Synthetic factor compositions and methods of use

Abstract

The present disclosure provides engineered synthetases and methods of using the same.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 479,993, filed on March 31, 2017, which is incorporated herein by reference in its entirety. Background Art

[0003] Manipulation of cells, particularly immune cells, to differentiate, develop specialized functions and to amplify in number is of great clinical significance. Many protein factors that affect these activities are known in the art, including in particular cytokines and chemokines. However, these signal transduction molecules also have pleiotropic effects on cells that are not targeted for manipulation, and therefore methods for selectively activating signal transduction in targeted cell populations are needed.

[0004] Cytokines, chemokines, growth factor agonists, etc., by making JAK / STAT; RTK connected; or death domain (TNF superfamily) receptor polymerization, such as generating homologous or heterologous dimers or advanced oligomers to activate the receptor by intracellular trans-phosphorylation to trigger signal transduction. The characteristics of the specific receptor chain in the polymer (such as dimer or trimer) determine signal transduction and functional response. In the case of cytokines, they act as dual-specific ligands, to specify which receptors are included in the dimer by forming specific contacts with each of the two receptor extracellular domains, thereby playing the role of bridging or cross-linking dimer signal transduction complexes. Cytokine receptor dimerization leads to the activation of intracellular JAK / STAT signal transduction pathways, which are composed of four Janus kinases (JAK1-3, TYK2) and seven signal transduction factors and transcription activators (STAT1-6) proteins.

[0005] Although the ligand is specific to the extracellular domain of its receptor, the JAK / TYK / STAT signal transduction module exists in multiple combinations in the endogenous receptor signal transduction complex, and is therefore capable of extensive cross-talk. The ligands of RTK receptors (such as EGF, VEGF, etc.) also force signal transduction through receptor dimerization, although the molecular mechanism may be completely different from that of cytokines. In two cases: JAK / STAT cytokines and RTK ligands, their role is to induce their specific receptor subunits to be localized into dimers, so that the intracellular kinase domain is in an orientation and proximity that can trans-phosphorylate both kinases and receptor intracellular domains. The sequence requirements (i.e., substrate specificity) of these tyrosine kinases can be quite degenerate, thereby increasing the possibility that these enzymes can be redirected by alternative receptor dimerization ligands to phosphorylate receptor substrates other than the receptor substrates that are usually presented with them in nature.

[0006] Given that ligands determine the composition of receptor dimers, and the intracellular kinase degeneracy of JAK / TYK and RTK enzymes, the number of cytokine and growth factor receptor dimer pairs that exist in nature accounts for only a small fraction of the total number of signaling-competent receptor pairs that the system theoretically allows. For example, the human genome encodes approximately forty different JAK / STAT cytokine receptors. In principle, approximately 1,600 unique homo- and hetero-dimeric cytokine receptor pairs can be generated, with the potential for signaling through different JAK / TYK / STAT combinations. However, the human genome encodes less than 50 different cytokine ligands, limiting the range of cytokine receptor dimers to those that can be assembled by natural ligands. Similar arguments can be made for the RTK receptor and ligand families. In addition, given that it has been demonstrated that death receptors are capable of signaling as dimers or trimers, this concept can also be extended to this family.

[0007] The ability to selectively activate a target signaling pathway is of great interest. The present invention provides compositions and methods for this purpose. Summary of the invention

[0008] Engineered synthetic signaling molecules are provided, referred to herein as "synthekines". Synthekines are genetically engineered, dual-specific ligands for cell surface receptors, wherein the synthekine specifically binds with high affinity to the extracellular domain of at least one, often two, different cell surface receptor polypeptides. Cell surface receptors are characterized by activation of signal transduction upon multimerization. In some embodiments, the generation of receptor multimers by binding to the synthekine results in intracellular trans-phosphorylation of the receptor. Synthekines include, but are not limited to, small organic molecules and polypeptides.

[0009] Synthetic factors can also be trispecific ligands of cell surface receptors, or higher specific ligands, wherein the synthetic factors specifically bind to the extracellular domains of at least three different cell surface receptor polypeptides with high affinity. Cell surface receptors are characterized by activating signal transduction when multimerized. In some embodiments, the generation of receptor multimers by binding to synthetic factors leads to intracellular transphosphorylation of receptors. Synthetic factors include, but are not limited to, small organic molecules and polypeptides.

[0010] In some embodiments, the cell surface receptor polypeptide is one or more of the following: (i) a cytokine receptor that activates the JAK / STAT pathway in the cell; (ii) a receptor tyrosine kinase; or (iii) a TNFR superfamily member. In some embodiments, each multimeric receptor polypeptide is naturally expressed in the targeted single cell. In some embodiments, the target cell is engineered to express the one or more multimeric receptor polypeptides.

[0011] In some embodiments, the synthetic factors specifically bind to two or more different cytokine receptors, which, when activated by multimerization, undergo trans-phosphorylation and signaling through JAK / STAT and other pathways (including but not limited to ERK, AKT and other signaling messengers). In some embodiments, the cytokine receptor is selected from, but not limited to, βc, γc, IL-3Rα, βIL-3R, GM-CSFRα, IL-5Rα, CNTFα, CRLF1, LIFRα, gp130, IL-6Rα, IL-11Rα, OSMRβ, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-9Rα, IL-13Rα, IL-15Rα, IL-21Rα, IFNAR2, IL-23R, EpoR, IL-12Rβ, IFNAR1, IFNAR2, G-CSFR, c-MPLR. In some embodiments, the synthetic factors bind to two such receptors and activate JAK / STAT signaling. In some embodiments, the synthetic factors bind to three such receptors and activate JAK / STAT signaling. Typically, the synthetic factors activate pathways that are different from the pathways by which natural cytokines activate receptors.

[0012] In some embodiments, the synthetic factor binds to two or more different receptor tyrosine kinase proteins that are activated by transphosphorylation when the proteins are polymerized. In some of these embodiments, the RTK receptor is selected from but not limited to EGFR, ErbB2, ErbB3, ErbB4, InsR, IGF1R, InsRR, PDGFRα, PDGFRβ, CSF1R / Fms, cKit, Flt-3 / Flk2, VEGFR1, VEGFR2, VEGFR3, FGFR1, FGFR2, FGFR3, FGFR4, PTK7 / CCK4, TrkA, TrkB, TrkC, Ro r1, Ror2, MuSK, Met, Ron, Axl, Mer, Tyro3, ​​Tie1, Tie2, EphA1-8, EphA10, EphB1-4, EphB6, Ret, Ryk, DDR1, DDR2, Ros, LMR1, LMR2, LMR3, ALK, LTK, SuRTK106 / STYK1, Activin-R, BMP-R, TGF-β-R, Noggin-R. In some embodiments, synthetic factors bind to two such receptors and activate signal transduction. Typically, the pathway activated by the synthetic factor is different from the pathway by which the natural ligand activates the receptor.

[0013] In some embodiments, the synthetic factor binds to two or more different TNFR superfamily polypeptides, and the TNFR superfamily polypeptides are activated when the proteins are multimerized. In some embodiments, the receptor is selected from TNFR1 (TNFRSF1A), TNFR2 (TNFRSF1B; TNFRSF2), 41-BB (TNFRSF9), AITR (TNFRSF18), BCMA (TNFRSF17), CD27 (TNFRSF7), CD30 (TNFRSF8), CD40 (TNFRSF5), death receptor 1 (TNFRSF10C), death receptor-3 (TNFRSF25), death receptor 4 (TNFRSF10A), death receptor 5 (TNFRSF10B), death receptor-6 (TNFRSF21), Decoy receptor-3 (TNFRSF6B), Decoy receptor 2 (TNFRSF10D), EDAR, Fas (TNFRSF6), HVEM (TNFRSF14), LTβ-R (TNFRSF3), OX40 (TNFRSF4), RANK (TNFRSF11A), TACI (TNFRSF13B), Troy (TNFRSF19), XEDAR (TNFRSF27), osteoprotegerin (TNFRSF11B), TWEAK receptor (TNFRSF12A), BAFF receptor (TNFRSF13C), NGF receptor (TNFRSF16). In some embodiments, synthetic factors bind to two or more of these receptors and activate signal transduction. Typically, the pathway activated by the synthetic factor is different from the pathway by which the natural ligand activates the receptor.

[0014] In other embodiments, the synthetic factors bind to two or more mixed classes of receptors, such as a JAK / STAT receptor in combination with a TNFRSF and / or RTK receptor; an RTK receptor in combination with a TNFRSF receptor, and the like.

[0015] In some embodiments of the invention, the synthetic factor is a polypeptide that may contain a separate or continuous binding domain or element that binds to each receptor extracellular domain (ECD) polypeptide. The polypeptide synthetic factor may be a single chain, a dimer, or a higher order multimer. The binding domain / element of each receptor may be directly connected, or may be separated by a linker, such as a polypeptide linker or a non-peptide linker. In some embodiments, the synthetic factor does not activate the natural receptor configuration. For example, the synthetic factor binding domain may bind to one chain of a natural receptor, but may not bind to the second chain of a natural receptor. Such binding domains include, but are not limited to, dominant negative mutants of cytokines.

[0016] In polypeptide embodiments, the receptor binding domain can be selected from any domain that binds to the extracellular domain of a desired receptor with high affinity, e.g., a Kd of no more than about 1 x 10 -7 M, not more than about 1x 10 -8 M, not more than about 1x 10 -9 M, or no more than about 1x 10 -10 M. Suitable binding domains include, but are not limited to, binding proteins designed from scratch, binding proteins derived from antibodies, such as scFv, Fab, etc., and other parts of antibodies that specifically bind to one or more receptor ECD sequences; binding domains derived from nanoantibodies; engineered scaffolds based on knottin; norrin and engineered binding fragments derived from norrin, naturally occurring binding domains, etc. Naturally occurring binding domains, such as cytokines, growth factors, etc., are usually engineered to prevent activation of the activity of natural receptors. The binding domain can be affinity selected to enhance binding to the desired ECD; and / or mutagenesis can be performed to prevent binding to unwanted ECDs.

[0017] Synkine polypeptides may be fused, linked or alternatively co-administered with agents to enhance receptor activation.Synkine polypeptides may be fused, linked or alternatively co-administered with target cytokines, chemokines or growth factors.

[0018] The binding domains can be continuous within a globular domain, or separated by linkers, such as polypeptide linkers or non-peptide linkers, etc. The length of the linker, and therefore the spacing between the binding domains, can be used to adjust the signal strength and can be selected based on the desired use of the synthetic factor. The mandatory distance between the binding domains can vary, but in certain embodiments can be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, or less than about 50 angstroms.

[0019] In some embodiments, the linker is a rigid linker, in other embodiments, the linker is a flexible linker. In the case where the linker is a peptide linker, the length of the linker can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids, and has enough length and amino acid composition to force the distance between the binding domains. In some embodiments, the linker comprises one or more glycine and / or serine residues or is composed of one or more glycine and / or serine residues.

[0020] Synth factors can be multimerized, for example, via an Fc domain, via concatenation, coiled coil, polypeptide zipper, biotin / avidin or streptavidin multimerization, etc. Synth factors can also be linked to moieties such as PEG, Fc, etc., as known in the art, to enhance in vivo stability.

[0021] The composition of interest includes, but is not limited to, an effective dose of a synthetic factor in a pharmaceutically acceptable excipient. The composition may include other agents, such as adjuvants, etc. The synthetic factor may be produced synthetically; as known in the art, produced by various suitable recombinant methods, etc.

[0022] In some aspects of the invention, a method for activating, increasing or enhancing JAK / STAT, DD and / or RTK signal transduction selected in a cell is provided. In such methods, cells expressing a homologous receptor polypeptide of a target synthetic factor are contacted with a certain concentration of synthetic factors, and the concentration of synthetic factors effectively increases signal transduction, for example, relative to signal transduction in the absence of synthetic factors, signal transduction is increased by 25%, 50%, 75%, 90%, 95% or more. Such signal transduction activation can induce JAK / STAT or RTK signal transduction pathways, and includes but is not limited to immune response, regulation of growth factor response, induction of death domain response, etc. In some methods, cells expressing receptors are contacted in vitro. In other embodiments, cells expressing receptors are contacted in vivo.

[0023] In some aspects of the invention, a method for treating or preventing a disease or condition in a subject in need thereof is provided, the method comprising providing an effective amount of a synthetic factor to the subject. In certain embodiments, the subject suffers from an immune disease or dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The invention is best understood when the following detailed description is read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Instead, the sizes of the various features are arbitrarily expanded or reduced for clarity. The following figures are included in the drawings.

[0025] Figure 1A-1B : Dimerization of non-natural receptor pairs by engineered synthetic factor ligands. Figure 1A A schematic diagram of the dimerization of a novel cytokine receptor pair by a synthetase is detailed. The putative synthetase recruits receptors A and D to form a novel ternary complex that is distinct from the ternary complex formed by cytokines X and Y. Figure 1BSchematic representation of IL-1 mediated IL-1R1 and IL-1R1AcP chimeric receptor complexes. The intracellular domains of the cytokine receptors specified in the table to the right were grafted onto the extracellular domains of IL-1R1 or IL-1R1AcP. The JAKs and STATs activated by each receptor are specified in the table.

[0026] Figure 2A-2I : Non-natural cytokine receptors activate signaling. Figure 2A Depend on Figure 1B Heat map representation of STAT molecules activated by 100 different cytokine receptor pair combinations generated by the chimeric receptor matrix described in. In Western blot analysis, results were binary coded as 1 (band present) or 0 (band absent). Figure 2B Schematic diagram of the designed IL-1 inducible chimeric receptor (left). Alanine insertion mutagenesis of the EpoR juxtamembrane domain is detailed in the center. 251 Alanine residues (1A, 2A, 3A or 4A) were then inserted. The projection of the α-helical wheel with twisted rows introduced by the addition of alanine residues is presented at the bottom right. Each residue adds 109° of rotation, and the insertion of 3A residues brings the rows close to their original position. Figure 2C Phospho-STAT3 (pSTAT3) and pSTAT5 levels measured by Western blot in IL-1 activated Jurkat cells expressing the indicated chimeric receptor pairs. Insertion of two alanines restores signaling through the IL-1R1-EpoR / IL-1R1AcP-γc receptor pair. Total levels of TYK2 are presented as a loading control. The Western blots presented are representative examples of two independent experiments. Figure 2D Cell surface expression of chimeric receptors in Jurkat cells. Flow cytometric dot plots of surface expression levels of IL-1R1 and IL-1R1Acp chimeric receptors in Jurkat cells 24 hours after transfection. Figure 2E Signaling profiles of chimeric receptor activation in Jurkat cells. pSTAT1, pSTAT2, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 levels measured by Western blot in IL-1 activated Jurkat cells expressing the indicated chimeric receptor pairs. Total levels of TYK2 are presented as a loading control. Figure 2F Cell surface expression of EpoR chimeric receptors in Jurkat cells. Flow cytometric dot plots showing cell surface levels of the indicated chimeric receptors in Jurkat cells 24 hours after transfection. Figure 2G , Figure 2H , Fig.2I Alanine insertion failed to restore signaling through IL-23R-IL-12Rβ, IL-2Rβ-IL2Rβ, and EpoR-EpoR chimeric receptors. Figure 2G, Figure 2H , Fig.2I ) STAT activation upon IL-1 stimulation measured by Western blot (left panel) and cell surface expression of chimeric receptors with the indicated alanine insertions in Jurkat cells measured by flow cytometry (right panel).

[0027] Figures 3A-3F : Synthetic factors that dimerize non-natural cytokine receptor pairs to activate signaling. Figure 3A Synthetic factor layout and complex formation. Two dominant negative cytokine variants were genetically fused via a Gly4 / Ser linker to generate novel molecules that induce heterodimer formation of non-native cytokine receptors. Figure 3B-3D The expression of IL-4, Super-2 (an affinity matured variant of IL-2), and IFNω cytokines in Hut78 T cells was measured by flow cytometry. Figure 3B ), dominant negative cytokine variants IL-4DN, IL-2DN and IFNDN ( Figure 3C ) or SY1 SL, SY1 LL and SY2 cytokines ( Figure 3D ) Activated pSTAT1, pSTAT3, pSTAT5 and pSTAT6 levels. Data (mean + / - SD) are from two independent replicates. Figure 3E , Figure 3F . Signaling profiles activated by stimulation with Super-2 / IL-4 and IL-4 / IFN cytokine combinations. ( Figure 3A-3B ) in Hut78 cells measured by flow cytometry by treatment with indicated concentrations of Super-2 / IL-4 ( Figure 3A ) and IL-4 / IFN cytokine combination stimulation for 15 minutes. Data (mean + / - SD) are from three independent experiments.

[0028] Figures 4A-4D : Synthetic factors and genomically encoded cytokines activate different signaling programs. Figure 4A Bubble plot representation of signaling pathways activated by the indicated ligands in Hut78 T cells after 15, 60, or 120 minutes of stimulation. The size of the bubble indicates the intensity of the activated signal. Figure 4B Illustration of a filled radar plot of signaling molecules activated by genomically encoded cytokines and synthokines in Hut78 cells after 15 minutes of stimulation. The signaling molecules activated by the ligands are shown at the circumference, and their respective activation potency is represented by the radius of the circle. The different shapes of the filled radar plots exhibited by different ligands define their different signaling characteristics. Figure 4CSTAT activation ratios of cytokines and synthetic factors after Hut78 cells were stimulated for 15 minutes. Each column represents the total STAT activation level of each ligand normalized to 100%. The relative activation efficacy of each STAT was corrected accordingly. The different distributions of STAT activation levels of various ligands indicate the differential STAT usage between genomic encoded cytokines and synthetic factors. Data (mean values) are from two independent replicates. Figure 4D Unsupervised clustering of signaling programs participated by cytokines and synthokines. Principal component analysis (PCA) of signaling programs participated by genomically encoded cytokines and synthokines after 15 and 60 minutes of stimulation in Hut78 cells. Genomically encoded cytokine and synthokine signatures are spatially spaced equidistantly, indicating that synthokine signaling programs are as distinct from the parental cytokines as they are from each other.

[0029] Figures 5A-5C : Synthetic factors elicit cellular signatures and immune activities that are distinct from those of genome-encoded cytokines. Figure 5A Heat map representation of activation levels of six signaling effectors induced by saturating doses of the indicated ligands in 29 immune cell types analyzed from PBMCs measured by mass cytometry (CyTOF). Data (mean) are from two independent replicates. Figure 5B Detailed analysis of the secretion profile of 63 cytokines from PBMCs stimulated with the indicated ligands. Cytokines secreted more than 2-fold above background are labeled. Data (mean + / - SD) are from two independent replicates. Figure 5C Immune Cell Profiling for Identification of Signaling Signatures Activated by Cytokines and Synkine Treatments. Mass cytometry-mediated gating scheme for identification of 29 distinct immune cell subsets in peripheral blood mononuclear cells (PBMCs) isolated from whole blood for assessment of signaling effector activation responses to cytokine and synthokine treatments.

[0030] Figures 6A-6G : Synthetic factor that dimerizes cytokine receptors and tyrosine kinase receptors to activate signal transduction. Fig. 6A Schematic representation of IL-1-mediated complexing of IL1-R1-EGFR and IL-1R1AcP-cytokine receptor chimeras. Figure 6B Phospho-EGFR (pY EGFR), pSTAT3 and pSTAT5 levels measured by Western blot analysis in Jurkat cells expressing the indicated chimeric receptor pairs activated by IL-1. Total levels of Erk are presented as loading controls. The Western blots presented are representative examples of two independent experiments. Figure 6CLayout and complex formation of synthetic factors that dimerize cytokine receptors and tyrosine kinase receptors. Two scFvs that bind to cytokine receptors and tyrosine kinase receptors, respectively, are genetically fused to acidic or basic leucine zippers to generate new molecules that are able to form heterodimeric receptor complexes that do not exist in nature. Fig.6D Phospho-cKit Y703 and pJAK2 levels measured by Western blot in Mo7E cells after stimulation with synthetic factors that dimerize TpoR and cKit (SY3, SY4, and SY5) for the indicated time periods. Total levels of Lamin are presented as a loading control. The Western blots presented are representative examples of two independent experiments. Fig. 6E Erk (left panel) and STAT5 (right panel) phosphorylation activated in Mo7e cells by stimulation with indicated doses of SCF, TPO or indicated anabolic factors for 10 min measured by flow cytometry. Data (mean + / - SD) are from three independent replicates. Fig. 6F , Figure 6G Functional characterization of tyrosine kinase receptor / cytokine receptor dimerization synthases. Fig. 6F Flow cytometric graphs representing the surface expression levels of the indicated chimeric receptors in Jurkat cells 24 hours after transfection. Figure 6G Erk activation response in Mo7e cells to 10 min stimulation with indicated doses of SCF, TPO and SY5 + / - JAK2 inhibitor as measured by flow cytometry. Data (mean + / - SD) are from three independent experiments.

[0031] Figures 7A-7C : Synthetic factors that dimerize cytokine receptors and tyrosine kinase receptors activate signaling programs that are different from those of their natural ligands. Fig. 7A Bubble plot representation of signaling pathways activated by the indicated ligands in Mo7e cells after 10, 60, and 120 minutes of stimulation. The size of the bubble indicates the intensity of the activated signal. Figure 7B Stacked bar graphs of signaling molecules engaged by SCF, TPO, and SY5 after 10 minutes of stimulation in Mo7e cells. For each molecule, the combined activation level of the three ligands was normalized to 100%, and the relative contribution of each ligand was corrected accordingly. Some molecules were better activated by SCF, others by TPO, and still others by SY5. The data presented in Figures A and B represent the average of two independent experiments performed in triplicate. Figure 7C pPLCG2, pErk and pLCK levels induced by the indicated ligands in Mo7e cells after 10, 60 and 120 min of stimulation. Data (mean + / - SD) are from three independent replicates.

[0032] Figure 8 :Designed a trimerization factor (SY3) that linked IL-2 to a scFv that specifically binds to IL-4Rα via a gly4ser linker. Thus, the trimerization factor binds to three receptor polypeptides, γc, IL-2Rβ, and IL-4Rα.

[0033] Fig. 9 : Trimeric synthetic factors induce a distinct pSTAT activation profile from their wild-type counterparts.

[0034] Fig.10 : Trimeric synthetic factors exhibit a distinct signaling activation profile from their wild-type counterparts.

[0035] Fig.11 : Trimeric synthetic factors induce a cytokine secretion profile that is very different from their wild-type counterparts.

[0036] Fig.12 : Trimeric synthetic factors differentiate monocytes into a previously uncharacterized population of dendritic cells.

[0037] Fig.13 : Dendritic cells differentiated by trimerized synthetic factors exhibit a high degree of phagocytosis.

[0038] Fig.14 : Dendritic cells differentiated by trimerized synthetic factors exhibit a high degree of phagocytosis.

[0039] Fig.15 : Differentiation markers on dendritic cells differentiated by trimerized synthetic factors are different from those of native cell populations.

[0040] Figure 16. IL-2λ synthesis factor. Synthesis factor (SY6) is a hybrid interferon that dimerizes type I and type III IFN receptors. A) Table of STATs activated when IFN receptors, their associated JAKs, and receptor dimerization. B) SY6 is a hybrid interferon that dimerizes IFNAR1 and IFNλR1 receptors and their corresponding JAKs. C) The Emax of phospho-STAT1 activation of SY6 is equal to that of type I IFN and is twice the signal induced by type III IFN. Error bars represent ± SEM (n = 3). D) SY6 effectively induces antiproliferative effects, while type I IFN, type III IFN, or a combination of type I and type III IFN treatment is ineffective. Error bars represent ± SEM (n = 3). Phospho-STAT1 signaling and antiproliferative assays were performed in Hap1 cells that are naturally responsive to both type I and type III IFN.

[0041] Fig.17. IL-4DN-IFNβDN2 (SY7) connects IL-4DN to IFNβ2DN2 via a gly / ser linker. Lymphocytes were isolated from spleen / LN of C57BL / 6 mice and activated for 48 hours with plate-bound anti-CD3 (2.5μg / ml) + soluble anti-CD28 (5μg / ml). The cells were then left to rest overnight in 10IU / ml mIL2, then serum starved for 4 hours, and then stimulated for 20' with the specified cytokines / synthetic factors. Cell signaling was terminated and the cells were fixed with PFA, permeabilized with PermIII buffer (BD) and stained with phosphoSTAT6 (Y641) antibody (BD). The sequence is provided as SEQ ID NO: 2. DETAILED DESCRIPTION

[0042] In order to make the present disclosure more readily understood, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be read in light of what those skilled in the art would know at the time of the invention.

[0043] definition

[0044] Before describing the methods and compositions of the present invention, it should be understood that the present invention is not limited to the specific methods or compositions described, and thus can certainly be varied. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

[0045] When providing a range of values, it should be understood that, unless the context clearly indicates otherwise, each median value (to one tenth of the lower limit unit) between the upper and lower limits of the range is also clearly disclosed. Each smaller range between any specified value or median value in the specified range and any other specified value or median value in the specified range is included in the present invention. The upper and lower limits of these smaller ranges can be independently included in the range or excluded, and in the case where any limit, both limits are not or both limits are included in the smaller range, each range is also included in the present invention, based on any clearly excluded limit in the specified range. When the specified range includes one or two limits, the scope excluding any one or two of those included limits is also included in the present invention.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or test of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials related to the cited publications. It should be understood that, in the event of a conflict, the present disclosure replaces any disclosure of the incorporated publications.

[0047] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, such as polypeptides known to those skilled in the art, and so forth.

[0048] The publications discussed herein are provided only for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to precede such publications by virtue of prior invention. In addition, the publication dates provided may differ from the actual publication dates and may require independent confirmation.

[0049] "Comprising" means that the listed elements are required in the composition / method / kit, but other elements may also be included to form a composition / method / kit, etc. within the scope of the claim. For example, a composition comprising a wnt synthesis factor is a composition that may include other elements in addition to the synthesis factor, such as a functional portion that binds (e.g., covalently binds) to the wnt synthesis factor, such as a polypeptide, a small molecule, or a nucleic acid; an agent that promotes the stability of the wnt synthesis factor composition, an agent that promotes the solubility of the wnt synthesis factor composition, an adjuvant that will be readily understood in the art, but excluding elements covered by any negative conditions.

[0050] "Consisting essentially of" means that the scope of the described composition or method is limited to the specified materials or steps, and such limitation does not materially affect the basic and novel characteristics of the subject invention. For example, a wnt synthetase that "consists essentially of" a disclosed sequence has an amino acid sequence of a disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence, based on the sequence from which it is derived, such as about 5 residues, 4 residues, 3 residues, 2 residues, or about 1 residue less than the recited boundary amino acid residues, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited boundary amino acid residues.

[0051] "Consisting of" means excluding any elements, steps or ingredients not specified in the claims from the composition, method or kit. For example, a wnt synthesis factor "consisting of a disclosed sequence" consists only of a disclosed amino acid sequence.

[0052] "Functional moiety" or "FM" means a polypeptide, small molecule, or nucleic acid composition that confers functional activity to the composition. Examples of functional moieties include, but are not limited to, therapeutic moieties, binding moieties, and imaging moieties.

[0053] "Therapeutic moiety" or "TM" means a polypeptide, small molecule or nucleic acid composition that imparts therapeutic activity to the composition. Examples of therapeutic moieties include cytotoxins (e.g., small molecule compounds), protein toxoids, and radiosensitizing moieties (i.e., radionuclides that are intrinsically harmful to cells, etc.); agents that alter cellular activity, such as small molecules, peptide mimetics, cytokines, chemokines; and moieties that target ADCC or CDC-dependent death of cells, such as the Fc component of an immunoglobulin.

[0054] "Imaging moiety" or "IM" means a non-cytotoxic agent that can be used to localize and optionally visualize cells (eg, cells that have been targeted by a composition of the subject application).

[0055] The term "treatment" and the like are generally used herein to mean obtaining the desired pharmacological and / or physiological effects. This effect can be preventive in terms of completely or partially preventing a disease or its symptoms, and / or can be therapeutic in terms of partially or completely curing a disease and / or side effects attributable to the disease. "Treatment" as used herein includes any treatment for a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) alleviating the disease, i.e., causing the disease to regress. The therapeutic agent can be administered before, during, or after the disease or injury occurs. In the case of treatment stabilization or reduction of adverse clinical symptoms in patients, the treatment of persistent diseases is particularly interesting. It is expected that such treatment will be performed before complete loss of function in the affected tissue. The subject therapy can be administered during the disease symptom period, and in some cases after the disease symptom period.

[0056] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy.

[0057] General methods in molecular and cellular biochemistry can be found in standard textbooks such as: Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., CSH Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (Ausubel et al., eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al., eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy, eds., Academic Press 1995; Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosure of which is incorporated herein by reference. The reagents, cloning vectors and kits for genetic manipulation mentioned in this disclosure are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich and ClonTech.

[0058] Natural receptor and ligand pairs include, but are not limited to, the following receptors:

[0059]

[0060]

[0061]

[0062] Synthetic factors can be engineered to bind any of the receptor polypeptide combinations in the table above, but will generally not activate the same receptor polypeptide combinations as the natural ligands listed above. For example, while LIF activates a heterodimer of LIFR and cd130, synthetic factors may activate LIFR and γc, or LIFR and βc, etc. The receptor polypeptide combinations activated by synthetic factors may be naturally expressed in the target cells, or the cells may be engineered to express the desired receptor polypeptide combination.

[0063] JAK / STAT pathway and receptors. Receptors that activate the JAK / STAT pathway when dimerized include, but are not limited to, βc, γc, IL-3Rα, βIL-3R, GM-CSFRα, IL-5Rα, CNTFα, CRLF1, LIFRα, gp130, IL-6Rα, IL-11Rα, OSMRβ, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-9Rα, IL-13Rα, IL-15Rα, IL-21Rα, IFNAR2, IL-23R, EpoR, IL-12Rβ, IFNAR1, G-CSFR, c-MPLR.

[0064] The JAK-STAT signaling pathway transmits information from extracellular chemical signals to the cell nucleus, causing the transcription and expression of genes involved in immunity, proliferation, differentiation, apoptosis and tumorigenesis. The JAK-STAT signaling cascade consists of three main components: cell surface receptors, Janus kinases (JAKs) and two signal transduction factors and transcription activators (STAT) proteins as disclosed above. Disruption or dysregulation of JAK-STAT function can lead to immunodeficiency syndromes and cancer.

[0065] Cytokine binds to receptors on the cell surface, leading to the activation of receptor-associated tyrosine kinase JAK. Once activated, JAK is mutually trans-phosphorylated, thereby producing docking sites for signal transduction factors and transcription activators (STAT) molecules. After binding, STAT is activated by JAK-mediated tyrosine phosphorylation and forms homologous or heterodimers, which are translocated to the nucleus, where they regulate transcription. There are four different JAK kinases (JAK1, 2, 3 and TYK2) and seven different STAT proteins (STAT1, 2, 3, 4, 5A, 5B and 6). A cytokine can activate more than one JAK, and each JAK targets more than one STAT protein. This multi-layered and complex activation pattern sometimes produces a fine phenotype. Reviews of Jak-STAT signaling include, for example, Villarino et al. (2017) Nat. Immunol. 18(4):374-384; Majoros et al. (2017) Front Immunol. 8:29; Bannarjee et al. (2017) Drugs 77(5):521-546; Pencik et al. (2016) Cytokine 87:26-36, each of which is expressly incorporated herein by reference.

[0066] Receptor tyrosine kinase pathways. RTK receptor polypeptides include, but are not limited to, EGFR, ErbB2, ErbB3, ErbB4, InsR, IGF1R, InsRR, PDGFRα, PDGFRβ, CSF1R / Fms, cKit, Flt-3 / Flk2, VEGFR1, VEGFR2, VEGFR3, FGFR1, FGFR2, FGFR3, FGFR4, PTK7 / CCK4, TrkA, TrkB, TrkC, Ror1, Ror2, MuSK, Met, Ron, Axl, Mer, Tyro3, ​​Tie1, Tie2, EphA1-8, EphA10, EphB1-4, EphB6, Ret, Ryk, DDR1, DDR2, Ros, LMR1, LMR2, LMR3, ALK, LTK, and SuRTK106 / STYK1.

[0067] Receptor tyrosine kinases (RTKs) are high affinity cell surface receptors for many polypeptide growth factors, cytokines and hormones. See, e.g., Robinson et al. (2000). Oncogene 19(49):5548–57, which is expressly incorporated herein by reference. There are 58 known RTKs in humans, which belong to twenty subfamilies. All RTKs have a similar molecular structure, with a ligand binding region in the extracellular domain, a single transmembrane helix, and a cytoplasmic region containing a protein tyrosine kinase (TK) domain plus an additional carboxyl (C-) terminal and a proximal membrane regulatory region.

[0068] In general, growth factor binding activates RTK by inducing receptor dimerization, but a subgroup of RTK forms oligomers even in the absence of an activating ligand. Ligand binding activates receptors by stabilizing individual receptor molecules of an active polymer configuration. Typically, one of the polypeptide chains then phosphorylates one or more tyrosines, and the phosphorylated receptor is active in assembling and activating intracellular signal transduction proteins. The dimerization of the RTK extracellular region induced by the ligand leads to the activation of an intracellular tyrosine kinase domain (TKD).

[0069] The first major substrate of RTK phosphorylation is the receptor itself. The autophosphorylation site in the kinase domain itself plays an important regulatory role in most RTKs. Then in other cytoplasmic regions of most RTKs, other tyrosine autophosphorylation. The phosphotyrosine produced plays the role of a specific site of downstream signal transduction molecule assembly, which is recruited on the receptor and activated in response to growth factor stimulation. Autophosphorylation is carried out in trans, and the autophosphorylation site is phosphorylated in an accurate order. By making the cis-autoinhibition interaction unstable, each successive event has a significant impact on the catalytic properties.

[0070] The cellular response to RTK autophosphorylation is to recruit and activate many downstream signal transduction molecules. These molecules contain SH2 or PTB domains that specifically bind to phosphotyrosine. They can be directly recruited into the phosphotyrosine in the receptor, or they can be indirectly recruited by binding to the docking proteins of the RTK phosphorylation associated with them. These docking proteins include FRS2, IRS1 (insulin receptor substrate-1) and Gab1 (Grb2 related binding protein). Docking proteins usually contain a membrane targeting site at its amino terminal, followed by a series of tyrosine phosphorylation sites, which are used as binding sites for downstream signal transduction proteins of different groups of libraries. Although many docking proteins (such as Gab1) are recruited by multiple RTKs, other docking proteins are limited to specific receptor subgroups. In most receptors, there are multiple phosphotyrosines and many docking proteins are involved, and the activated RTK can recruit and affect a large number of different signal transduction molecules. Reviews of signaling pathways include, for example, Lemmon and Schlesinger (2010) Cell 141(7): 1117-34, which is expressly incorporated herein by reference.

[0071] TNFRSF pathway. TNFRSF polypeptides include, but are not limited to, TNFR1 (TNFRSF1A), TNFR2 (TNFRSF1B; TNFRSF2), 41-BB (TNFRSF9), AITR (TNFRSF18), BCMA (TNFRSF17), CD27 (TNFRSF7), CD30 (TNFRSF8), CD40 (TNFRSF5), death receptor 1 (TNFRSF10C), death receptor-3 (TNFRSF25), death receptor 4 (TNFRSF10A), death receptor 5 (TNFRSF10B), death receptor-6 (TNFRSF21), Decoy receptor-3 (TNFRSF6B), decoy receptor 2 (TNFRSF10D), EDAR, Fas (TNFRSF6), HVEM (TNFRSF14), LTβ-R (TNFRSF3), OX40 (TNFRSF4), RANK (TNFRSF11A), TACI (TNFRSF13B), Troy (TNFRSF19), XEDAR (TNFRSF27), osteoprotegerin (TNFRSF11B), TWEAK receptor (TNFRSF12A), BAFF receptor (TNFRSF13C), NGF receptor (TNFRSF16).

[0072] The tumor necrosis factor receptor (TNFR) superfamily consists of 29 transmembrane receptors with significant homology in their extracellular domains, characterized by the presence of up to 6 cysteine-rich domains (CRDs), which define their ligand specificity. The members of this family are type I transmembrane proteins with a C-terminal intracellular tail, a transmembrane region, and an N-terminal domain that binds to the extracellular ligand. The members of TNFR contain an extracellular domain responsible for ligand binding and an intracellular domain that mediates activation of signal transduction pathways. The TNF homology domain (THD) triggers the formation of non-covalent homotrimers. TNFR can be divided into two categories: activating receptors and death receptors (DR).

[0073] DR includes eight members, such as TNFR1 and Fas, which have a protein interaction module called death domain (DD) in the intracellular region, which mediates cell death induced by extrinsic signals. Binding to ligands leads to receptor aggregation and recruitment of adaptor proteins, which in turn trigger a proteolytic cascade by recruiting and activating the initiator caspases 8 and 10. Death receptors trigger multiple signaling pathways, including regulation of cell proliferation and differentiation, chemokine production, inflammatory responses, apoptosis, and tumor-promoting activities.

[0074] Death receptors are activated by their cognate ligands, which are a class of complementary cytokines that belong to the TNF family of proteins. Cytotoxic signaling through death receptors occurs through: 1) binding to cognate ligands; 2) recruitment of adaptor / docking proteins, which in turn recruit initiator caspases 8 and 10; and 3) discrete signaling pathways that depend on the stoichiometry of various adaptor proteins and caspases 8 and 10, as well as cellular internalization events. A number of non-cytotoxic signaling pathways may also be involved, mediated primarily by activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) from the receptor / adapter protein complex.

[0075] Most TNF receptors require specific adaptor proteins such as TRADD, TRAF, RIP, and FADD for downstream signaling and may ultimately function to activate NF-κB. For example, upon binding to TNFα, the intracellular DD of TNFR1 recruits TNF receptor-associated DD protein (TRADD), which in turn recruits receptor-interacting protein kinase 1 (RIP1), cell inhibitor of apoptosis proteins 1 and 2 (cIAP1 and 2), and TNF receptor-associated factor 2. TRADD is important for TNF-induced NF-κB signaling pathways, as IκB phosphorylation and degradation are completely abolished in TRADD-deficient MEFs.

[0076] Expression constructs: In this method, the synthetic factors can be produced by recombinant methods. The synthetic factors can be introduced into the cells to be engineered on expression vectors. The DNA encoding the synthetic factors can be obtained from various sources designed during the engineering process.

[0077] As described herein, amino acid sequence variants are prepared by introducing appropriate nucleotide changes into the coding sequence. Such variants represent insertions, substitutions and / or specified deletions of the residues. Any combination of insertions, substitutions and / or specified deletions is produced to obtain the final construct, provided that the final construct has the desired biological activity as defined herein.

[0078] The nucleic acid encoding the synthetic factor is inserted into a replicable vector for expression. Many such vectors are available. Vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integration vectors, etc.

[0079] Cytokines can not only be directly recombinantly produced, but also can be produced as fusion polypeptides with heterologous polypeptides, such as signal sequences or other polypeptides with specific cleavage sites at the N-terminus of mature proteins or polypeptides. In general, the signal sequence can be a component of the vector, or can be a part of the coding sequence inserted into the vector. The heterologous signal sequence preferably selected is a signal sequence recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, natural signal sequences can be used, or other mammalian signal sequences may also be suitable, such as signal sequences of secretory polypeptides from the same or related species, and viral secretory leaders, such as herpes simplex gD signal sequences.

[0080] Expression vectors usually contain selection genes, also referred to as selectable markers. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in selective medium. Host cells that are not transformed with a vector containing a selection gene will not survive in the medium. Typical selection gene encoded proteins (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) supplement auxotrophic defects, or (c) provide key nutrients that cannot be obtained from complex medium.

[0081] The expression vector will contain a promoter that is recognized by the host organism and operably connected to the synthetic factor coding sequence. A promoter is a non-translated sequence located upstream (5') of the start codon of a structural gene (usually within about 100 to 1000bp), which controls the transcription and translation of a specific nucleic acid sequence that is operably connected thereto. Such promoters are generally divided into two categories, inducible and constitutive. Inducible promoters are promoters that trigger an increase in the level of DNA transcription that is controlled by them in response to some changes in culture conditions, such as the presence or absence of nutrients or temperature changes. A large number of promoters that are recognized by various potential host cells are well known.

[0082] For example, transcription from the vector in a mammalian host cell can be controlled by promoters obtained from the genome of viruses such as polyoma virus, fowl pox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus (such as murine stem cell virus), hepatitis B virus, and most preferably Simian virus 40 (SV40), from heterologous mammalian promoters, e.g., actin promoter, PGK (phosphoglycerate kinase) or immunoglobulin promoter, and from heat shock promoters, provided such promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus can be conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.

[0083] By inserting enhancer sequences into vectors, transcription of higher eukaryotic organisms can often be increased. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on promoters to increase their transcription. Enhancers are relatively independent of direction and position and have been found at 5' and 3' of the transcription unit, in introns, and in the coding sequence itself. Many enhancer sequences are now known to come from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, enhancers from eukaryotic cell viruses are usually used. Examples include SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers, and adenovirus enhancers on the rear side of the replication origin. Enhancers can be spliced ​​into the 5' or 3' position of the coding sequence in the expression vector, but are preferably located at the 5' site of the promoter.

[0084] Expression vectors for eukaryotic host cells will also contain sequences necessary for termination of transcription and stabilization of mRNA. Such sequences are commonly available from 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. The construction of suitable vectors containing one or more of the above components employs standard techniques.

[0085] A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if the DNA of a signal sequence is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA of the signal sequence is operably linked to the DNA of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or if a ribosome binding site is positioned to facilitate translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader, continuous and in reading phase. However, enhancers do not have to be continuous.

[0086] The synthetic factor produced by recombinant production can be recovered from the culture medium as a secreted polypeptide, but can also be recovered from the host cell lysate. Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), can also be used to inhibit proteolytic degradation during purification, and antibiotics can be included to prevent the growth of foreign contaminants. Various purification steps are known in the art and can be used, such as affinity chromatography. Affinity chromatography utilizes highly specific binding sites that are usually present in biomacromolecules to separate molecules according to their ability to bind specific ligands. Covalent bonds attach ligands to insoluble porous support media in a way that ligands are clearly presented to protein samples, thereby using the natural biospecific binding of a molecular species to separate and purify a second species from a mixture. Antibodies are commonly used in affinity chromatography. Size selection steps can also be used, such as gel filtration chromatography (also referred to as size exclusion chromatography or molecular sieve chromatography) for separating proteins according to the size of the protein. In gel filtration, a protein solution is passed through a column filled with a semipermeable porous resin. The semipermeable resin has a range of pore sizes, which determines the size of the protein that can be separated by the column. Also of interest is cation exchange chromatography.

[0087] The final synthetic factor composition can be concentrated, filtered, dialyzed, etc. using methods known in the art. For therapeutic applications, the synthetic factors can be administered to mammals containing an appropriate combination of receptor polypeptides. Administration can be as a bolus or by continuous infusion over a period of time and intravenous administration. Alternative routes of administration include intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, local or inhalation routes. The synthetic factors are also suitable for administration by intratumoral, peritumoral, intralesional or perilesional routes, or to lymphatic administration, to exert local and systemic therapeutic effects.

[0088] Physiologically acceptable carriers are encompassed in such dosage forms, and the carrier itself is nontoxic and non-therapeutic. Examples of such carriers include ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based materials and PEG. Carriers for topical or gel-based polypeptide forms include polysaccharides (such as sodium carboxymethyl cellulose or sodium methyl cellulose), polyvinyl pyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG and wood wax alcohol. For all administrations, conventional reservoir forms are suitable for use. Such forms include, for example, microcapsules, nanocapsules, liposomes, plasters, inhalation forms, nasal sprays, sublingual tablets and sustained release formulations. The polypeptide is typically formulated in such a vehicle at a concentration of about 0.1 μg / ml to 100 μg / ml.

[0089] If a synthetase is "substantially pure," it may be at least about 60% by weight (dry weight) of the polypeptide of interest (e.g., a polypeptide comprising the amino acid sequence of the synthetase). For example, the polypeptide may be at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% by weight of the polypeptide of interest. Purity may be measured by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0090] In another embodiment of the present invention, a product containing materials that can be used to treat the above-mentioned symptom is provided. The product includes a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be formed of a variety of materials, such as glass or plastic. The container holds a composition effective for treating the symptom and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The active agent in the composition is a synthetic factor. The label on or attached to the container indicates that the composition is used to treat the selected symptom. Other containers can be provided for the product, which can hold, for example, pharmaceutically acceptable buffers, such as phosphate buffered saline, Ringer's solution or dextrose solution. The product can also include other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes and package inserts containing instructions for use.

[0091] As used herein with respect to polypeptides or DNA sequences, the term "identity" refers to the subunit sequence identity between two molecules. When a subunit position in two molecules is occupied by the same monomer subunit (e.g., the same amino acid residue or nucleotide), the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so as to obtain the highest order match. If necessary, publicly available techniques and widely available computer programs such as (Devereux et al., Nucleic Acids Res. 12: 387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215: 403, 1990) can be used to calculate identity. Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705) with its default parameters.

[0092] The terms "polypeptide," "protein" or "peptide" refer to any chain of amino acid residues, regardless of its length or post-translational modifications (eg, glycosylation or phosphorylation).

[0093] By "protein variant" or "variant protein" or "variant polypeptide" herein is meant a protein that differs from a wild-type protein due to at least one amino acid modification. A parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified form of a WT polypeptide. A variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or an amino sequence encoding the polypeptide. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, for example, about 1 to about 10 amino acid modifications compared to the parent, preferably about 1 to about 5 amino acid modifications.

[0094] As used herein, "parent polypeptide", "parent protein", "precursor polypeptide" or "precursor protein" means an unmodified polypeptide that is subsequently modified to produce a variant. The parent polypeptide can be a wild-type (or native) polypeptide, or a variant or engineered form of a wild-type polypeptide. The parent polypeptide can refer to the polypeptide itself, a composition comprising the parent polypeptide, or an amino acid sequence encoding the polypeptide.

[0095] As used herein, "wild type" or "WT" or "native" means an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0096] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. Preferably, the mammal is a human.

[0097] As used herein, "therapeutically effective amount" refers to an amount of a therapeutic agent, such as an adoptive T cell and an orthogonal cytokine combination, sufficient to treat or control a disease or condition. A therapeutically effective amount may refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of a disease, such as to delay or minimize the spread of a cancer, or an amount that effectively reduces or increases signaling from a target receptor. A therapeutically effective amount may also refer to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment or control of a disease. In addition, a therapeutically effective amount with respect to a therapeutic agent of the present invention means an amount of a therapeutic agent that provides a therapeutic benefit in the treatment or control of a disease, alone or in combination with other therapies.

[0098] As used herein, the term "preventing" or "prevention" refers to the prevention of the recurrence or onset of one or more symptoms of a disorder in a subject as a result of administration of a prophylactic or therapeutic agent.

[0099] As used herein, the term "combination" refers to the use of more than one preventive and / or therapeutic agent. The use of the term "combination" does not limit the order in which the preventive and / or therapeutic agent is administered to a subject suffering from a condition. The first preventive or therapeutic agent can be administered to a subject suffering from a condition before the second preventive or therapeutic agent is administered (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks ago), simultaneously or afterwards (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks later) is administered.

[0100] Composition

[0101] Synthetic factors and methods of using the same are provided. The synthetic factors cause a measurable increase in the level of signaling of targeted pathways such as Jak / STAT, ERK, AKT, NF-κB, etc., provided that a different signaling profile is activated relative to the natural ligand. These and other objects, advantages and features of the present invention will become apparent to those skilled in the art upon reading the details of the compositions and methods described more fully below.

[0102] Synth factor molecules are defined by their physical and biological properties. The main feature is that the synthetine binds specifically to one or more, usually 2 or more different extracellular domains of cell surface receptors, which are characterized by activation by ligand-induced multimerization (often ligand-induced dimerization), and in many cases activation by trans-phosphorylation. Synth factors activate non-natural combinations of receptors, which usually do not activate combinations of receptors activated by natural (i.e., genome-encoded) ligands. Target receptors include receptors that activate JAK-STAT signaling, exemplified by the cytokine receptors described herein; receptor tyrosine kinases, exemplified by cytokine and growth factor receptors, and TNF receptors.

[0103] Synthetic factors can be any molecule with the desired binding properties, such as a protein or a drug. Small molecules that may be less than about 15Kd are of interest and can be developed by compound screening as described herein. Polypeptides are also of interest. In addition, some synthetic factors may comprise both polypeptide regions or domains and non-polypeptide regions or domains.

[0104] Synthetic factors can be polypeptides in which the binding domains of two different receptor extracellular domains are connected. Polypeptide synthetic factors can be single chains, dimers or higher-order multimers. The binding domains can be directly connected or can be separated by a linker, such as a polypeptide linker or a non-peptide linker.

[0105] In some embodiments, one or all of the binding domains comprise a binding domain of a natural ligand, i.e., IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-4, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-α, IFN-β, IFN-γ, LT-β, ​​TNF-α, TNF-β, 4-1BBL, CD70, CD153, CD178, TGF-β1, TGF-β2, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF, MSP; wherein the binding domain does not activate the natural receptor for the ligand. For example, a binding domain may comprise a targeted amino acid substitution that results in a lack of binding to one but not another of a native receptor polypeptide. Many such modified binding domains are known in the art and may, for example, generate dominant negative mutations relative to the native receptor configuration.

[0106] In various other embodiments, the binding domain can be an antibody, or a binding portion derived from an antibody, that specifically binds to one chain of the receptor.

[0107] The term "specific binding" refers to the binding that occurs between paired species such as enzymes / substrates, receptors / ligands, antibodies / antigens, and lectins / carbohydrates, which can be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. Therefore, "specific binding" occurs between paired species, where there is an interaction between the two, resulting in a binding complex with the characteristics of an antibody / antigen or ligand / receptor interaction. By measuring the level of activity in a functional assay after in vivo administration, the biological activity of the wnt synthesis factor in the composition can be determined, such as accelerated bone regeneration, enhanced stem cell proliferation, etc., nuclear localization of β-catenin, increased transcription of wnt response genes; etc.

[0108] Each binding domain can be a small molecule or a polypeptide and can be selected from any domain that binds to the extracellular domain of the desired receptor with high affinity, e.g., K D is at least about 1 x 10 -7 M, at least about 1x 10 -8 M, at least about 1x 10 -9 M, at least about 1x 10 -10M. Suitable binding domains include, but are not limited to, binding proteins designed from scratch, binding proteins derived from antibodies, such as scFv, Fab, etc., and other parts of antibodies that specifically bind to one or more proteins; binding domains derived from nanobodies; engineered scaffolds based on knottin; and the like.

[0109] The binding domain can be subjected to affinity selection to enhance the combination with the required extracellular domain. The affinity selection method for this purpose can optionally utilize one or more rounds of selections carried out in the following manner: introduce targeted amino acid changes and produce a candidate coding sequence library, transform a cell population with the candidate coding sequence, for example, transform into a yeast cell, select (for example, using paramagnetic microbeads) required specificity. Usually multiple rounds of selections will be carried out, and the resulting vector will be sequenced and used as the basis of protein engineering. For example, a binding domain can be selected, including but not limited to a domain, an engineered protein, etc., in modified cytokines, antibodies or nanoantibodies, to selectively bind to the target cell extracellular domain.

[0110] Variants. The binding domain may also include derivatives, variants, and biologically active fragments of the above polypeptides, such as variants of natural ligands. A "variant" polypeptide means a biologically active polypeptide having less than 100% sequence identity with a provided sequence as defined below. Such variants include polypeptides comprising one or more amino acid modifications (e.g., insertions, deletions, or substitutions) compared to the provided sequence, for example, wherein one or more amino acid residues are added to the N-terminus or C-terminus of the native sequence or within it; about 1 to 40 amino acid residues are deleted, optionally substituted with one or more amino acid residues; and derivatives of the above polypeptides, wherein the amino acid residues have been covalently modified so that the resulting product has non-naturally occurring amino acids. Typically, a biologically active variant will have an amino acid sequence having at least about 90% amino acid sequence identity, preferably at least about 95%, and more preferably at least about 99% amino acid sequence identity with a native sequence polypeptide.

[0111] A "functional derivative" of a sequence is a compound that has qualitative biological properties in common with the original sequence. "Functional derivatives" include, but are not limited to, fragments of a sequence and derivatives of a sequence, provided that they have a common biological activity. The term "derivative" encompasses amino acid sequence variants of a polypeptide and covalent modifications thereof.

[0112] The binding domains used in the subject compositions and methods can be modified using common molecular biology techniques and synthetic chemistry to improve their resistance to protein degradation, or to optimize solubility, or to make them more suitable as therapeutic agents. Analogs of such polypeptides include analogs containing residues other than naturally occurring L-amino acids (e.g., D-amino acids or non-naturally occurring synthetic amino acids). D-amino acids can replace some or all of the amino acid residues.

[0113] Synthetic factors can be fused or bonded to other polypeptide sequences. Examples include immunoadhesins that combine synthetic factors with immunoglobulin sequences, particularly Fc sequences, and epitope tag polypeptides that contain natural inhibitor polypeptides or portions thereof fused to "tag polypeptides". Tag polypeptides have enough residues to provide epitopes against which antibodies can be produced, but are short enough that they do not interfere with the biological activity of natural inhibitor polypeptides. Suitable tag polypeptides typically have at least 6 amino acid residues, typically between about 6-60 amino acid residues. Synthetic factors can also be fused or combined in formulations, or co-administered with agents that enhance activity, such as cytokines, growth factors, chemotherapeutic agents, immunosuppressants, etc.

[0114] Linker. The binding domain can be separated by a linker, such as a polypeptide linker or a non-peptide linker. The amino acid linker of the linking domain can play an important role in the structure and function of the multi-domain protein. There are many examples of proteins that require a suitable linker composition for catalytic activity. In general, it has been confirmed that changing the linker length of the linking domain can affect protein stability, folding rate and inter-domain orientation (see George and Hering (2003) Prot. Eng. 15: 871-879). The length of the linker in the synthetic factor and the interval between the binding domains can be used to adjust the signal intensity of the synthetic factor, and can be selected according to the desired use of the synthetic factor. The mandatory distance between the binding domains of the synthetic factor can vary, but in certain embodiments can be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, less than about 50 angstroms.

[0115] In some embodiments, the linker is a rigid linker, in other embodiments, the linker is a flexible linker. In some embodiments, the linker portion is a peptide linker. In some embodiments, the peptide linker comprises 2 to 100 amino acids. In some embodiments, the peptide linker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 98, 99, but not more than 100 amino acids. In some embodiments, the peptide linker is 5 to 75, 5 to 50, 5 to 25, 5 to 20, 5 to 15, 5 to 10, or 5 to 9 amino acids in length. Exemplary linkers include linear peptides with at least two amino acid residues such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, Gly-Gly-Gly-Gly-Ser. Suitable linear peptides include polyglycine, polyserine, polyproline, polyalanine and the oligopeptides consisting of alanyl and / or seryl and / or prolyl and / or glycyl amino acid residues. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of Gly9, Glu9, Ser9, Gly5-Cys-Pro2-Cys, (Gly4-Ser)3, Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn, Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn, Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys, and Gly9-Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn. In one embodiment, the linker comprises the amino acid sequence GSTSGSGKSSEGKG or (GGGGS)n, wherein n is 1, 2, 3, 4, 5, etc.; however many such linkers are known and used in the art and may be used for this purpose.

[0116] The synthetic factor can be provided in a single chain form, which means that the binding domains are linked by peptide bonds through a linker peptide. In other embodiments, the binding domains are single peptides and can be linked by non-peptide linkers.

[0117] Chemical groups that can be used to attach binding domains include carbamates, amides (amines plus carboxylic acids), esters (alcohols plus carboxylic acids), thioethers (alkyl halides plus sulfhydryls; maleimides plus sulfhydryls), Schiff bases (amines plus aldehydes), ureas (amines plus isocyanates), thioureas (amines plus isothiocyanates), sulfonamides (amines plus sulfonyl chlorides), disulfides, hydrazones, lipids, and the like, as known in the art.

[0118] The connection between the binding domains may include a spacer, such as an alkyl spacer, which may be a straight chain or branched chain, is typically a straight chain, and may include one or more unsaturated bonds; typically has 1 to about 300 carbon atoms; more typically has about 1 to 25 carbon atoms; and may be about 3 to 12 carbon atoms. This type of spacer may also contain heteroatoms or functional groups, including amines, ethers, phosphodiesters, and the like. Specific target structures include: (CH2CH2O)n, where n is 1 to about 12; (CH2CH2NH)n, where n is 1 to about 12; [(CH2)n(C=O)NH(CH2) m ] z , wherein n and m are from 1 to about 6, and z is from 1 to about 10; [(CH2)nOPO3(CH2) m ] z , wherein n and m are from 1 to about 6, and z is from 1 to about 10. Such linkers may include polyethylene glycol which may be linear or branched.

[0119] The binding domains can be connected by a homo- or hetero-bifunctional linker having a group at one end capable of forming a stable connection with a hydrophilic head group and a group at the opposite end capable of forming a stable connection with a targeting moiety. Illustrative entities include: azidobenzohydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), disulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, N-γ-maleimidobutyryloxysuccinimidyl ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MAL; 4- [N-maleimidomethyl] cyclohexane-1-carboxylic acid succinimidyl ester; 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimidyl ester (SPDP); N,N'-(1,3-phenylene) bismaleimide; N,N'-ethylene-bis-(iodoacetamide); or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimidyl ester (SMCC); m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS) and 4-(p-maleimidophenyl) butyric acid succinimidyl ester (SMPB), which is a chain-extended analog of MBS. The succinimidyl group of these crosslinkers reacts with primary amines, and the thiol-reactive maleimide forms a covalent bond with the thiol of the cysteine ​​residue.

[0120] Other reagents used for this purpose include: p,p'-difluoro-m,m'-dinitrodiphenyl sulfone (which forms irreversible crosslinks with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4-disulfonyl chloride (which reacts primarily with amino groups); hexamethylene diisocyanate or diisothiocyanate, or azobenzene-p-diisocyanate (which reacts primarily with amino groups); bis-nitrobenzidine (which reacts primarily with tyrosine and histidine); o-phenyltriazolyloxytetramethyluronium hexafluorophosphate (HATU), dicyclohexylcarbodiimide, bromo-tris(pyrrolidinyl)phosphonium bromide (PyBroP); N,N-dimethylaminopyridine (DMAP); 4-pyrrolidinylpyridine; N-hydroxybenzotriazole; etc. Homobifunctional crosslinking agents include bismaleimidohexane ("BMH").

[0121] Antibody: As used herein, the term "antibody" refers to a polypeptide comprising classical immunoglobulin sequence elements sufficient to confer specific binding to a specific target antigen. As known in the art, a complete antibody produced in nature is a tetrameric agent of approximately 150 kD, consisting of two identical heavy chain polypeptides (each about 50 kD) and two identical light chain polypeptides (each about 25 kD), which are associated with each other into a substance generally referred to as a "Y-shaped" structure. Each heavy chain consists of at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the top of the Y structure), followed by three constant domains: CH1, CH2, and carboxyl-terminal CH3 (located at the base of the Y stem). A short region called a "switch" connects the heavy chain variable region and the constant region. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in the hinge region connect the two heavy chain polypeptides in the complete antibody to each other. Each light chain consists of two domains - an amino-terminal variable (VL) domain, followed by a carboxyl-terminal constant (CL) domain, separated from each other by another "switch". A complete antibody tetramer consists of two heavy chain-light chain dimers, in which the heavy chain and the light chain are connected to each other by a single disulfide bond; two additional disulfide bonds connect the heavy chain hinge regions to each other, so that the dimers are connected to each other and form a tetramer. Naturally produced antibodies are usually also glycosylated on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold", which is formed by two β folds (e.g., 3-, 4- or 5-chain folds) that are packaged relative to each other into a compressed antiparallel β barrel. Each variable domain contains three hypervariable loops called "complementarity determining regions" (CDR1, CDR2 and CDR3) and four "framework" regions (FR1, FR2, FR3 and FR4) that are somewhat invariant. When a native antibody folds, the FR regions form beta sheets that provide a structural framework for the domains, and the CDR loop regions from both the heavy and light chains come together in three dimensional space to create a single hypervariable antigen binding site located at the apex of a Y structure.

[0122] The Fc region of naturally occurring antibodies binds to elements of the complement system and also binds to receptors on effector cells (including, for example, effector cells that mediate cytotoxicity). As is known in the art, the affinity and / or other binding properties of the Fc region for Fc receptors can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized according to the present invention include glycosylated Fc domains, including Fc domains having such glycosylation that has been modified or engineered.

[0123] Any polypeptide or polypeptide complex comprising sufficient immunoglobulin domain sequences found in natural antibodies can be referred to and / or used as an "antibody", whether such polypeptides are produced naturally (e.g., by an organism that reacts to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, as known in the art, antibody sequence elements can be humanized, primatized, chimeric, etc.

[0124] Furthermore, the term "antibody" as used herein may refer in appropriate embodiments (unless otherwise specified or clear from the context) to any construct or format known or developed in the art to utilize the structural and functional characteristics of antibodies in alternative presentations. For example, in one embodiment, the format of an antibody utilized according to the present invention is selected from, but not limited to, complete IgG, IgE and IgM, bispecific or multispecific antibodies (e.g., etc.), single-chain Fv, Fab, small modular immunopharmaceuticals (“SMIPs TM ”), single-chain or tandem diabodies VHH, Minibody Ankyrin repeat protein or DART, TCR-like antibodies, Microprotein, and In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if it were produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other side groups [e.g., polyethylene glycol, etc.].

[0125] In many embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementary determining regions (CDRs); in some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR substantially identical to the CDR found in the reference antibody (e.g., at least one heavy chain CDR and / or at least one light chain CDR). In some embodiments, the included CDR is substantially identical to the reference CDR because it is identical in sequence or contains 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because it exhibits at least 96%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because at least one amino acid in the included CDR is deleted, added or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because 1-5 amino acids in the included CDR are deleted, added or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because at least one amino acid in the included CDR is substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that 1-5 amino acids within the included CDR are deleted, added or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0126] Small molecule compositions. Synthetic factors also include organic molecules, preferably small organic compounds with a molecular weight greater than 50 and less than about 20,000 Daltons. Useful synthetic factors are identified, for example, by screening assays in which high affinity binding of the molecule to one or two target ECDs is determined. The molecule can provide a binding moiety that will be linked to another binding moiety, or to a binding domain as described above for a polypeptide agent.

[0127] Candidate synthesis factors contain functional groups necessary for structural interaction with the receptor ECD, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two chemical functional groups. Candidate synthesis factors often contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found in biomolecules, including peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0128] Candidate synthesis factors can be obtained from a variety of sources, including synthetic or natural compound libraries. For example, many methods can be used for random and directional synthesis of a variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, natural compound libraries in the form of bacterial, fungal, plant and animal extracts can be obtained or easily produced. In addition, natural or synthetically produced libraries and compounds are easy to modify by conventional chemical, physical and biochemical methods, and can be used to produce combinatorial libraries. Known pharmacological agents can be directed or randomly chemically modified, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs. Test agents can be obtained from libraries, such as natural product libraries or combinatorial libraries. Many different types of combinatorial libraries and methods for preparing such libraries have been described, including, for example, PCT publications WO93 / 06121, WO 95 / 12608, WO 95 / 35503, WO 94 / 08051 and WO95 / 30642, each of which is incorporated herein by reference.

[0129] In the case where the screening assay is a binding assay, one or more molecules can be attached to a label, wherein the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescent agents, chemiluminescent agents, enzymes, specific binding molecules, particles (e.g., magnetic particles), etc. Specific binding molecules include pairs such as biotin and streptavidin, digoxin and anti-digoxin. For specific binding members, complementary members will generally be labeled with molecules that provide detection according to known procedures.

[0130] A variety of other reagents may be included in the screening assay. These include reagents such as salts, neutral proteins (e.g., albumin), detergents, etc., which are used to promote optimal protein-protein binding and / or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay may be used, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. The mixture of components is added in any order that provides the necessary binding. Incubation is performed at any suitable temperature, typically between 4 and 40°C. The incubation period is selected to obtain optimal activity, but it can also be optimized to facilitate rapid high-throughput screening. Typically 0.1 to 1 hour is sufficient.

[0131] Preliminary screening can be performed by screening compounds that can bind to the target receptor polypeptide. Binding assays generally involve contacting the receptor ECD with one or more test compounds, and allowing the protein and test compound to have enough time to form a binding complex. Any binding complex formed can be detected using any of many established analytical techniques. Protein binding assays include but are not limited to measuring coprecipitation, co-migration on non-denaturing SDS-polyacrylamide gels and co-migration on Western blotting methods (see, for example, Bennet, JP and Yamamura, HI (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, HI et al., ed.), 61-89 pages.

[0132] Certain screening methods involve screening for compounds that modulate signal transduction activity. Such methods may involve performing a cell-based assay in which a test compound is contacted with one or more cells, expressing and then detecting an increase in the expression of a response gene, detecting changes in various adaptor proteins Jak, STAT, etc.

[0133] The level of expression or activity can be compared with the baseline value. As mentioned above, the baseline value can be the value of the control sample or the statistical value of the expression level representing the control population. The expression level of cells that do not express the receptor can also be measured as a negative control. Such cells are generally substantially genetically identical to the test cells in addition. Various controls can be performed to ensure that the observed activity is real, including parallel reactions with cells lacking a reporter gene construct or not contacting the cells carrying the reporter gene construct with the test compound. Compounds can also be further verified as described below.

[0134] Compounds initially identified by any of the aforementioned screening methods can be further tested to verify apparent activity. The basic form of such methods involves administering the lead compound identified during the initial screening to an animal or a cell culture model used as a human model. The animal model used in the validation study is typically a mammal. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats.

[0135] The active test agent identified by the screening method described herein can be used as a lead compound for synthesizing similar compounds. Generally, similar compounds are synthesized to have electronic configurations and molecular conformations similar to the lead compound. Similar compounds can be identified by using techniques such as self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode kinetic analysis. Computer programs for implementing these techniques can be used. See, for example, Rein et al., (1989) Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York).

[0136] Pharmaceutical composition

[0137] For therapeutic use, the synthetic factors are administered to mammals (preferably humans) in physiologically acceptable dosage forms, including those that can be administered to humans intravenously as a bolus or by continuous infusion over a period of time. Alternative routes of administration include topical, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes. The synthetic factors are also suitable for administration by intratumoral, peritumoral, intralesional or perilesional routes, or to lymphatic administration, to exert local and systemic therapeutic effects.

[0138] Pharmaceutical compositions may also comprise a combination of molecules of the invention and cells, including stem cells, progenitor cells, immune effector cells, etc. In such combinations, cells may be pre-treated with the molecules of the invention prior to use, e.g., immune effector cells are treated ex vivo with synthetic factors; cells may be administered simultaneously with the molecules of the invention in separate or combined formulations; cells may be provided to an individual prior to treatment with the molecules of the invention, etc.

[0139] Depending on the desired formulation, the pharmaceutical composition may include a pharmaceutically acceptable non-toxic diluent carrier, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may include other carriers, adjuvants, or non-toxic non-therapeutic, non-immunogenic stabilizers, excipients, and the like. The composition may also contain other substances that approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, wetting agents, and detergents.

[0140] The composition may also include any of a variety of stabilizers, such as antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide may be complexed with various known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifications or complexing agents include sulfates, gluconates, citrates, and phosphates. The polypeptide of the composition may also be complexed with molecules that enhance its in vivo properties. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0141] Further guidance on formulations suitable for various types of administration may be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of drug delivery methods, see Langer, Science 249: 1527-1533 (1990).

[0142] The pharmaceutical compositions can be administered for prophylactic and / or therapeutic treatment. Toxicity and therapeutic efficacy of the active ingredients can be determined in cell cultures or experimental animals according to standard pharmaceutical procedures, including, for example, determination of LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as LD 50 / ED 50 Ratios. Compounds that exhibit large therapeutic indices are preferred.

[0143] The data obtained from cell culture and / or animal studies can be used to formulate a range of dosage for use in humans. The dose of the active ingredient is generally within a range that includes the ED with low toxicity. 50The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0144] For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. The active ingredient can be encapsulated in a gelatin capsule with an inactive ingredient and a powdered carrier, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum powder, magnesium carbonate. Examples of other inactive ingredients that can be added to provide desired color, taste, stability, buffering capacity, dispersibility or other known desired characteristics are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide and edible white ink. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be made into sustained release products to provide continuous release of drugs within a few hours. Compressed tablets can have sugar coating or film coating to cover any unpleasant taste and protect tablets from atmospheric influences, or have enteric coating to selectively disintegrate in the gastrointestinal tract. Liquid dosage forms for oral administration can contain colorants and flavorings to increase patient acceptance.

[0145] The active ingredients, alone or in combination with other suitable ingredients, can be made into aerosol formulations to be administered by inhalation (ie, they can be "nebulized"). Aerosol formulations can be placed in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen.

[0146] Formulations suitable for parenteral administration, such as by intra-articular (in a joint), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and include aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickening agents, stabilizers and preservatives.

[0147] The components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, usually at least analytical grade, more usually at least pharmaceutical grade). Moreover, compositions intended for in vivo use are generally sterile. In the sense that a given compound must be synthesized prior to use, the resulting product is generally substantially free of any potentially toxic agents, particularly any endotoxins, that may be present during the synthesis or purification process. Compositions for parenteral administration are also sterile, substantially isotonic and prepared under GMP conditions.

[0148] The effective amount of the therapeutic composition to be administered to a particular patient will depend on a variety of factors, several of which will vary from patient to patient. The formulation may be provided, for example, in unit doses. A competent clinician will be able to determine the effective amount of the therapeutic agent to be administered to the patient. The dosage of the synthetic factor will depend on the treatment, the route of administration, the nature of the therapeutic agent, the sensitivity of the disease to the therapeutic agent, etc. 50 Animal data and other available information, clinicians can determine the maximum safe dose for an individual based on the route of administration. Compositions that are rapidly cleared from the body can be administered at higher doses or in repeated doses to maintain therapeutic concentrations. Using common techniques, competent clinicians will be able to optimize the dose of a specific therapeutic or imaging composition during routine clinical trials. Typically, the dose is 0.001 to 100 mg of the agent per kg of subject body weight.

[0149] The composition may be administered to a subject more than once. For therapeutic compositions, it is sometimes necessary to administer regularly (e.g., every 2-3 days), or it may be necessary to administer regularly to reduce toxicity. For therapeutic compositions to be used in repeated dosage regimens, it is preferred that the portion that does not induce an immune response.

[0150] In another embodiment of the present invention, a product containing materials that can be used to treat the conditions described herein is provided. The product includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed of a variety of materials, such as glass or plastic. The container holds a composition effective for treating the condition and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The active agent in the composition is a synthetic factor. The label on or attached to the container indicates that the composition is used to treat the selected condition. Other containers can be provided for the product, which can hold, for example, pharmaceutically acceptable buffers, such as phosphate buffered saline, Ringer's solution, or dextrose solution. The product can also include other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use.

[0151] As used herein, the term "therapeutically effective amount" means an amount sufficient to treat the disease, disorder and / or condition when administered to a population suffering from or susceptible to the disease, disorder and / or condition according to a therapeutic agent dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder and / or condition, stabilizes one or more characteristics thereof and / or delays its onset. One of ordinary skill in the art will recognize that the term "therapeutically effective amount" does not actually require successful treatment to be achieved in a particular individual. Instead, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a considerable number of subjects when administered to a patient in need of such treatment.

[0152] For example, in some embodiments, the term "therapeutically effective amount" refers to an amount that, when administered to an individual in need thereof in the context of the present therapies, will block, stabilize, attenuate or reverse a disease process occurring in the individual.

[0153] How to use

[0154] Synthetic factors can be used for preventive and therapeutic purposes. Therefore, as used herein, the term "treatment" is used to refer to the prevention of disease and the treatment of pre-existing conditions. In some cases, prevention refers to the inhibition or delay of the onset of a disease or condition in a patient identified as being at risk of developing a disease or condition. The treatment of persistent diseases to stabilize or improve the patient's clinical symptoms is a particularly important benefit provided by the present invention. It is expected that such treatment will be performed before loss of function in the affected tissue; therefore, the preventive treatment benefits provided by the present invention are also important. Evidence of efficacy can be any reduction in the severity of the disease. The efficacy can be measured based on clinical results, or can be determined by immunological or biochemical tests. The patient being treated can be a mammal, such as a primate, including humans, can be an experimental animal, such as a rabbit, rat, mouse, etc., and particularly for the evaluation of therapy, is a horse, dog, cat, farm animal, etc.

[0155] The dosage of a therapeutic formulation (e.g., a pharmaceutical composition) can vary widely, depending on the nature of the condition, the frequency of administration, the mode of administration, the clearance of the agent from the host, etc. In certain embodiments, the initial dose may be larger, followed by smaller maintenance doses. In certain embodiments, the dose may be administered infrequently, weekly or biweekly, or more often divided into smaller doses and administered daily, semiweekly, or otherwise as needed to maintain an effective dosage level.

[0156] In some embodiments of the present invention, the administration of the composition or formulation comprising the synthetic factor is carried out by local administration. As used herein, limited administration may refer to local administration, but also refers to injection at the treatment site or otherwise introduced into the body. Examples of such administration include intramuscular injection, subcutaneous injection, intraperitoneal injection, etc. In other embodiments, the composition or formulation comprising the synthetic factor is systemically administered, such as orally or intravenously. In one embodiment, the composition or formulation comprising the synthetic factor is administered by infusion, such as continuously infused over a period of time, such as 10 minutes, 20 minutes, 3 minutes, 1 hour, 2 hours, 3 hours, 4 hours or longer.

[0157] In some embodiments of the invention, the composition or formulation is administered in a short period of time, such as a single administration, or in a series of administrations, such as 1, 2, 3 or more days, up to 1 or 2 weeks, in order to obtain a rapid, significant increase in activity. The size of the administered dose must be determined by the physician and will depend on many factors, such as the nature and severity of the disease, the age and health of the patient, and the patient's tolerance to the drug itself.

[0158] In certain methods of the invention, an effective amount of a composition comprising a synthetic factor is provided to a cell, for example, by contacting the cell with an effective amount of the composition to achieve a desired effect, such as enhanced signaling, diffusion, etc. In certain embodiments, the contact is performed in vitro, ex vivo, or in vivo. In certain embodiments, the cell is derived from or present in a subject in need of increased signaling.

[0159] In some methods of the invention, an effective amount of the subject composition is provided to enhance signal transduction in a cell. Biochemically, an effective amount or effective dose of a synthetic factor is an amount that increases signal transduction in a cell by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% relative to signal transduction in the absence of the synthetic factor. The modulated amount of cellular activity can be determined in a variety of ways known to those of ordinary skill in the biological field.

[0160] In clinical terms, an effective dose of a synthetic factor composition is a dose that, when administered to a subject for a suitable period of time, such as at least about one week, possibly about two weeks or longer, up to about 4 weeks, 8 weeks or longer, will show a change in symptoms associated with a lack of signaling. In some embodiments, an effective dose will not only slow or stop the progression of the disease condition, but will also induce a reversal of the condition. One skilled in the art will appreciate that an initial dose may be administered for such a period of time, followed by a maintenance dose, which in some cases will be a reduced dose.

[0161] The calculation of the effective amount or effective dose of the synthetic factor composition to be administered is within the skill of those of ordinary skill in the art and will be routine for those skilled in the art. Needless to say, the final amount administered will depend on the route of administration and the nature of the disorder or condition to be treated.

[0162] Cells suitable for the subject method are cells comprising one or more receptors. The cells to be contacted can be in vitro, i.e. in culture, or they can be in vivo, i.e. in a subject. The cells can be from / in any organism, but preferably from mammals, including humans, livestock and farm animals, and zoos, laboratories or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebrafish, fruit flies, worms, etc. Preferably, the mammal is a human. The cells can be from any tissue. The cells can be frozen, or can be fresh. They can be primary cells, or can be cell lines. The cells are typically primary cells used in vivo, or primary cells treated in vitro before being introduced into the recipient.

[0163] In vitro cells can be contacted with a composition comprising a synthetic factor by any of many well-known methods in the art. For example, the composition can be provided to cells in the culture medium of the subject cell being cultivated. The nucleic acid encoding the synthetic factor can be provided to the subject cell or the cell co-cultured with the subject cell on a carrier under conditions (e.g., electroporation, calcium chloride transfection, and lipofection) that promote its uptake as known in the art. Alternatively, the nucleic acid encoding the synthetic factor can be provided to the subject cell or the cell co-cultured with the subject cell by a virus, even if the cell is contacted with a viral particle comprising a nucleic acid encoding a peptide synthetic factor polypeptide. Retroviruses, such as slow viruses, are particularly suitable for the method of the present invention because they can be used to transfect non-dividing cells (see, e.g., Uchida et al. (1998) PNAS 95 (20): 11939-44). Commonly used retroviral vectors are "defective", i.e., can not produce the viral proteins required for productive infection. On the contrary, the replication of the vector needs to grow in a packaging cell line.

[0164] Likewise, cells in vivo may be contacted with the subject synthetase compositions by any of a number of well-known methods in the art for administering peptides, small molecules, or nucleic acids to a subject. The synthetase compositions may be incorporated into a variety of formulations or pharmaceutical compositions, which in some embodiments will be formulated in the absence of detergents, liposomes, etc., as has been described for formulation of the full-length protein.

[0165] In some embodiments, the compounds of the invention are administered for treating diseased or damaged tissue, for tissue regeneration and for cell growth and proliferation, and / or for tissue engineering. Specifically, the invention provides wnt synthesis factors or compositions comprising one or more synthesis factors according to the invention for treating tissue loss or damage due to aging, trauma, infection or other pathological conditions.

[0166] In some embodiments, the synthetic factor acts on immune effector cells and regulates immune response. For example, the pathways involved in inflammatory diseases can be targeted. Inflammation is the process in which the immune system responds to infection or tissue damage. Inflammatory diseases are caused by the activation of the immune system, which causes illness in the absence of infection or tissue damage, or at the level of response that causes illness. Inflammatory diseases include autoimmune diseases, which are caused by immunity, any disease that is misdirected in healthy cells and / or tissues of the body. Autoimmune diseases are characterized by abnormal targeting of self-proteins, polypeptides, peptides and / or other self-molecules by T and B lymphocytes, resulting in damage and / or dysfunction of organs, tissues or cell types (e.g., pancreas, brain, thyroid or gastrointestinal tract) in the body and causing the clinical manifestations of the disease. Autoimmune diseases include diseases that affect specific tissues and diseases that can affect multiple tissues, which depends in part on whether the response is directed to antigens that are limited to specific tissues or antigens that are widely distributed in the body.

[0167] The immune system employs a highly complex mechanism designed to generate responses to protect mammals from a variety of foreign pathogens while preventing responses to self-antigens. In addition to deciding whether to respond (antigen specificity), the immune system must also select the appropriate effector functions to deal with each pathogen (effector specificity). Target inflammatory diseases include, but are not limited to, secondary progressive multiple sclerosis (SPMS); primary progressive multiple sclerosis (PPMS); neuromyelitis optica (NMO); psoriasis; systemic lupus erythematosus (SLE); ulcerative colitis; Crohn's Disease; ankylosing spondylitis (see, e.g., Mei et al. (2011) Clin. Rheumatol. 30:269-273; type 1 (IDDM); asthma; chronic obstructive pulmonary disease (COPD); chronic hepatitis; amyotrophic lateral sclerosis (ALS); Alzheimer's Disease (AD); Parkinson's Disease; frontotemporal lobar degeneration (FTLD); atherosclerosis / cardiovascular disease and obesity / metabolic syndrome.

[0168] In other embodiments, the synthetic factor activates immune effector cells to treat cancer, or activates the pathway of inducing cancer cell death or reducing cancer cell growth. As used herein, the term "cancer" refers to a variety of conditions caused by abnormal, uncontrolled cell growth. Cells that can cause cancer, called "cancer cells", have characteristic properties, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or some typical morphological features. Cancer can be detected in any of many ways, including but not limited to detecting the presence of a tumor or multiple tumors (e.g., by clinical or radiological means), examining cells in the tumor or from another biological sample (e.g., from a tissue biopsy), measuring blood markers indicating cancer, and detecting the genotype indicating cancer. However, the negative results of one or more of the above-mentioned detection methods do not necessarily indicate that there is no cancer, for example, patients who have shown a complete response to cancer treatment may still suffer from cancer, as demonstrated by subsequent recurrence.

[0169] The term "cancer" as used herein includes cancers, (e.g., carcinoma in situ, invasive cancer, metastatic cancer) and pre-malignant conditions, i.e., new metastatic changes that are unrelated to their histological origin. The term "cancer" is not limited to any stage, grade, histomorphological characteristics, invasiveness, aggressiveness or malignancy of the affected tissue or cell aggregation. Specifically, it includes stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer and primary cancer.

[0170] Cancers and cancer cells that can be treated include, but are not limited to, hematological cancers, including leukemias, lymphomas, and myelomas, and solid cancers, including, for example, brain tumors (glioblastoma, medulloblastoma, astrocytoma, oligodendroglioma, ependymoma), carcinomas (e.g., lung cancer, liver cancer, thyroid cancer, bone cancer, adrenal cancer, spleen cancer, kidney cancer, lymph node cancer, small intestine cancer, pancreatic cancer, colon cancer, stomach cancer, breast cancer, endometrial cancer, prostate cancer, testicular cancer, ovarian cancer, skin cancer, head and neck cancer, and esophageal cancer).

[0171] The synthetic factors of the present invention also have wide application in non-therapeutic methods, such as in vitro research methods. The synthetic factors can be administered directly to cells in vivo, administered to patients orally, intravenously or by other methods known in the art, or administered to cells ex vivo. In some embodiments where the synthetic factors of the present invention are administered to cells ex vivo, these cells can be transplanted into the patient before, after or during administration of the synthetic factors.

[0172] Now that the invention is fully described, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

[0173] experiment

[0174] Synthetic factors: synthetic cytokines and growth factor agonists that force signaling through non-native receptor dimers

[0175] Cytokines and growth factor ligands are usually signaled by homologous or heterodimer cell surface receptors via trans-phosphorylation mediated by JAK / TYK or RTK. However, the number of such receptor pairings occurring in nature is limited to those driven by endogenous ligands encoded in our genome. We have engineered synthetic cytokines (synthetic factors), which drive the formation of cytokine receptor pairings that are not formed by endogenous cytokines, and activate different signal transduction programs. We have confirmed that a wide range of non-natural cytokine receptor heterodimers can carry out signal transduction. Our engineered synthetic factor assembly is not naturally occurring IL-2Rβ / IL-4Rα or IL-4Rα / IFNAR2 receptor heterodimers, triggering signal transduction and functional responses different from signal transduction and functional responses activated by natural IL-2, IL-4 and IFN cytokines. In addition, the hybrid synthetic factor ligands that dimerize JAK / STAT cytokine receptors with receptor tyrosine kinases (RTKs) also trigger signal transduction outputs. The synthetic factors represent a new family of synthetic "orphan" ligands to open up the full combinatorial range of dimeric signaling receptors encoded within the human genome.

[0176] For JAK / STAT cytokine receptors, it has been previously demonstrated in several systems that genetically modified chimeric receptors in which the extracellular domain (ECD) of a cytokine receptor has been fused to the intracellular domain (ICD) of an unrelated receptor activate signaling in a ligand-dependent manner. However, in order for this concept to be practically useful, soluble ligands that can selectively mate endogenous receptors and assemble non-natural dimers on unmodified cells and tissues are required. This can be achieved by synthetic cytokines or synthetic factors that drive the formation of cytokine receptor pairs that are not formed by natural endogenous cytokines.

[0177] Compared to genomic-encoded cytokines, synthetic factors can activate new signaling programs and elicit unique immunomodulatory activities, providing an almost unlimited supply of ligands with new functions. Previous studies have reported the engineering of cytokine variants that promote new activities by genetically fusing two cytokines via a polypeptide linker, thereby generating dimers of native receptor signaling dimers. An important claim to this approach is that the two linked cytokines are fully active on their own and produce an additive combination of two native cytokine signaling dimers. Therefore, they can activate signaling in cells expressing only one pair of cognate receptors as well as in cells expressing four receptor subunits engaged by the two cytokines, resulting in a mixed phenotype. Therefore, the molecular basis for the differential activities exhibited by these linked ligands is unclear.

[0178] Engineering cytokine ligands (synthetic factors) that dimerize two different cytokine receptors at the surface of responding cells in a typically molecularly defined 1:1 stoichiometry provides an alternative approach that will produce unique, rather than additive, signaling outputs. The formation of defined dimeric complexes, such as those formed by genomically encoded cytokines, allows precise mechanistic insight into the nature of the signaling complexes that initiate new signaling programs and activities engaged by these ligands. Signaling initiated by synthetic factors will be more uniform and targeted than that of linked cytokines, thereby reducing pleiotropic effects and potential toxicity caused by off-target effects. The synthetic factor approach allows one to explore non-natural cytokine receptor pairs and determine whether they activate signaling.

[0179] To explore the generality of this idea, we expressed and characterized a 10×10 matrix of chimeric cytokine receptor pairs using orthogonal extracellular domains as common dimerization units fused to cytokine receptor intracellular domains, allowing the evaluation of signaling of 100 different cytokine receptor dimers. Most cytokine receptor pairs sampled in this chimeric receptor matrix activated signaling. In a second step, we genetically fused two antagonist forms of IL-2, IL-4, and interferon (IFN) to a polypeptide linker to engineer synthetic factors that dimerize non-natural cytokine receptor pairs on the cell surface. Stimulation of cell lines and peripheral blood mononuclear cells (PBMCs) with engineered synthetic factors showed signaling and cellular characteristics different from those of the parental cytokines.

[0180] We extend the synthetic factor concept to dimerize c-kit, a tyrosine kinase receptor, and thrombopoietin receptor (TpoR), a JAK / STAT cytokine receptor, to generate measurable signaling outputs that are qualitatively different from those induced by their respective endogenous ligands. Our results are proof of concept that dimerization of non-native JAK / STAT and RTK receptor pairs is a viable approach to generate novel signaling programs and activities that can be explored for their functional consequences as if they were newly discovered orphan cytokine ligands.

[0181] First we wanted to determine the potential for JAK / STAT crosstalk between a large number of forced non-native cytokine receptor dimers ( Figure 1A We generated a series of chimeric receptors in which the extracellular domain (ECD) of the IL-1 receptor (IL-1R1 and IL-1R1Acp) was fused to the transmembrane (TM) and intracellular domains (ICD) of ten different cytokine receptors, generating a 10·10 matrix of possible receptor pair combinations ( Figure 1B We used the IL-1 system for two reasons: 1) IL-1 binds to its receptor with very high affinity, allowing signal activation even at low receptor expression levels; and 2) IL-1 does not signal through the classical JAK / STAT pathway, eliminating the background activity generated by endogenous IL-1 receptor dimerization. Jurkat cells expressing all JAKs and STATs except STAT4 were electroporated with the indicated chimeric receptor combinations and analyzed by flow cytometry for IL-1R1 and IL-1R1Acp surface expression ( Figure 2D ), and IL-1-dependent signaling activation was analyzed by Western blot ( Figure 2A and Figure 2E ).

[0182] Although most IL-1 receptor combinations showed robust cell surface expression, very low levels of expression were detected for some receptor pairs, but this did not significantly affect the detection of signaling resulting from IL-1 stimulation ( Figure 2E ). Figure 2A A binary heat map depicting the phosphorylation of the six STATs is presented in Figure 2. Red squares indicate signaling and black squares indicate no signaling. As expected, STAT2 and STAT4 were not activated by any combination of receptors due to low STAT2 expression and lack of STAT4 expression in Jurkat cells ( Figure 2ASTAT1 and STAT6 proteins are activated only by chimeric receptor pairs containing IFNAR2 and IL-4Rα, respectively, consistent with the specific activation of these two STATs by IFN and IL-4 and IL-13 cytokines ( Figure 2A ).

[0183] In contrast, STAT3 and STAT5 proteins are activated combinatorially by many chimeric receptor pairs, consistent with the more potent use of these two STATs by cytokines ( Figure 2A ). While most receptor pairs activated signaling in combination, we also found receptor pairs that did not induce productive signaling despite robust surface expression ( Figure 2D ), such as IL-2Rβ homodimers and IL-13Rα1 homodimers ( Figure 2A and Figure 2E ). Overall, our data show that most receptor dimer combinations tested activated STAT proteins, revealing a high plasticity of the cytokine-cytokine receptor system.

[0184] Signal activation by the JAK2 / JAK3 cytokine receptor pair. A surprising observation from our chimeric receptor studies was that the chimeric receptor combination pairing JAK2 and JAK3 (i.e., erythropoietin receptor (EpoR) / γc and IL-23R / γc) failed to activate signaling. Interestingly, the JAK2 / JAK3 pairing is not found in nature, raising the question of whether signal activation due to the lack of this pairing could be due to steric clashes or incompatible geometries between the two kinase molecules that would prevent cross-activation.

[0185] To test this hypothesis, we inserted alanine residues into the juxtamembrane domain of EpoR, which has been shown to modulate signaling by altering the juxtamembrane domain of the receptor. 251 Then insert 1 to 4 alanine amino acids ( Figure 2B These EpoR mutants were fused to IL-1R1 ECD and co-transfected with IL-1R1Acp-IFNAR2 (positive control) or IL-1R1Acp-γc in Jurkat cells ( Figure 2F Insertion of one, three, or four alanines into the juxtamembrane domain of EpoR did not affect its signaling ability when paired with IFNAR2, but insertion of two alanines prevented signaling by this receptor pair ( Figure 2C Insertion of one or three alanines in the juxtamembrane domain of EpoR did not restore signaling of the EpoR / γc (JAK2 / JAK3) receptor pair, insertion of four alanines more or less restored signaling, and insertion of two alanines fully restored the signaling activity of the receptor pair ( Figure 2C ).

[0186] "These results suggest that there are structural constraints between JAK2 and JAK3 that prevent these two kinases from triggering signaling in our chimeric receptor system. However, this experiment clearly demonstrates that altering the ligand-receptor geometry in non-native receptor pairs using JAK2 and JAK3 is a viable option to restore signaling."

[0187] We have previously demonstrated that altering the dimer geometry of the EpoR with synthetic factor ligands results in differential signaling outputs, so synthetic factors may also exploit this parameter. In addition to the JAK2 / JAK3 pairing, we observed other chimeric receptor pairs that failed to activate signaling. We asked whether insertion of alanine in the juxtamembrane domain would also restore signaling by these receptors. Insertion of alanine in the juxtamembrane domains of IL-2Rβ, IL-12Rβ, and EpoR did not restore signaling by the IL-2Rβ-IL-2Rβ, IL-12Rβ-IL-23R, and EpoR-EpoR pairs ( Figure 2G ).

[0188] Strikingly, the IL-12Rβ-IL-23R and EpoR-EpoR pairs represent receptor dimers engaged by IL-23 and EPO, respectively. We believe that it is most likely that the extracellular orientation and proximity of the IL-1 receptor is unfavorable for certain natural and non-natural cytokine receptor pairs. This is a technical limitation of the chimeric receptor strategy we used, and the lack of signaling for some pairings is not due to the inability of a particular JAK / TYK / STAT combination to function on its own. The convergent result of the chimeric IL-1 receptor experiments is that many, if not most, non-natural cytokine receptor pairs can signal through one or more STATs, but some pairs will have different dimer orientations and proximity necessary, which will depend on the synthetic factors.

[0189] Signaling activation profiles induced by synthetic factors. We wished to explore whether dimerization of non-native cytokine receptor pairs by synthetic factors would activate signaling in unmodified cells ( Figure 3A). We used a bispecific strategy in which we fused together two cytokines, each of which can bind only to one of its two receptors, thereby creating a defined 1:1 receptor dimer. To achieve this approach, we engineered antagonist or “dominant negative (DN)” forms of IL-4, IL-2, and IFN that retain binding to their high-affinity receptor subunits (IL-4Rα for IL-4, IL-2Rβ for IL-2, and IFNAR2 for IFN), but in doing so disrupt binding to their low-affinity receptor subunits (L-13Rα1 and γc for IL-4, γc for IL-2, and IFNAR1 for IFN). These “DN” cytokines act as high-affinity binding modules that have no signaling activity themselves. As expected, wild-type cytokines activated signaling in the Hut78 T cell line ( Figure 3B ), but the dominant negative mutant failed to promote the designation of Super-2, which has a 200-fold enhanced affinity for the IL-2Rβ receptor subunit ( Figure 3B ).

[0190] We then genetically fused pairs of dominant negative cytokine mutants via a Gly4 / Ser linker to generate novel ligands that induce the formation of non-native receptor dimers of IL-2Rβ-IL-4Rα and IL-4Rα-IFNAR2 ( Figure 3A When added to unmodified cell lines, these bispecific DN synthetases activated a signaling profile that was qualitatively and quantitatively different from that induced by the parental cytokine ( Figure 3D ).

[0191] Stimulation of Hut78 cells with SY1 SL, which promotes dimerization of IFNAR2 and IL-4Rα, leads to activation of STAT5 and STAT6 ( Figure 3D Notably, extending the peptide linker connecting the two dominant negative proteins by 10 residues (SY1LL) increased the signaling potency exhibited by the synthetic factor ( Figure 3D ). Stimulation of Hut78 cells with SY2 synthetase, which dimerizes IL-2Rβ and IL-4Rα, resulted in activation of STAT1 and, to a lesser extent, STAT3, STAT5, and STAT6 ( Figure 3D In all cases, the maximal responses (Emax) elicited by the synthetic factors were significantly lower than those activated by the genomically encoded cytokines.

[0192] Importantly, the distinct signaling programs activated by the synthetic factors are not simply the result of additive effects from the two parental cytokines, as the signaling programs induced by simultaneous addition of the parental cytokine pairs differ from those induced by the corresponding synthetic factors ( Figure 3E To determine whether the signaling program activated by synthezoids is different from that activated by IL-2, IL-4, and IFN, we investigated the role of synthezoids in CD4 + The activation of 120 different signaling molecules was studied by phospho-flow cytometry in the T cell line Hut78 (Figure 4). Of the 120 molecules studied, 20 were activated by ligands. The natural cytokine profile was as expected: Super-2 strongly activated the STAT5 and PI3K pathways ( Figure 4A and Figure 4B ); IL-4 stimulation robustly induced activation of STAT6 and PI3K pathways ( Figure 4A and Figure 4B ); and IFN leads to strong activation of all STAT molecules ( Figure 4A and Figure 4B ).

[0193] When Hut78 cells were stimulated with different synthetic factors, we could detect new signaling programs in which these engineered ligands participate. Stimulation with SY1 LL (herein referred to as SY1) strongly activated STAT5 and STAT6, and also to a lesser extent STAT1 and STAT3 ( Figure 4A and Figure 4B In addition, the synthetic factor induces a strong activation of the PI3K pathway (i.e., GSK3B, Akt, RPS6) ( Figure 4A SY2 stimulation resulted in an overall weaker signal than other ligands, preferentially activating STAT1 ( Figure 4A and Figure 4B Moreover, the ratios of STAT activation triggered by cytokines and anabolic factors were significantly different, with SY1 showing a STAT5 / STAT6 preference and SY2 showing a STAT1 preference ( Figure 4C ). Principal component analysis of the signaling programs triggered by genomically encoded cytokines and synthokines further confirmed that synthokines activate distinct signaling programs and not just a subset of the original program engaged by the parental cytokine ( Figure 4D ).

[0194] Cellular and signaling signatures induced by synthokines. We analyzed the responses to synthokine treatment in 31 cell populations profiled from human PBMCs by mass cytometry (CyTOF). Figure 5A and Figure 5C). Natural cytokines behaved as expected: Super-2 strongly activated STAT5 and, to a lesser extent, STAT1, STAT3, Erk, and S6R, showing a clear T cell preference ( Figure 5A ); IL-4 stimulation produces potent activation of STAT6 and homologous signaling trajectories in T cells and monocytes, consistent with ubiquitous expression of IL-4Rα and γc receptor subunits ( Figure 5A IFN stimulation promoted strong activation of STAT5 and STAT6 and weaker activation of STAT1 and STAT3, consistent with previous observations ( Figure 5A IFN-induced STAT activation profiles were mapped to two distinct cell clusters, with T cells inducing stronger STAT5 and STAT6 activation, and B cells and monocytes showing strong STAT6 activation but weak STAT5 activation ( Figure 5A The cell signaling patterns triggered by synthetic factors differ from those triggered by endogenous cytokines ( Figure 5A SY1 synthetase induced strong STAT5 activation in T cells but failed to activate signaling in NK, B cells, and monocytes ( Figure 5A SY2 synthetase elicited weak signaling activation of each signaling effector studied in all cell populations, with a small bias toward STAT1 activation ( Figure 5A ).

[0195] Cytokine secretion profiles induced by synthetic factors. Following ligand stimulation, the levels of cytokines secreted in the extracellular environment are often used to define the nature of the immune response produced by a given cytokine. We investigated the cytokine secretion profiles induced by synthetic factors versus natural cytokines. PBMCs were stimulated with IL-2, IL-4, IFN or synthetic factors, and the levels of 63 different analytes were measured by bead-based immunoassays 24 hours after stimulation ( Figure 5B ). Super-2 and the two synthetic factors increased cytokine secretion, IFN had a neutral effect, and IL-4 reduced the amount of cytokine secreted by PBMC ( Figure 5B ).

[0196] More detailed data analysis revealed that, as expected, stimulation of PBMCs with Super-2 promoted the secretion of high levels of LIF, IL-13, and IFNγ ( Figure 5B In addition, Super-2 induces PBMC to secrete IL-22 and CD40L ( Figure 5B ). In addition, consistent with previous reports, IFN stimulation induced the secretion of IL-27, whereas IL-4 stimulation led to the downregulation of cytokine secretion by resting PBMCs, with IFNγ being the most potent downregulatory cytokine ( Figure 5B ). The stimulation profiles of the two synthetic factors differed from those induced by natural cytokines. SY1 stimulation induced the secretion of many cytokines: IL-17F, IL-27, IL-13, IL17A, IFNγ, BDNF, IL-23, FGFβ, PDGFBB, and ENA78, and SY2 stimulation resulted in marginal levels of eotaxin, BDNF, and PDGFBB secretion ( Figure 5B ).

[0197] Synthetic factors that dimerize RTKs with JAK / STAT receptors activate signal transduction. JAK / STAT cytokine receptors represent only a subset of transmembrane receptors that signal by dimerization-induced kinase activation. Receptor tyrosine kinases (RTKs) (e.g., EGFR [epidermal growth factor receptor], c-Kit, etc.) represent another large family of dimeric cell surface receptors that signal by trans-phosphorylation of their intracellular kinase domains. We wondered if we could expand the range of synthetic factors to include molecules that would force heterodimerization and activation between JAK / STAT cytokine receptors and RTKs.

[0198] To evaluate the potential for JAK / STAT receptor crosstalk with RTKs, we fused the TM and ICD of the epidermal growth factor receptor (EGFR) to the ECD of IL-1R1 and transfected this construct in Jurkat cells along with our panel of IL-1R1AcP-cytokine receptor ICDs ( Fig. 6A and Figure 6B All 10 cytokine receptor / EGFR pairs are expressed on the surface of Jurkat cells ( Fig. 6F ). Stimulation with IL-1 resulted in varying degrees of phosphorylation of EGFR and, to a lesser extent, STAT3 and STAT5 proteins ( Figure 6B ), demonstrating that these cytokine and tyrosine kinase receptors are capable of trans-phosphorylation when forced into close proximity. This is consistent with previous studies showing that instances of such crosstalk exist on native cells. However, one caveat to these chimeric receptor studies is that overexpression of kinase-linked receptors can result in aberrant, spurious phosphorylation events. Therefore, we sought to force heterodimerization of JAK / STAT and RTK-mediated receptors normally expressed on native cells using synthetic factor ligands in the absence of overexpression.

[0199] We generated a synthetic factor to force dimerization of cKit, a tyrosine kinase receptor, and thrombopoietin receptor (TpoR), a cytokine receptor. To generate synthetic factor dual-specific ligands, we identified sequences of antibodies that bind to either cKit or TpoR ECD with high affinity. We reformatted them into single-chain variable fragments (scFv) and synthesized them by fusing each to complementary acidic and basic leucine zippers ( Figure 6C ) and applied them to acute megakaryoblastic leukemia Mo7e cells, which are known to express cKit and TpoR, to force their heterodimerization. SY4 and SY5 synthetic factors induced modest phosphorylation of cKit above background in Mo7e cells, but only SY5 induced detectable phosphorylation of TpoR-associated JAK2 above background (albeit very weakly). Fig.6D SY5 induced measurable Erk activation (50% Emax compared with the natural ligand stem cell factor [SCF]) but only weak STAT5 activation, consistent with the apparent asymmetric activation of cKit over TpoR ( Fig. 6E Indeed, inhibition of JAK2 using a small molecule inhibitor of JAK2 resulted in loss of Erk activation by SY5, suggesting that the signaling program engaged by these synthetic factors is at least partially dependent on JAK2 activity ( Figure 6G ). Thus, there appears to be an asymmetry in the efficiency of trans-phosphorylation within the TpoR / cKit heterodimer. We performed high-throughput phosphorylation flow cytometry-based studies to analyze the signaling responses of 120 signaling molecules in stimulated Mo7e cells.

[0200] like Fig. 7A As shown, 54 of the 120 different signaling molecules were activated above the significance threshold by different ligands. Interestingly, the signaling signature elicited by SY5 appears to elicit qualitatively different outputs with SCF, TPO, or a combination of both ligands; this depends on the pathway effector being studied ( Figure 7B and Figure 7C Together, the chimeric receptor and synthetic factor studies showed that, although less efficiently than their natural ligands, JAK / TYK- and RTK-mediated signaling receptors are capable of crosstalk, consistent with previous studies showing that JAK and RTK components are able to phosphorylate each other's natural substrates.

[0201] In this study, we used synthetic ligands that can be imprecisely analogized to “orphan” or “synthetic” cytokines to expand the scope of kinase-linked dimeric receptor signaling on native cells. This approach can exploit the full combinatorial potential of JAK / TYK / STAT and RTK signaling through receptor dimers. A compelling reason for us to explore this approach is that despite their immunotherapeutic potential, relatively few cytokines are clinically useful, in large part due to their pleiotropic and off-target effects. In recent years, cytokine variants with more defined activities and reduced toxicity have been engineered. However, an inherent limitation of this approach is that the engineered cytokines exhibit a subset of activities within the bioactive space occupied by the parent cytokine.

[0202] We have performed a series of proof-of-concept experiments to demonstrate that synthetic cytokines (synkines) engineered to dimerize non-native cytokine receptor pairs can achieve activation of distinct signaling programs and, by extension, immune activity. Synkines can exploit the high plasticity of cytokine receptor pairing to elicit novel signaling activities, paving the way for "designer" ligands to specifically target biological processes relevant to health and disease. Our data show that synthetic factors activate distinct and novel signaling programs and induce secretion of novel cytokine signatures by stimulated PBMCs.

[0203] First, although most synthetic factors trigger signals that are partially similar to the signals of the parental cytokines, the ratio of activated STATs differs significantly between cytokines and synthetic factors. For example, SY1 triggers a STAT6>STAT5>STAT1>STAT3 pattern instead of the STAT6>STAT1>STAT3>STAT5 pattern seen with IL-4 plus IFN, while SY2 triggers a STAT1>STAT6>STAT5>STAT3 pattern instead of the STAT6>STAT5>STAT3>STAT1 pattern seen with IL-4 plus IL-2. Changes in the STAT activation ratio can alter the biological response induced by cytokines.

[0204] Second, given that synthetic factors induce dimerization of non-naturally occurring cytokine receptor pairs, they may also alter the abundance of STAT heterodimers and induce the formation of new STAT heterodimer pairings, leading to the induction of entirely new gene expression programs and activities. Synthetic factor biology can be tested in mouse systems and disease models.

[0205] The physiological roles of synthetic factors will be no less complex than those of natural cytokines, but knowledge of the activities of the parent receptor chains used to form non-natural dimers may be predictive of the activities of synthetic factors. For example, we anticipate that in some cases the physiological roles and disease applications may be similar or related to those of one of the parent receptor chains and in other cases be completely different.

[0206] The synthetic factor design paradigm covers several key considerations: 1) Select two cytokine receptor subunits that are expressed simultaneously in the same cell. Cellular responses to cytokines are strictly regulated by the surface expression patterns of cytokine receptor subunits. Therefore, there are many cytokine receptor pair combinations that, although compatible with signaling, are not relevant in vivo due to the lack of naturally occurring cell subpopulations that express both receptor subunits simultaneously. 2) Select the type of cytokine receptor subunit to be dimerized by the synthetic factor. From our chimeric receptor studies, we infer that most cytokine receptor pair combinations will activate signaling to some extent. However, other parameters such as structural properties may affect the extent and nature of signaling activation. For example, cytokine receptors can be subdivided into two categories based on ICD length. Receptors with long ICDs typically bind their ligands with high affinity, pair with JAK1 or JAK2, encode STAT binding sites, and drive signal activation. In contrast, receptors with short ICDs typically bind their ligands with lower affinity, pair with TYK2 or JAK3, and minimally contribute to STAT recruitment and activation.

[0207] Interestingly, many of the receptor pairs that did not activate signaling in our chimeric receptor studies contained short ICD receptors, suggesting that synthetic factors that dimerize two short ICD receptor subunits will elicit weaker and less diverse signaling activation programs than those that dimerize long ICD receptor subunits. In addition, our results show that synthetic factors can be generated that dimerize receptors from two different cell surface receptor families, specifically the cytokine receptor and tyrosine kinase receptor families.

[0208] A very important aspect of the synthetic factors that we have engineered is that they dimerize cytokine receptor pairs into defined 1:1 molecular entities, which enables signaling pathways to be unambiguously attributed to receptor dimers. The formation of molecularly defined surface complexes by engineered ligands is critical for characterizing the signaling and phenotypic programs activated by these ligands, as well as for predicting potential toxicities caused by off-target effects and / or cellular interactions. Previous attempts to engineer synthetic ligands with novel activities by linking fully functional cytokines have produced ligands that can form a variety of independently acting receptor complexes ranging from dimers to tetramers, depending on the abundance and relative ratios of the receptor subunits expressed by a given cell type. While these ligands elicit novel biologically active programs, the heterogeneity of the complexes they form makes it difficult to assign signaling or activity signatures to specific complexes or to predict toxic side effects that might result from systemic administration.

[0209] In contrast, our targeted approach using dominant-negative cytokine mutants to dimerize surface receptors allowed us to interrogate the activity of specific dimer pairs. A consistent conclusion from our studies was that the engineered synthetic factors were relatively inefficient at activating signaling compared with the cytokines encoded by the parental genome; at best we could detect 60% of the signaling amplitude induced by IL-2, IL-4, or IFN. Synthetic factors that elicited signaling from cKit / TpoR heterodimers exhibited even weaker activation properties.

[0210] One explanation for this observation is that the structure of the cytokine-receptor complex is a determinant of signal potency. It is possible that the receptor binding topology induced by engineered synthetic factors is suboptimal, and signaling strength can be improved by altering the conformation of these molecules.

[0211] Materials and Methods

[0212] Protein expression and purification. Human IL-4, Super-2, IFN dominant negative cytokines and synthesis factors were expressed and purified using a baculovirus expression system, as described in (Laporte et al., 2005). The sequence of the Super-2 variant of IL-2 is provided in (Levin et al., 2012). SY1 SL and LL synthesis factors are produced by genetically fusing IL-4DN and IFNDN proteins via a single (SY1 SL) or two (SY1 LL) Gly4Ser joints. SY2 synthesis factors are produced by genetically fusing IL-2DN and IL-4DN proteins via Gly4Ser joints. IL-4DN is produced by introducing the previously described R121D / Y124D mutations on site II, which disrupt the combination with common gamma chains (Wenzel S et al., The Lancet, 2007). By introducing mutations F63A and R120E on the IFN-IFNAR1 binding interface, the combination with IFNAR1 is destroyed, and IFNDN is produced. IL-2DN (also known as IL-2RETR) was previously described in Mitra et al., Immunity, 2015. The single-chain variable fragments (scFv) used to engineer SY3, SY4, and SY5 were similarly expressed and purified in a baculovirus system by transferring their variable regions into the pAcGP67A vector (BD Biosciences) with an N-terminal gp67 signal peptide and a C-terminal hexahistidine tag. H ) chain and variable light (V L ) chains are separated by a 12-amino acid (Gly4Ser)3 linker fused to an acidic or basic leucine zipper for dimerization. All proteins contained a C-terminal hexahistidine tag and were separated by nickel chromatography and further purified by size exclusion chromatography on a Superdex 200 column (GE Healthcare) equilibrated in 10 mM HEPES (pH 7.3) and 150 mM NaCl to a homogeneity of >98%.

[0213] Chimeric receptor generation. In order to produce a 10x10 signal transduction matrix, the ICD of 10 different parental cytokine receptors was fused with IL-1R1 and IL-1R1Acp ECD. In the IL-1R1 ECD form, the nucleotide sequence encoding the HA tag was inserted between the end of the natural signal sequence and the first residue of the IL-1R1 ECD. Each ICD was fused to the 3' end of the IL-1R1 sequence. In addition to using the V5 tag, the IL-1R1Acp chimera was cloned in the same manner. The boundaries of the mature protein and the transmembrane span were depicted using SignalP and TMHMM network servers. The DNA sequence for IL-1R1 was codon optimized to be expressed and synthesized (Integrated DNA Technologies) in Homo sapiens as an organism (jcat.de). The chimeric receptor was cloned into the pcDNA3.1+ vector (Invitrogen) using NheI and KpnI restriction sites (NEB).

[0214] Tissue culture. Jurkat cells were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS, 2mM L-glutamine and penicillin-streptomycin (Gibco)). Hut78 cells were cultured in RPMI complete medium (RPMI 1640 medium supplemented with 10% FBS, 2mM L-glutamine and penicillin-streptomycin (Gibco)). Mo7e cells were cultured in IMEM complete medium (IMEM medium supplemented with 10% FBS, 2mM L-glutamine, 10nM GM-SCF and penicillin-streptomycin (Gibco)). Prior to stimulation, Mo7e cells were starved overnight in a modified growth medium lacking FBS and GM-CSF. All cell lines were maintained in a humidified atmosphere containing 5% CO2 at 37°C.

[0215] Hut78 and Mo7e intracellular signal transduction studies. 5Hut78 or Mo7e cells were placed in 96-well plates, washed with PBSA buffer (phosphate buffered saline (PBS) pH 7.2, 1% BSA), and resuspended in PBSA containing serial dilutions of the specified ligands. The cells were stimulated at 37°C for the specified time and immediately fixed by adding formaldehyde to 1.5%, followed by incubation at room temperature for 10 minutes. The cells were then permeabilized with 100% ice-cold methanol at 4°C for 30 minutes. The fixed and permeabilized cells were washed twice with PBSA and incubated with fluorescent 22-labeled detection antibodies diluted in PBSA for 1 hour at room temperature. pSTAT3, pSTAT5 and pSTAT6 antibodies were purchased from BD Biosciences. pSTAT1, pErk, pcKit, pEGFR antibodies were purchased from Cell Signaling Technology. The cells were then washed twice in PBSA buffer and the mean fluorescence intensity (MFI) was quantified on an Accuri C6 flow cytometer. Dose-response curves were fitted to a logistic model, and EC was calculated in GraphPad Prism data analysis software after subtracting the MFI of unstimulated cells and normalizing to the maximal signal intensity induced by wild-type cytokine stimulation. 50 value.

[0216] Peripheral blood mononuclear cells (PBMC) were isolated from human whole blood. Peripheral blood mononuclear cells (PBMC) were isolated from human whole blood (Stanford Blood Bank) according to the manufacturer's protocol using a gradient of Ficoll-Paque Plus (GE Healthcare). Freshly isolated PBMC were used for mass spectrometry flow cytometry studies and bead-based immunoassays. Prior to stimulation, PBMC were left to stand in RPMI complete medium at 37°C, 5% CO2 for 1 hour.

[0217] Mass cytometry immune cell signaling analysis. This assay was performed at the Human Immune Monitoring Center at Stanford University. Freshly isolated PBMCs were plated at 5 10 5Cells were seeded in 96-well plates and stimulated with serial dilutions of designated ligands in complete RPMI at 37°C, 5% CO2 for 20 minutes. Cells were then fixed by incubation in paraformaldehyde (1.5% final concentration) for 10 minutes at room temperature. The cells were washed and resuspended in CyFACS buffer (PBS supplemented with 2% BSA, 2mM EDTA and 0.1% sodium azide) containing anti-surface antigen antibodies labeled with metal chelating polymers for 30 minutes at room temperature. The antibodies were labeled from purified unconjugated, carrier-free stock solutions from BD Biosciences, Biolegend or Cell Signaling Technology, and polymers and metal isotopes were from DVS Sciences. The cells were washed once in CyFACS buffer and then permeabilized overnight in methanol at -80°C. The next day, the cells were washed once in CyFACS buffer and resuspended in CyFACS buffer containing anti-intracellular antigen antibodies labeled with metal chelating polymers for 30 minutes at room temperature. Cells were washed twice in PBS (phosphate buffered saline pH 7.2), resuspended in iridium-containing DNA intercalators (1:200 dilution in PBS, DVS Sciences) and incubated on ice for 20 minutes. The cells were then washed three times in MilliQ water, then diluted in MilliQ water with a total volume of 700 μL, and then injected into a CyTOF instrument (DVS Sciences). FlowJo (CyTOF device) was used to gate cells based on iridium isotopes from intercalators, and then a line graph of iridium isotopes of an intercalator relative to cell length was used to gate the complete single peak, followed by cell subpopulation specific gating for data analysis. The signal intensity of each condition was reported as the 90th percentile intensity minus the intensity of the unstimulated control sample. TM4 microarray software suite (Dana-Farber Cancer Institute) (Saeed et al., 2003) was used to generate a heat map of the response to the maximum concentration of each treatment, wherein the signal intensity was normalized to the maximum signal effector response in each cell type. The mass cytometry experiments were repeated two independent times and obtained similar results.

[0218] Bead-based immunoassay cytokine secretion studies. The assay was performed at the Human Immune Monitoring Center at Stanford University. Freshly isolated PBMCs (1.5·105 per well) were stimulated with the indicated ligands in the presence of 1 μg / mL phytohemagglutinin (PHA) in complete RPMI and incubated for 24 h at 37°C, 5% CO2. The cells were then pelleted by centrifugation and lysed using The supernatant was harvested using the Biotek ELx405 platform (Luminex Corporation) for bead-based immunoassay analysis. The human 63-plex kit was purchased from eBiosciences / Affymetrix and used according to the manufacturer's recommendations with the modifications described below. Briefly: Beads were added to a 96-well plate and washed in a Biotek ELx405 washer. The harvested supernatant was added to a plate containing mixed antibody-linked beads and incubated at room temperature for 1 hour, followed by shaking overnight at 4°C. Low temperature and room temperature incubation steps were performed on an orbital shaker at 500-600 rpm. After overnight incubation, the plate was washed in a Biotek ELx405 washer, and biotinylated detection antibody was added and kept at room temperature with shaking for 75 minutes. The plate was washed again as described above and streptavidin-PE (Invitrogen) was added. After incubation at room temperature for 30 minutes, a final wash was performed as above, and read buffer was added to the wells. The plates were analyzed on a 200-well instrument with a minimum of 50 beads per sample per cytokine. Custom assay control beads (Radix Biosolutions) were added to all wells. Each sample was measured in duplicate and the raw MFI was averaged from the two replicates. Results are presented as the fold change in the MFI of the treated cells relative to control cells stimulated with PHA alone.

[0219] Primity Bio Pathway Phenotyping. Hut78 or Mo7e cells were stimulated with saturating concentrations of the indicated ligands for 15, 60, and 120 minutes and fixed with 1% PFA for 10 minutes at room temperature. Fixed cells were prepared for antibody staining according to standard protocols (Krutzik and Nolan, 2003). Briefly, fixed cells were permeabilized in 90% methanol for 15 minutes. Cells were then stained with a panel of antibodies specific for the indicated markers (Primity Bio Pathway Phenotyping service) and analyzed on an LSRII flow cytometer (Becton Dickinson, San Jose, CA). The MFI of the stimulated samples divided by the log2 ratio of the unstimulated control samples was calculated as a measure of the response.

[0220] Western blot analysis. Cells were lysed in 1% NP-40 lysis buffer and 30 μg of protein was analyzed as described in (Marijanovic et al., 2007). The following polyclonal antibodies were used: anti-phospho-Tyk2, anti-phospho-STAT1; anti-phospho-STAT2; anti-phospho-STAT3; anti-phospho-STAT4; anti-phospho-STAT5; anti-phospho-STAT6; anti-phospho-EGFR; anti-phospho-cKit pY703 (Cell Signaling Technology, Beverly, MA). Signals were visualized using ECL enhanced chemiluminescence western blotting reagent, Western Lightning Chemiluminescence Reagent Plus (PerkinElmer).

[0221] Electroporation. Place 15-20x10 6 Maintained at 0.5-1.0·10 6 Jurkat cells with a density of 10 cells / ml were washed twice with RPMI culture medium (sterile) and resuspended in 0.25ml of Ingenio electroporation solution (Mirusbio). 5-30 μg DNA (no more than 15% of the total volume) was added to the resuspended Jurkat cells, and the mixture was transferred to a 4mm gap cuvette (Invitrogen) and incubated at room temperature for 15-20 minutes. The cells were electroporated in a Biorad electroporator set to 0.28kV and 960μF. After electroporation, the cells were transferred to a pre-warmed culture medium (without penicillin / streptomycin) and cultured normally. Protein expression was monitored after 24 hours.

[0222] Cell surface receptor staining. Surface receptor levels were monitored using fluorescently labeled monoclonal antibodies specific for HA and V5 tags (Cell Signaling Technology) as described (Marijanovic et al., 2007). Electroporated Jurkat cells were resuspended in cold PBS containing 3% fetal bovine serum and incubated with the indicated antibodies for 1 hour. Samples were analyzed on an Accuri C6 flow cytometer (BD Biosciences).

[0223] Example 2

[0224] Trimerization factor

[0225] Using a Gly / Ser peptide linker, a cytokine (IL-2) was linked to a scFv (monovalent antibody) that binds to IL-4Rα. This new molecular entity binds to 3 cytokine receptor peptides: γc, IL-2Rβ, and IL-4Rα. The signaling profile elicited by the trimerizing synthetase differs from that activated by IL-2, IL-4, or a combination of IL-2 and IL-4 treatment. In addition, this new cytokine induced the differentiation of monocytes into an uncharacterized subset of dendritic cells with high phagocytic activity, such as Figure 8-15 In some embodiments, novel compositions of synthetic factors are provided. In some embodiments, a population of monocytes is contacted with an effective dose of trimerized synthetic factors in vitro or in vivo. In some embodiments, a population of phagocytic cells differentiated from monocytes using trimerized synthetic factors is provided.

[0226] Example 3

[0227] Synthetic factor that dimerizes type I and type III IFN receptors.

[0228] A synthetic factor (SY6) comprising an IFNλR1 binding sequence (H11DN) and an IFNAR1 binding sequence (IFNWDN2) was generated. The complete synthetic factor sequence is provided in SEQ ID NO: 1. The synthetic factor thus generated is a hybrid interferon that dimerizes IFNAR1 and IFNλR1 receptors and their corresponding JAKs. Fig.15 As shown, the Emax of phospho-STAT1 activation by SY6 is equal to that of type I IFN and twice that of the signal induced by type III IFN. Error bars represent ± SEM (n=3). Fig.15 As shown in D, SY6 effectively induced antiproliferative effects, whereas type I IFN, type III IFN, or a combination of type I and type III IFN treatment was ineffective. Error bars represent ± SEM (n=3). Phospho-STAT1 signaling and antiproliferative assays were performed in Hap1 cells that are naturally responsive to both type I and type III IFN.

[0229] It is also important to note that combinations of type I and type III interferons do not provide this activity unless linked in a hybrid polypeptide such as SY6. The activity and specificity of the synthetic factors provide potent agents with antiproliferative and antiviral activity that provide selectivity of action and thereby avoid the adverse side effects of type I interferons.

[0230] Example 4

[0231] mIL4DN-mIFNβDN2 synthesis factor

[0232] The synthetic factor (SY7) is produced by genetically fusing mouse IL-4DN and mIFNβDN2 proteins via a Gly4Ser linker. The sequence is shown in Figure 16. Lymphocytes are isolated from the spleen / LN of C57BL / 6 mice and activated for 48 hours with plate-bound anti-CD3 (2.5 μg / ml)+soluble anti-CD28 (5 μg / ml). The cells are then left to stand overnight (O / N) in 10IU / ml mIL2, then serum starved for 4 hours, and then stimulated for 20' with specified cytokines / synthetic factors. Cell signaling is terminated and the cells are fixed with PFA, permeabilized with PermIII buffer (BD) and stained with phosphorylated STAT6 (Y641) antibody (BD).

[0233] Table 1

[0234] Experimentally generated synthetic factors

[0235]

[0236] Antagonists or "dominant negative (DN)" forms of IL-4, IL-2, and IFN were engineered that retain binding to their high affinity receptor subunits (IL-4Rα for IL-4, IL-2Rβ for IL-2, and IFNAR2 for IFN), but to do so by disrupting binding to their low affinity receptor subunits (L-13Rα1 and γc for IL-4, γc for IL-2, and IFNAR1 for IFN). These "DN" cytokines act as high affinity binding modules that have no signaling activity themselves. Pairs of dominant negative cytokine mutants were fused via Gly4 / Ser linkers to generate new ligands that induce the formation of non-natural receptor dimers of IL-2Rβ-IL-4Rα and IL-4Rα-IFNAR2, as shown in the table above for SY1 and SY2. IL-4DN is produced by introducing the previously described R121D / Y124D mutation at site II, which disrupts the binding to the common γ chain (Wenzel S et al., The Lancet, 2007). IFNDN is produced by disrupting the binding to IFNAR1 by introducing mutations F63A and R120E at the IFN-IFNAR1 binding interface. IL-2DN (also referred to as IL-2RETR) has been previously described in Mitra et al., Immunity, 2015.

[0237] Expression of single chain variable fragments (scFv) for engineering SY3, SY4 and SY5, which can be variable (V H ) chain and variable light (V L) chains are separated by a 12-amino acid (Gly4Ser)3 linker fused to an acidic or basic leucine zipper for dimerization. The SY4 and SY5 constructs utilize antibodies that bind to cKit or TpoR ECD with high affinity. We reformatted them into single-chain variable fragments (scFv) and forced their heterodimerization by fusing each to complementary acidic and basic leucine zippers.

[0238] The SY3 protein uses a Gly / Ser peptide linker to connect a cytokine (IL-2) to a scFv (monovalent antibody) that binds to IL-4Rα. This new molecular entity binds to three cytokine receptor peptides: γc, IL-2Rβ, and IL-4Rα.

[0239] The SY6 sequence is provided as SEQ ID NO: 1, comprising residues 1-163 from a variant form of human IFNλ, with amino acid substitutions at residues corresponding to Q26A, Q99A, H102A, H131R, T161A, and V174E of a reference human IFNλ3 sequence (Genbank reference XP_005258822.1), wherein the variant sequence is truncated by lacking residues 1-11 of the reference IFNλ3 protein. Residues 164-168 are gly / ser linkers, and residues 169-342 comprise a variant form of human IFNω.

[0240] The SY7 sequence is provided as SEQ ID NO: 2, wherein residues 1-17 are the signal peptide, residues 18-138 are the mIL-4 DN, and have amino acid substitutions at residues corresponding to Q116D and Y119D, such as Fig.17 As shown; residues 139-143 are gly / ser linkers; and residues 144-304 correspond to mIFNβ DN2, with amino acid substitutions at residues corresponding to R15A, L30A, R33A, R147A. Sequence Listing <110> The Board of Trustees-Leland Stanford Junior University Gonzalez, Ignacio Moraga Garcia, Kenan Christopher <120> Synthetic factor compositions and methods of use <130> STAN-1402WO <150> 62 / 479,993 <151> 2017-03-31 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 351 <212> PRT <213> Artificial sequence <220> <223> Synthesis of multiple peptides <400> 1 Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Ala Glu 1 5 10 15 Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu 20 25 30 Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp 35 40 45 Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu 50 55 60 Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro 65 70 75 80 Ala Leu Gly Asp Val Leu Asp Ala Pro Leu Ala Thr Leu His His Ile 85 90 95 Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro 100 105 110 Arg Thr Arg Gly Arg Leu His Arg Trp Leu His Arg Leu Gln Glu Ala 115 120 125 Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn 130 135 140 Leu Phe Arg Leu Leu Ala Arg Asp Leu Asn Cys Val Ala Ser Gly Asp 145 150 155 160 Leu Cys Glu Gly Ser Gly Ser Gly Leu Gly Cys Asp Leu Pro Gln Asn 165 170 175 His Gly Leu Leu Ser Ala Asn Thr Leu Val Leu Leu His Gln Met Arg 180 185 190 Arg Ile Ser Pro Phe Leu Cys Ala Lys Asp Ala Arg Asp Phe Arg Phe 195 200 205 Pro Gln Glu Met Val Lys Gly Ser Gln Leu Gln Lys Ala His Val Met 210 215 220 Ser Val Leu His Glu Met Leu Gln Gln Ile Phe Ser Leu Phe His Thr 225 230 235 240 Glu Arg Ser Ser Ala Ala Trp Asn Met Thr Leu Leu Asp Gln Leu His 245 250 255 Thr Gly Leu His Gln Gln Leu Gln His Leu Glu Thr Cys Leu Leu Gln 260 265 270 Val Val Gly Glu Gly Glu Ser Ala Gly Ala Ile Ser Ser Pro Ala Leu 275 280 285 Thr Leu Arg Arg Tyr Phe Gln Gly Ile Arg Val Tyr Leu Lys Glu Lys 290 295 300 Lys Tyr Ser Asp Cys Ala Trp Glu Val Val Arg Met Glu Ile Met Ala 305 310 315 320 Ser Leu Phe Leu Ser Thr Asn Met Gln Glu Arg Leu Arg Ser Lys Asp 325 330 335 Arg Asp Leu Gly Ser Ser Ala Ala Ala His His His His His His 340 345 350 <210> 2 <211> 313 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 2 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala 1 5 10 15 Gly Ser His Ile His Gly Cys Asp Lys Asn His Leu Arg Glu Ile Ile 20 25 30 Gly Ile Leu Asn Glu Val Thr Gly Glu Gly Thr Pro Cys Thr Glu Met 35 40 45 Asp Val Pro Asn Val Leu Thr Ala Thr Lys Asn Thr Thr Glu Ser Glu 50 55 60 Leu Val Cys Arg Ala Ser Lys Val Leu Arg Ile Phe Tyr Leu Lys His 65 70 75 80 Gly Lys Thr Pro Cys Leu Lys Lys Asn Ser Ser Val Leu Met Glu Leu 85 90 95 Gln Arg Leu Phe Arg Ala Phe Arg Cys Leu Asp Ser Ser Ile Ser Cys 100 105 110 Thr Met Asn Glu Ser Lys Ser Thr Ser Leu Lys Asp Phe Leu Glu Ser 115 120 125 Leu Lys Ser Ile Met Asp Met Asp Asp Ser Gly Ser Gly Ser Gly Ile 130 135 140 Asn Tyr Lys Gln Leu Gln Leu Gln Glu Arg Thr Asn Ile Ala Lys Cys 145 150 155 160 Gln Glu Leu Leu Glu Gln Leu Asn Gly Lys Ile Asn Ala Thr Tyr Ala 165 170 175 Ala Asp Phe Lys Ile Pro Met Glu Met Thr Glu Lys Met Gln Lys Ser 180 185 190 Tyr Thr Ala Phe Ala Ile Gln Glu Met Leu Gln Asn Val Phe Leu Val 195 200 205 Phe Arg Asn Asn Phe Ser Ser Thr Gly Trp Asn Glu Thr Ile Val Val 210 215 220 Arg Leu Leu Asp Glu Leu His Gln Gln Thr Val Phe Leu Lys Thr Val 225 230 235 240 Leu Glu Glu Lys Gln Glu Glu Arg Leu Thr Trp Glu Met Ser Ser Thr 245 250 255 Ala Leu His Leu Lys Ser Tyr Tyr Trp Arg Val Gln Arg Tyr Leu Lys 260 265 270 Leu Met Lys Tyr Asn Ser Tyr Ala Trp Met Val Val Arg Ala Glu Ile 275 280 285 Phe Ala Asn Phe Leu Ile Ile Arg Arg Leu Thr Arg Asn Phe Gln Asn 290 295 300 Ala Ala Ala His His His His His His 305 310

Claims

1. Use of a synthetase polypeptide in the preparation of a medicament for treating a disease, wherein the synthetase polypeptide binds to the extracellular domains of two or more cytokine receptor subunit polypeptides in a non-natural combination on the surface of a cell, in, The synthetase polypeptide comprises at least a first binding domain and a second binding domain, wherein The first binding domain is a first monovalent antibody that specifically binds to the extracellular domain of a first cytokine receptor subunit; The second binding domain is a second monovalent antibody that specifically binds to the extracellular domain of a second cytokine receptor subunit; wherein the first and second cytokine receptor subunits do not naturally multimerize in response to contact of the cell with a naturally occurring cytokine ligand; wherein contacting the cell expressing the first and second cytokine receptor subunits with a synthokine polypeptide causes multimerization of the cytokine receptor subunits and activation of JAK / STAT-mediated signaling in the cell; and wherein the first cytokine receptor subunit is selected from interleukin 2 receptor β (IL-2Rβ), interleukin 4 receptor α (IL-4Rα), interferon α receptor 2 (IFNAR2), glycoprotein 130 (gp130), interleukin 23 receptor (IL-23R), erythropoietin receptor (EpoR), interleukin 12 receptor β (IL-12Rβ), interferon α / β receptor α chain (IFNAR1), interleukin 13 receptor α1 (IL-13Rα1) and γ chain (γc); and Wherein, the second cytokine receptor subunit is selected from interleukin 2 receptor β (IL-2Rβ), interleukin 4 receptor α (IL-4Rα), interferon α receptor 2 (IFNAR2), glycoprotein 130 (gp130), interleukin 23 receptor (IL-23R), erythropoietin receptor (EpoR), interleukin 12 receptor β (IL-12Rβ), interferon α / β receptor α chain (IFNAR1), interleukin 13 receptor α1 (IL-13Rα1) and γ chain (γc).

2. The method of claim 1, wherein the synthetic factor comprises an ECD having an affinity for the receptor subunit of less than 1 x 10 -7 The binding domain of M.

3. The use of claim 1, wherein the binding domains are directly linked.

4. The method of claim 1, wherein the binding domains are connected via a linker.

5. The use according to claim 1, wherein the monovalent antibody is VHH.

6. The use according to claim 1, wherein the binding domains are connected via a peptide linker comprising 2-100 amino acids.

7. The use of claim 1, wherein the binding domains are connected via a non-peptide linker.

8. A synthetic factor comprising: at least two binding domains, wherein each binding domain specifically binds to the extracellular domain of a first cytokine receptor subunit polypeptide expressed on the cell surface, wherein the first binding domain is a first monovalent antibody that specifically binds to the extracellular domain of a first cytokine receptor subunit, wherein the first cytokine receptor subunit has an intracellular domain comprising at least one JAK / STAT binding sequence; and the second binding domain is a second monovalent antibody that specifically binds to the extracellular domain of a second cytokine receptor subunit, wherein the second cytokine receptor subunit has an intracellular domain that comprises at least one JAK / STAT binding sequence; in, Contact of the cell with the synthokine polypeptide results in multimerization of the non-natural combination of cytokine receptor subunits and activation of JAK / STAT-mediated signaling in the cell; and wherein the first cytokine receptor subunit is selected from interleukin 2 receptor β (IL-2Rβ), interleukin 4 receptor α (IL-4Rα), interferon α receptor 2 (IFNAR2), glycoprotein 130 (gp130), interleukin 23 receptor (IL-23R), erythropoietin receptor (EpoR), interleukin 12 receptor β (IL-12Rβ), interferon α / β receptor α chain (IFNAR1), interleukin 13 receptor α1 (IL-13Rα1) and γ chain (γc); and Wherein, the second cytokine receptor subunit is selected from interleukin 2 receptor β (IL-2Rβ), interleukin 4 receptor α (IL-4Rα), interferon α receptor 2 (IFNAR2), glycoprotein 130 (gp130), interleukin 23 receptor (IL-23R), erythropoietin receptor (EpoR), interleukin 12 receptor β (IL-12Rβ), interferon α / β receptor α chain (IFNAR1), interleukin 13 receptor α1 (IL-13Rα1) and γ chain (γc).

9. The synthetic factor of claim 8, wherein the at least first binding domain and the second binding domain are directly linked.

10. The synthetic factor of claim 8, wherein the at least first binding domain and the second binding domain are connected by a linker.

11. The synthetic factor of claim 8, wherein the antibodies are independently selected from single chain Fv and Nanobodies®.

12. The synthetic factor of claim 8, wherein the monovalent antibody is a VHH.

13. A nucleic acid sequence encoding the synthesis factor according to any one of claims 8 to 12. A recombinant vector comprising the nucleic acid sequence according to claim 13 . A recombinant host cell comprising the nucleic acid sequence of claim 13 .

16. Use of a synthetic factor as claimed in any one of claims 8 to 12 in the preparation of a medicament for treating or preventing a disease or condition in a subject in need thereof, the use of the medicament comprising: An effective amount of the synthetic factor is provided to the subject.

17. The use according to claim 16, wherein the subject suffers from an immune disease or dysfunction.

18. The use according to any one of claims 16-17, wherein the subject is a human.

19. The use according to claim 18, wherein the drug is administered by bolus intravenous injection or by continuous infusion at a dose of 0.001 to 100 mg of the drug per kg of subject body weight.

20. The use of claim 19, wherein the subject suffers from tissue loss or damage, inflammatory disease and / or cancer.

21. The use of claim 20, wherein the inflammatory disease is selected from the group consisting of: secondary progressive multiple sclerosis; primary progressive multiple sclerosis; neuromyelitis optica; psoriasis; systemic lupus erythematosus; ulcerative colitis; Crohn's disease; ankylosing spondylitis; asthma; chronic obstructive pulmonary disease; chronic hepatitis; amyotrophic lateral sclerosis; Alzheimer's disease; Parkinson's disease; frontotemporal lobar degeneration; atherosclerosis / cardiovascular disease and obesity / metabolic syndrome.

22. The use according to claim 20, wherein the cancer is selected from the group consisting of: hematological cancer and solid cancer.

23. The method of claim 22, wherein the hematological cancer comprises leukemia, lymphoma and myeloma, and the solid cancer comprises glioblastoma, medulloblastoma, astrocytoma, oligodendroglioma, ependymoma, lung cancer, liver cancer, thyroid cancer, bone cancer, adrenal cancer, spleen cancer, kidney cancer, lymph node cancer, small intestine cancer, pancreatic cancer, colon cancer, stomach cancer, breast cancer, endometrial cancer, prostate cancer, testicular cancer, ovarian cancer, skin cancer, head and neck cancer and esophageal cancer.

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