is based on Foldikine 10-22_3 in which a group of five additional amino acid substitutions were made in an attempt to increase the stability of the IL-22 split 3D domain to improve the IL-22 activity of the protein (SEQ ID NO: 76). Heterodimer10-22. A control in which the engineered monomeric IL-10 described in (Josephson et al., 2000) is fused to IL-22 by a short gly-ser linker of 6 amino acids joining the C-terminus of IL-10 to the N-terminus of IL-22. Heterodimer22-10. A control in which engineered monomeric IL-22 is fused to monomeric IL-10 as described in (Josephson et al., 2000) by a short gly-ser linker of 6 amino acids joining the C-terminus of IL-22 to the N-terminus of IL-10.

[0408] Construction and Design We began by modeling the IL-22 dimer structure (3dlq.pdb) on the IL-10 swapped domain structure (1y6k.pdb). To form the "Sewing1022" construct (SEQ ID NO:74), we removed the IL-22 loop between helices C and D of the native IL-22 sequence (truncating after residues Leu129 and before Arg142), as well as the loop between Lys88 and Cys114 of IL-10 (helices C and D), and connected Leu129 of IL-22 to Lys88 of IL-10 with an engineered DPKAAFKS linker (SEQ ID NO:314) and Cys114 of IL-10 to Arg143 of IL-22 via a DSKVNR linker (SEQ ID NO:315). At the same time, the N- and C-termini of IL-10 were closed by an engineered peptide linker, NFGGLDY (SEQ ID NO: 316). In italics, we indicate the residues corresponding to IL-22, and in underline, the residues of IL-10. Bold italics indicate the two central linkers introduced to join the two halves of IL-22 to IL-10. Bold non-italics is the linker peptide connecting the N- and C-termini of IL-10. >sewing1022 / Foldikine 10-22 (SEQ ID NO: 74) CRLDKSNFQQPYITNRTFMLAKEASLADNNTTDVRLIGEKLFHGVSMSERCYLMKQVLNFTL hhhhhhhhhhhhhhhhhhh hhh hhhhhhhhhhhhh HA-IL22 HB-IL22 EEVLFPQSDRFQPYMQEVVPFLARLSNRLDPKAAFKS KAVEQVKNAFNKLQEKGIYKAMSE hhhhhhhhhhhhhhhhhh HC HD-IL10 HE-IL10 FDIFINYIEAYMTM NFGGLDY LPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG hhhhhhhhhhh NtCt hhhhhhhhhh hhhhhh HF-IL10 HA-IL10 YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPC DSKVN hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh HB-IL10 HC-IL10 RRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI Hhhhhhhhhhhhh hhhhhhh hhhhhhhhhhh HD-IL22 HE_IL22 HF-IL22

[0409] This initial sawing pattern of the foldikine-10 / 22 molecule demonstrated IL-10 activity in assays but not significant IL-22 activity. The same was observed with foldikine 10-22_3 / chimera 3 (SEQ ID NO: 75), in which the first Cys residue was mutated to Ala. This result may be due to a lack of stability in the IL-22 split-domain or problems with the designed linker. To investigate and solve this issue, we generated a set of mutations to increase the stability of the hydrophobic core of the split-domain IL-22 in an attempt to rescue functionality (foldikine 1022_5 / chimera 5; SEQ ID NO: 76). In the table below, we list the set of mutations made. Mutations are shown in bold and underlined in SEQ ID NOs: 75 and 76 below.

[0410] > Foldikine 10-22_3 (SEQ ID NO: 75) ARLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMLQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLDPKAAFKCKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNFGGL DYLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCLCLSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCISKVLRRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI*

[0411] >Foldikine 10-22_5 (SEQ ID NO: 76) ARLDKSNFQQPYITNR L FMLAKEASL M DNNTDVRLIGEKLFHGVSMSERCYLMLQ I LNF F LEEVLFPQSDRFQPYMQEVVPFLARLSNRLDPKAAFKCKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMNFGGLDYLPNMLRDLRDAFSRVKTFFQMKDQ LDNLLLKESLLEDFKGYLGCLCLSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCISKVLRRNVQKLKDTVKKLGESGEIKAIGELDLLFM L LRNACI The mutations in this molecule are as follows:

[0412] [Table 16]

[0413] As a control, we generated two different protein fusions: an engineered monomeric IL-10 (underlined below) described in (Josephson et al., 2000) fused to IL-22 (italics) via a short 6-amino acid gly-ser linker (heterodimer10-22) (SEQ ID NO: 77). In parallel, we generated the reverse construct (heterodimer22-10) in which the C-terminus of IL-22 (italics) was fused to the N-terminus of IL-10 (underlined) via a short 6-amino acid gly-ser linker (SEQ ID NO: 78). The linker is indicated in bold and underlined in SEQ ID NO: 78 below.

[0414] >heterodimer10-22 (SEQ ID NO: 77) MSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGNGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGS APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI

[0415] &g...

Claims

1. A single-chain dimeric cytokine polypeptide comprising (a) a first cytokine monomer domain or functional portion thereof and (b) a second cytokine monomer domain or functional portion thereof, wherein the sequence of one of the cytokine monomer domains or functional portions is not contiguous but is interrupted by the sequence of the other cytokine monomer domain or functional portion thereof.

2. 2. The single-chain dimeric cytokine polypeptide of claim 1, comprising: (a) a first cytokine monomer domain or a functional portion thereof; and (b) a second cytokine monomer domain or a functional portion thereof, wherein the sequence of the second cytokine monomer domain or a functional portion thereof is contiguous, while the sequence of the first cytokine monomer domain is such that a first sequence portion of the first cytokine monomer domain is disposed N-terminally to the second cytokine monomer domain or a functional portion thereof and a second sequence portion of the first cytokine monomer domain is disposed C-terminally to the second cytokine monomer domain or a functional portion thereof.

3. 3. The single-chain dimeric cytokine polypeptide of claim 2, wherein the first sequence portion of the first cytokine monomer domain corresponds to an N-terminal portion of a naturally occurring cytokine monomer domain and the second sequence portion of the first cytokine monomer domain corresponds to a C-terminal portion of the naturally occurring cytokine monomer domain.

4. 4. A single-chain dimeric cytokine polypeptide according to claim 2 or 3, i) a split domain comprising the free N- and C-termini of the single-chain dimeric cytokine polypeptide and the first and second sequence portions of the first cytokine monomer domain in their natural sequence order; and ii) a contiguous domain comprising the second cytokine monomer domain or a functional part thereof, constructed as a first sequence portion and a second sequence portion of the second cytokine monomer domain, the first and second sequence portions being in reverse order compared to their natural sequence order. Including, In the split domain, the sequence of the first and second sequence portions of the first cytokine monomer domain are separated by the sequence of the contiguous domain. Single-chain dimeric cytokine polypeptide.

5. i) an N-terminal portion of a first class I cytokine monomer comprising α-helices A and B of the first class I cytokine monomer; ii) a linker peptide bridging α-helix B of the first class I cytokine monomer with α-helix C of a second class I cytokine monomer; iii) a C-terminal portion of the second class I cytokine monomer comprising α-helices C and D of the second class I cytokine monomer; iv) a linker peptide bridging α-helix D of the second class I cytokine monomer with α-helix A of the second class I cytokine monomer.

5. The single-chain dimeric cytokine polypeptide of claim 3, which is a single-chain dimeric class I polypeptide comprising: a) a linker peptide linking α-helix B of the second class I cytokine monomer with α-helix C of the first class I cytokine monomer; v) an N-terminal portion of the second class I cytokine monomer comprising α-helices A and B of the second class I cytokine monomer; vi) a linker peptide bridging α-helix B of the second class I cytokine monomer with α-helix C of the first class I cytokine monomer; and vii) a C-terminal portion of the first class I cytokine monomer comprising α-helices C and D of the first class I cytokine monomer.

6. i) an N-terminal portion of a first class II cytokine monomer comprising α-helices A-C of the first class II cytokine monomer; ii) a linker peptide bridging α-helix C of the first class II cytokine monomer with α-helix D of a second class II cytokine monomer; iii) a C-terminal portion of the second class II cytokine monomer comprising α-helices D-F of the second class II cytokine monomer; iv) a linker peptide bridging α-helix F of the second class I cytokine monomer with α-helix A of the second class II cytokine monomer.

5. The single-chain dimeric cytokine polypeptide of claim 3, which is a single-chain dimeric class II polypeptide comprising: a car peptide; v) an N-terminal portion of the second class II cytokine monomer comprising alpha helices A-C of the second class II cytokine monomer; vi) a linker peptide bridging alpha helix C of the second class II cytokine monomer and alpha helix D of the first class II cytokine monomer; and vii) a C-terminal portion of the first class II cytokine monomer comprising alpha helices D-F of the first class II cytokine monomer.

7. A single-chain dimeric cytokine polypeptide according to any one of claims 3 to 4 and 6, i) a dimeric IL-22 comprising the sequence SEQ ID NO:325-(Xn1)-SEQ ID NO:326-(NtCt)-SEQ ID NO:327-(Xn2)-SEQ ID NO:328 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; ii) a dimeric IL-19 comprising the sequence of SEQ ID NO:195-(Xn1)-SEQ ID NO:196-(NtCt)-SEQ ID NO:197-(Xn2)-SEQ ID NO:198 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; iii) a dimeric IL-20 comprising the sequence SEQ ID NO:200-(Xn1)-SEQ ID NO:201-(NtCt)-SEQ ID NO:202-(Xn2)-SEQ ID NO:203 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; iv) a dimeric IL-26 comprising the sequence SEQ ID NO:210-(Xn1)-SEQ ID NO:211-(NtCt)-SEQ ID NO:212-(Xn2)-SEQ ID NO:213 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; v) dimeric IFNlambda2 comprising the sequence SEQ ID NO:215-(Xn1)-SEQ ID NO:216-(NtCt)-SEQ ID NO:217-(Xn2)-SEQ ID NO:218 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; vi) a dimeric IFNlambda3 comprising the sequence SEQ ID NO:220-(Xn1)-SEQ ID NO:221-(NtCt)-SEQ ID NO:222-(Xn2)-SEQ ID NO:223 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; vii) dimeric IFNlambda1 comprising the sequence SEQ ID NO:225-(Xn1)-SEQ ID NO:226-(NtCt)-SEQ ID NO:227-(Xn2)-SEQ ID NO:228 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; viii) dimeric IFNα1 / 13 comprising the sequence SEQ ID NO:230-(Xn1)-SEQ ID NO:231-(NtCt)-SEQ ID NO:232-(Xn2)-SEQ ID NO:233 or a sequence at least 80% identical thereto, and which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; ix) dimeric IFNα2 comprising the sequence SEQ ID NO:235-(Xn1)-SEQ ID NO:236-(NtCt)-SEQ ID NO:237-(Xn2)-SEQ ID NO:238 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; x) dimeric IFNβ comprising the sequence SEQ ID NO:240-(Xn1)-SEQ ID NO:241-(NtCt)-SEQ ID NO:242-(Xn2)-SEQ ID NO:243 or a sequence at least 80% identical thereto, and which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; xi) dimeric IFNΩ1 comprising the sequence of SEQ ID NO:245-(Xn1)-SEQ ID NO:246-(NtCt)-SEQ ID NO:247-(Xn2)-SEQ ID NO:248 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; xii) dimeric IFNε comprising the sequence of SEQ ID NO:250-(Xn1)-SEQ ID NO:251-(NtCt)-SEQ ID NO:252-(Xn2)-SEQ ID NO:253 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; or xiii) a dimeric IFN kappa comprising the sequence SEQ ID NO:255-(Xn1)-SEQ ID NO:256-(NtCt)-SEQ ID NO:257-(Xn2)-SEQ ID NO:258 or a sequence at least 80% identical thereto, which retains at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric class II cytokine; and (Xn1) and (Xn2) are peptide linkers; Single-chain dimeric cytokine polypeptide.

8. 8. The single chain dimeric cytokine polypeptide of any one of claims 3-4 and 6-7, which is a single chain dimeric IL-22 polypeptide comprising the sequence of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, or SEQ ID NO:288, or a sequence at least 80% identical thereto, and which retains at least the same stability and / or at least the same level of interaction with IL-22 receptor 1 and / or IL-22 receptor 2.

9. 7. A single chain dimeric cytokine polypeptide according to any one of claims 3 to 4 and 6, wherein a single chain chimeric IL-10-IL-22 polypeptide is provided which comprises or consists of the sequence of SEQ ID NO: 76 or a sequence at least 80% identical thereto and which retains at least the same stability and / or at least the same level of interaction with the IL-10 receptor and the IL-22 receptor.

10. 3. The single-chain dimeric cytokine polypeptide of claim 2, wherein the first sequence portion of the first cytokine monomer domain corresponds to a C-terminal portion of a naturally occurring cytokine monomer domain and the second sequence portion of the first cytokine monomer domain corresponds to an N-terminal portion of a naturally occurring cytokine monomer domain.

11. 11. A single-chain dimeric cytokine polypeptide according to claim 2 or 10, i) a split domain comprising the free N- and C-termini of the single-chain dimeric cytokine polypeptide and the first and second sequence portions of the first cytokine monomer domain in reverse orientation compared to their natural sequence order; and ii) a contiguous domain comprising said second cytokine monomer domain or a functional part thereof in its natural sequence. Including, In the split domain, the sequence of the first and second sequence portions of the first cytokine monomer domain are separated by the sequence of the contiguous domain. Single-chain dimeric cytokine polypeptide.

12. 12. A single-chain dimeric cytokine polypeptide according to claim 10 or 11, a) a linker peptide bridges the fourth alpha helix of a first class I cytokine monomer and the first alpha helix of a second class I cytokine monomer; b) a linker peptide bridges the fourth alpha helix of the second class I cytokine monomer and the first alpha helix of the first class I cytokine monomer; a single-chain dimeric class I polypeptide, wherein alpha helices A and B of said first class I cytokine monomer are not cross-linked, or alpha helices B and C of said first class I cytokine monomer are not cross-linked, or alpha helices C and D of said first class I cytokine monomer are not cross-linked. Single-chain dimeric cytokine polypeptide.

13. Based on short-chain class I cytokines, i) dimeric IL-2 comprising SEQ ID NO:89-(Xn1)-SEQ ID NO:90-(Xn2)-SEQ ID NO:91; ii) dimeric IL-4 comprising SEQ ID NO:93-(Xn1)-SEQ ID NO:94-(Xn2)-SEQ ID NO:95; iii) dimeric IL-3 comprising SEQ ID NO:97-(Xn1)-SEQ ID NO:98-(Xn2)-SEQ ID NO:99; iv) dimeric IL-7 comprising SEQ ID NO:103-(Xn1)-SEQ ID NO:104-(Xn2)-SEQ ID NO:105; v) dimeric IL-9 comprising SEQ ID NO:107-(Xn1)-SEQ ID NO:108-(Xn2)-SEQ ID NO:109; vi) dimeric IL-15 comprising SEQ ID NO:110-(Xn1)-SEQ ID NO:111-(Xn22)-SEQ ID NO:112; vii) dimeric IL-21 comprising SEQ ID NO:113-(Xn1)-SEQ ID NO:114-(Xn)-SEQ ID NO:115; viii) dimeric TSLP comprising SEQ ID NO:116-(Xn1)-SEQ ID NO:117-(Xn2)-SEQ ID NO:118; and ix) Dimeric GM-CSF (which may comprise or consist of SEQ ID NO:120-(Xn1)-SEQ ID NO:121-(Xn2)-SEQ ID NO:12 SEQ ID NO:101) or Sequences that are at least 80% identical thereto and that retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric short chain class I cytokine. and (Xn1) and (Xn2) are peptide linkers; A single-chain dimeric cytokine polypeptide according to any one of claims 10 to 12.

14. Based on long-chain class I cytokines, i) dimeric IL-6 comprising SEQ ID NO:135-(Xn1)-SEQ ID NO:136-(Xn2)-SEQ ID NO:137; ii) dimeric IL-11 comprising SEQ ID NO:139-(Xn1)-SEQ ID NO:140-(Xn2)-SEQ ID NO:141; iii) dimeric IL-12α comprising SEQ ID NO:143-(Xn1)-SEQ ID NO:144-(Xn2)-SEQ ID NO:145; iv) dimeric IL-23α comprising SEQ ID NO:147-(Xn1)-SEQ ID NO:148-(Xn2)-SEQ ID NO:149; v) dimeric IL-27α comprising SEQ ID NO:151-(Xn1)-SEQ ID NO:152-(Xn2)-SEQ ID NO:153; vi) dimeric IL-31 comprising SEQ ID NO:154-(Xn1)-SEQ ID NO:155-(Xn2)-SEQ ID NO:156; vii) dimeric CLCF1 comprising SEQ ID NO:158-(Xn1)-SEQ ID NO:159-(Xn2)-SEQ ID NO:160; viii) a dimeric ONCM comprising SEQ ID NO:161-(Xn1)-SEQ ID NO:162-(Xn2)-SEQ ID NO:163; ix) dimeric CTF1 comprising SEQ ID NO:165-(Xn1)-SEQ ID NO:166-(Xn2)-SEQ ID NO:167; x) dimeric CNTF comprising SEQ ID NO:169-(Xn1)-SEQ ID NO:170-(Xn2)-SEQ ID NO:171; xi) dimeric LIF comprising SEQ ID NO:173-(Xn1)-SEQ ID NO:174-(Xn2)-SEQ ID NO:175, and xii) Dimeric CSF3 comprising SEQ ID NO:177-(Xn1)-SEQ ID NO:178-(Xn2)-SEQ ID NO:179 or Sequences that are at least 80% identical thereto and that retain at least the same stability and / or at least the same level of interaction with the respective receptor(s) of said monomeric long chain class I cytokine. and (Xn1) and (Xn2) are peptide linkers; A single-chain dimeric cytokine polypeptide according to any one of claims 10 to 12.

15. 14. The single chain dimeric cytokine polypeptide of any one of claims 10 to 13, which is a dimeric IL-2 and comprises SEQ ID NO:89-(Xn1)-SEQ ID NO:90-(Xn2)-SEQ ID NO:91, wherein said peptide linkers Xn1 and Xn2 are identical and comprise or consist of SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, or SEQ ID NO:

275.

16. 12. A single-chain dimeric cytokine polypeptide according to claim 10 or 11, a) a linker sequence bridges the first alpha helix of a first class II cytokine monomer and the sixth alpha helix of a second class II cytokine monomer; b) a linker sequence bridges the sixth alpha helix of the first class II cytokine monomer and the first alpha helix of the second class II cytokine monomer; c) the first and second α-helices, the second and third α-helices, the third and fourth α-helices, the fourth and fifth α-helices, or the fifth and sixth α-helices of the first class II cytokine monomer are not cross-linked; is a single-chain dimeric class II polypeptide, Single-chain dimeric cytokine polypeptide.

17. 2. The single-chain dimeric cytokine polypeptide of claim 1, comprising: (a) a first class I cytokine monomer domain or functional portion thereof; and (b) a second class I cytokine monomer domain or functional portion thereof, wherein the sequence of the first class I cytokine monomer domain or functional portion thereof is contiguous, while the sequence of the second cytokine monomer domain is such that the first sequence portion of the second class I cytokine monomer located at the N-terminus of the first class I cytokine monomer or functional portion thereof corresponds to the N-terminal portion of a naturally occurring class I cytokine monomer, and the second sequence portion of the second class I cytokine monomer located at the C-terminus of the second class I cytokine monomer or functional portion thereof corresponds to the C-terminal portion of the naturally occurring class I cytokine monomer.

18. 20. The single-chain dimeric cytokine polypeptide of claim 1 or 17, i) a split domain comprising the free N- and C-termini of said single-chain dimeric class I cytokine polypeptide and a first and second sequence portion of said second class I cytokine monomer domain; and ii) a contiguous domain comprising the first class I cytokine monomer domain or a functional portion thereof; Including, In the split domain, the sequence of the first and second sequence portions of the first class I cytokine monomer domain are separated by the sequence of the contiguous domain. Single-chain dimeric cytokine polypeptide.

19. 19. A single-chain dimeric cytokine polypeptide according to claim 17 or 18, a) a linker peptide bridges the fourth alpha helix of a first class I cytokine monomer and the third alpha helix of said second class I cytokine monomer; b) a linker peptide bridges a first alpha helix of the first class I cytokine monomer and a second alpha helix of the second class I cytokine monomer; c) a linker peptide bridges a first α-helix (α-helix A) of the first class I cytokine monomer and a second α-helix of the second class I cytokine monomer; d) the α-helices B and C of the second class I cytokine monomer are not cross-linked; Single-chain dimeric cytokine polypeptide.

20. 20. The single-chain dimeric cytokine polypeptide of any one of claims 17 to 19, which is a single-chain dimeric IL-2 polypeptide comprising the sequence of SEQ ID NO: 296 or a sequence at least 80% identical thereto and which retains at least the same stability and / or at least the same level of interaction with the IL-2 receptor.

21. 2. The single-chain dimeric cytokine polypeptide of claim 1, comprising: (a) a first class II cytokine monomer domain or a functional portion thereof; and (b) a second class II cytokine monomer domain or a functional portion thereof, wherein the sequence of the second class II cytokine monomer domain or a functional portion thereof is contiguous and in reverse order compared to its native sequence order, while the sequence of the first class II cytokine monomer domain is such that a first sequence portion of the first class II cytokine monomer domain disposed N-terminal to the second class II cytokine monomer domain or a functional portion thereof corresponds to the N-terminal portion of a native class II cytokine monomer domain, and a second sequence portion of the first class II cytokine monomer domain disposed C-terminal to the second class II cytokine monomer domain or a functional portion thereof corresponds to the C-terminal portion of the native class II cytokine monomer domain.

22. 22. The single-chain dimeric cytokine polypeptide of claim 2 or 21, i) a split domain comprising the free N-terminus and C-terminus of the single-chain dimeric class II cytokine polypeptide and the first and second sequence portions of the first class II cytokine monomer domain in their natural sequence order; and ii) a contiguous domain comprising the second class II cytokine monomer domain or a functional portion thereof; and wherein the single-chain dimeric class II cytokine polypeptide comprises In the split domain, the sequence of the first and second sequence portions of the first class II cytokine monomer domain are separated by the sequence of the contiguous domain of the second class II cytokine monomer domain. Single-chain dimeric cytokine polypeptide.

23. 20. The single chain dimeric cytokine polypeptide of any one of claims 5 to 7, 12 to 16 and 19, wherein said linker peptide has a sequence of about 3 to about 20 amino acids, about 3 to about 16 amino acids, about 4 to about 12 amino acids, about 4 to about 8 amino acids, about 3 to about 8 amino acids or about 3 to about 6 amino acids, said linker peptide sequence comprises 5 or less, preferably 3 or less adjacent Gly and / or Ser residues, and optionally said linker peptide is a structured linker.

24. 2. The single-chain dimeric cytokine polypeptide of claim 1, comprising first and second cytokine monomer domains forming a swapped domain dimer that is a class I cytokine, comprising, from N-terminus to C-terminus, a first cytokine monomer domain comprising helices A-C of the second cytokine monomer and helix D of the first cytokine monomer, and a second cytokine monomer domain comprising helix D of the second cytokine monomer and helices A-C of the first cytokine monomer, wherein a linker peptide bridges helix D of the second cytokine monomer and helix A of the first cytokine monomer.

25. 25. The single-chain dimeric cytokine polypeptide of claim 24, which is a dimeric IL-5 polypeptide comprising the sequence SEQ ID NO: 101-(NtCt)-SEQ ID NO: 102, where (NtCt) is a peptide linker, or a sequence at least 80% identical thereto, and which retains at least the same stability and / or at least the same level of interaction with the IL-5 receptor.

26. 2. The single-chain dimeric cytokine polypeptide of claim 1, which forms a swapped domain dimer and comprises a first and a second cytokine monomer that are class II cytokines, and comprises, from N-terminus to C-terminus, a first cytokine monomer domain that comprises helices A-C of the second cytokine monomer and helices D-F of the first cytokine monomer, and a second cytokine monomer domain that comprises helices D-F of the second cytokine monomer and helices A-C of the first cytokine monomer, and a linker peptide bridging helix F of the second cytokine monomer and helix A of the first cytokine monomer.

27. 27. The single-chain dimeric cytokine polypeptide of claim 26, which is a single-chain dimeric IL-10 and comprises the sequence of SEQ ID NO: 11-(NtCt)-SEQ ID NO: 12, where (NtCt) is a peptide linker, or a sequence having at least 80% sequence identity thereto, and which retains at least the same stability and / or at least the same level of interaction with the IL-10 receptor.

28. 28. The single-chain dimeric IL-10 polypeptide of claim 27, comprising SEQ ID NO:9 or SEQ ID NO:10 or a sequence at least 80% identical thereto, and retaining at least the same stability and / or at least the same level of interaction with the IL-10 receptor.

29. 27. The single-chain dimeric cytokine polypeptide of claim 26, which is a single-chain dimeric IFNγ and comprises the sequence of SEQ ID NO: 14-(NtCt)-SEQ ID NO: 15, where (NtCt) is a peptide linker, or a sequence having at least 80% sequence identity thereto, and which retains at least the same stability and / or at least the same level of interaction with the IFNγ receptor.

30. 30. The single-chain dimeric polypeptide of claim 29, comprising SEQ ID NO: 18 or a sequence having at least 80% sequence identity thereto, and retaining at least the same stability and / or at least the same level of interaction with the IFNγ receptor.

31. A polypeptide comprising the single-chain dimeric polypeptide according to any one of claims 1 to 30.

32. A nucleic acid encoding a polypeptide as defined in any one of claims 1 to 31.

33. 33. An expression vector comprising a nucleic acid as defined in claim 32.

34. 34. A host cell comprising a nucleic acid as defined in claim 32 or an expression vector as defined in claim 33.

35. A pharmaceutical composition comprising a polypeptide as defined in any one of claims 1 to 31.

36. 32. A method of treating a disease comprising administering a polypeptide as defined in any one of claims 1 to 31 to a subject in need thereof, optionally wherein the subject would benefit from a reduction or increase in the rate of inflammatory response or cell proliferation.

37. 32. A polypeptide as defined in any one of claims 1 to 31 for use in the treatment of a disease, optionally the disease being a disease that would benefit from a reduction or increase in the rate of inflammatory responses or cell proliferation.

38. 32. A polypeptide as defined in any one of claims 1 to 31 for the manufacture of a medicament for the treatment of a disease, optionally the disease being a disease that would benefit from a reduction or increase in the rate of inflammatory responses or cell proliferation.

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