HK1261663A1 - Molecules that selectively activate regulatory t cells for the treatment of autoimmune diseases - Google Patents

Description

Molecules selectively activating regulatory T cells for the treatment of autoimmune diseases

Cross-referencing

This application claims priority from U.S. patent application No.15/002,144 filed on 20/1/2016, which is hereby incorporated by reference in its entirety.

Reference to sequence listing

The sequence listing of 88,573 bytes, produced on 3.5.2016, mechanically formatted IBM-PC, MS-Windows operating system file sequence listing-097584-.

Background

The immune system must be able to distinguish between self and non-self. When self/non-self differentiation fails, the immune system destroys cells and tissues of the body and thus leads to autoimmune diseases. Regulatory T cells actively suppress activation of the immune system and prevent pathological autoreactivity and the resultant autoimmune disease. The development of drugs and methods for selectively activating regulatory T cells for the treatment of autoimmune diseases is the subject of intensive research and has been largely unsuccessful until the development of the present invention.

Regulatory T cells (tregs) are a class of CD4+ CD25+ T cells that suppress the activity of other immune cells. Tregs are central to the homeostasis of the immune system and play a major role in maintaining tolerance to self-antigens and modulating immune responses to foreign antigens. A variety of autoimmune and inflammatory diseases, including type 1 diabetes (T1D), Systemic Lupus Erythematosus (SLE), and Graft Versus Host Disease (GVHD), have been shown to have defects in Treg cell number or Treg function. Therefore, there is great interest in developing therapies that enhance the number and/or function of Treg cells.

One therapeutic approach being investigated for autoimmune diseases is the transplantation of autologous, ex vivo expanded Treg cells (Tang, q. et al, 2013, Cold Spring harb. perspect.med.,3:1-15) although this approach has shown promise in animal models of treating disease and in several early human clinical trials, it requires personalized treatment with the patient's own T cells, is traumatic and technically complex, another approach is treatment with low doses of interleukin-2 (IL-2) Treg cells characteristically expressing high constitutive levels of high affinity IL-2 receptor (IL2R αβ γ) consisting of subunits IL2RA (CD25), IL2RB (CD122) and IL2RG (CD132), and Treg cell growth has been shown to be dependent on IL-2(Malek, t.r. et al, 2010, imnity 33: 55-153, sa2011. 25 (CD132) and in other clinical trials of autoimmune diseases, high efficacy of IL-2 (gv. 7, hiv-7, low doses of IL-2) and in clinical trials of autoimmune diseases, high efficacy has shown in various clinical trials of IL-2 (g-2) and in patients.

Proleukin (marketed by Prometous Laboratories, San Diego, Calif.) (recombinant form of IL-2 used in these experiments) is associated with high toxicity Proleukin is approved for the treatment of metastatic melanoma and metastatic kidney cancer, but its side effects are so severe that its use is recommended only in hospital settings with intensive care use (http:// www.proleukin.com/assets/pd/Proleukin. pdf) until the recent characterization of Treg cells, IL-2 is considered an immune system stimulator, activating T cells and other immune cells to eliminate cancer cells since Treg cells respond to lower concentrations of IL-2 than many other immune cell types because they express IL2R αβ γ, clinical trials of IL-2 in autoimmune diseases have taken lower doses of IL-2 to target Treg cells (Klatzmn D, Revmann. 15:283-94) even though these cells have taken a safer dose and more frequent administration of T.2015-2 therapy than daily therapy, and more frequent T.5-targeting of Treg cells to treat the disease than daily immune diseases, even if these cells are taken as a safer course of therapy and more resistant to the underlying immune diseases.

One approach to improving the therapeutic index of IL-2-based therapies is to use variants of IL-2 which are selective for Treg cells relative to other immune cells IL-2 receptor is expressed on a variety of different immune cell types including T cells, NK cells, eosinophils and monocytes, and this broad expression profile is likely to contribute to its pleiotropic effects on the immune system and high systemic toxicity IL-2 receptor exists in three forms, (1) low affinity receptor IL2, which does not signal, (2) medium affinity receptor (IL2 γ), consisting of IL2 and IL2, which is widely expressed on conventional T cells (Tcons), NK cells, eosinophils and monocytes, and (3) high affinity receptor (IL2 γ), consisting of IL2, IL2 and IL2, which is transiently expressed on activated T cells and expressed on Treg cells, which is transiently expressed and which is expressed on activated T cells and which is expressed on Treg 2 γ -2 variants (shanahlt, IL2 γ) which are more selective for IL-2 γ, IL-2 and which are shown to be expressed on human cells after the clinical studies of CD-2 γ + CD-2 cells, which are shown to be less selective for IL-2- γ -2- γ -binding and human cells- γ -factor and human tumor- γ - α - γ -factor, and human tumor- γ -2-factor, and tumor- γ -factor, and tumor- γ -2- γ -factor, and tumor- γ -factor, and tumor-factor, such as a factor, and tumor-2-factor, and tumor-factor, such as a factor, and tumor-factor, a factor, such as a factor, such as a factor.

An early study to improve the therapeutic index of IL-2-based therapies was to optimize the pharmacokinetics of this molecule to maximally stimulate Treg cells IL-2 action, indicating that IL-2 stimulation of human T cell proliferation in vitro requires exposure to an effective concentration of IL-2 for at least 5-6 hours (Cantrell, d.a. et al, 1984, Science,224:1312-1316) when administered to human patients, IL-2 has a very short plasma half-life of 85 minutes for intravenous administration and 3.3 hours for subcutaneous administration (Kirchner, g.i. et al, 1998, Br J Clin pharmacol.46:5-10) when administered to human patients, maintaining circulating IL-2 at or above the level necessary to stimulate T cell proliferation for the duration of time necessary to maintain the high dose (which results in peak IL-2 levels significantly higher than EC50 for T cells) as a result in frequent administration or require that these peak IL-2 levels can be significantly higher than the peak IL-2 administration of ex vivo IL-2 therapy, and thus the effect of these drugs can be more effectively improved by increasing the peak IL-2 administration in vivo than the other, the circulating IL-2-stimulating drugs which would be administered to achieve a much less effective or less effective therapeutic effect than the other, less effective IL-2-stimulating drugs administered to human patients (figure 1).

One way to increase the half-life of a therapeutic protein is to fuse a therapeutically active portion of the molecule to the Fc region of another protein, such as IgG, to increase the circulating half-life. Fusion of a therapeutic protein to IgG Fc achieves this by increasing the hydrodynamic radius of the protein (thus reducing renal clearance) and recycling of the fusion protein (thus extending the circulating half-life) mediated by the neonatal Fc receptor (FcRn). Fusion of therapeutic proteins with albumin (Sleep, d. et al, 2013, biochem biophysis acta.,1830:5526-34) and fusion with non-immunogenic amino acid polymer proteins (Schlapschy, m. et al, 2007, Protein Eng Des sel.20: 273-84; Schellenberger, v. et al, 2009, natbiotechnol.27:1186-90) have also been used to increase circulating half-life. However, the construction of such fusion proteins in a manner that ensures stable biological activity of IL 2-selective agonist partners may be unpredictable, especially in the case of IL 2-selective agonists (small proteins that are defective in binding to one of the receptor subunits and that must assemble a complex of three receptor subunits to activate the receptor) (Wang, X. et al, 2005, Science 310: 1159-63).

Other investigators have produced various IL-2 fusion proteins using wild-type IL-2 or IL-2 with a C125S substitution that promotes stability. Morrison and colleagues (Penichet, M.L. et al 1997, Hum antibodies.8:106-18) produced fusion proteins with IgG fused to wild-type IL-2 for the purpose of both increasing the circulating half-life of IL-2 and targeting IL-2 to a specific antigen for enhancing the immune response to that antigen. This fusion protein consists of a complete antibody molecule consisting of a heavy chain (H) and a light chain (L), wherein the N-terminal H chain portion is fused to the C-terminal IL-2 protein portion. This IgG-IL-2 fusion protein has Fc effector functions. Key effector functions of IgG Fc proteins are Complement Dependent Cytotoxicity (CDC) and Antibody Dependent Cellular Cytotoxicity (ADCC). IgG-IL-2 fusion proteins are highly active in IL-2 bioassays and are shown to have CDC activity. Thus, penechet et al teach the use of antibody-IL 2 fusion proteins to target IL-2 activity to an antigen recognized by the antibody for the purpose of enhancing humoral and cell-mediated immune responses to the antigen. In a similar manner, gilles and colleagues have constructed a variety of IgG-IL-2 fusion proteins for cancer immunotherapy that utilize the antibody portion of the fusion protein to target antigens, and the IL-2 portion to stimulate immune responses to tumor cells (reviewed in Sondel, p.m. et al, 2012, Antibodies,1: 149-71). These teachings are quite different from the technology of the present invention, in which an IL-2 selective agonist (which promotes the growth and activity of immunosuppressive Treg cells) is fused to an Fc protein moiety lacking effector function for the purpose of increasing systemic exposure.

Strom and colleagues have constructed fusion proteins with IL-2 fused to the N-terminus of an Fc protein for the purpose of eliminating activated T cells expressing high affinity IL-2 receptors (Zheng, X.X. et al, 1999, J Immunol.1999,163: 4041-8). This fusion protein was shown to inhibit the development of autoimmune diabetes in a T1D mouse model of T cell transfer. The IL2-Fc fusion protein was shown to inhibit pro-disease T cell function from T1D-susceptible female NOD mice when transplanted into less disease-susceptible male NOD mice. They also demonstrate that IL-2-Fc fusion proteins can kill cells expressing high affinity IL-2 receptors in vitro. These investigators further compared IL2-Fc fusion proteins constructed from Fc derived from IgG2bFc with effector function and mutated IgG2b Fc deficient in effector function. IL2-Fc fusion proteins comprising only Fc with effector functions are effective in preventing the onset of disease. Thus, these investigators teach that an IL2-Fc fusion protein with effector function can eliminate disease-causing activated T cells, and that Fc effector function is required for its therapeutic activity. These teachings are very different from the technology of the present invention, in which an IL-2 selective agonist (which promotes the growth and activity of immunosuppressive Treg cells) is fused to an effector function deficient Fc protein moiety for the purpose of increasing systemic exposure and optimizing Treg expansion. Other work by Strom and colleagues taught the use of IL2-Fc fusion proteins to promote graft tolerance (Zheng, X.X. et al, 2003, Immunity,19: 503-14). In this work, IL2-Fc fusion protein was used in "triple therapy" in which it was combined with IL15-Fc receptor antagonist and rapamycin. Moreover, these researchers teach that an IL2-Fc fusion protein must have Fc effector functions to be effective, and further teach that this IL-2-Fc fusion protein must be combined with two other molecules to be effective.

The present invention provides novel therapeutic agents, IL2 selective agonist-Fc fusion proteins having a 6-30 amino acid peptide linker. This configuration combines the high cell selectivity of IL2 selective agonists for Treg cells with a long circulating half-life. In the course of developing this molecule, there are surprising and unexpected findings which reveal the structural elements and design features of the protein necessary for biological activity, and which lead to the discovery of several novel proteins that meet the desired therapeutic characteristics.

Disclosure of Invention

The invention provides fusion proteins between an IL2 αβ gamma selective agonist protein (IL2 selective agonist) and an IgG Fc protein, wherein the IL2 agonist and the Fc protein are linked byThe Fc portion provides an extended circulatory half-life compared to the circulatory half-life of the IL-2 or IL2 selective agonist protein the Fc portion increases the circulatory half-life by increasing the molecular size of the fusion protein to greater than 60,000 daltons (which is an approximate cut-off for macromolecular glomerular filtration of the kidney) and by recycling the fusion protein through the neonatal Fc receptor (FcRn) protein (which binds and recycles IgG) thus extending its circulatory half-lifeThe stage of aging. The Fc portion is also deficient in Fc effector functions such as Complement Dependent Cytotoxicity (CDC) and Antibody Dependent Cellular Cytotoxicity (ADCC), thereby enabling the fusion protein to selectively activate tregs to enhance Treg function and expand Treg numbers. The two protein moieties are fused in a manner that maintains the stable biological activity of the IL2 selective agonist moiety and enables the Fc moiety to promote a prolonged circulatory half-life and thus effectively potentiate Treg function and number. This enhancement of tregs will suppress the overly vigorous autoimmune or inflammatory response and have the benefit of treating autoimmune or inflammatory diseases. The proteins of the invention may be monomeric or in dimeric form forming dimers through cysteine residues in the Fc portion or domain.

More specifically, the present invention provides a fusion protein comprising: an N-terminal human IL-2 variant protein portion and a C-terminal IgG Fc protein portion, wherein said IL-2 fusion protein has the ability to selectively activate a high affinity IL-2 receptor and thus selectively activate human regulatory T cells. Variants of IL-2 include those having substitutions relative to the human IL2 protein selected from: N88R, N88G, D20H, Q126L and Q126F. SEQ ID NO 1 is a variant IL-2/N88R, with numbering corresponding to wt IL 2. In addition, IL-2 variant proteins optionally include human IL-2 with the substitution C125S. It is preferred that the protein of the invention is fused, wherein both the IL-2 variant protein and the IgG Fc protein have an N-terminus and a C-terminus and the human IL-2 variant protein is fused at its C-terminus to the N-terminus of the IgG Fc protein. It is also disclosed that the activity of the IL-2 variant domain is greatly enhanced when a linker peptide is positioned between the IL-2 variant protein and the IgG Fc protein portion. The IgG Fc protein portion or domain optionally lacks Fc effector function or comprises one or more amino acid substitutions that reduce effector function of the Fc portion of the fusion protein.

An example of the invention is a protein comprising: IL-2 variant proteins with amino acid substitutions N88R and C125S relative to human IL-2(SEQ ID NO:1-N88R), linker peptides as set forth in SEQ ID NO:15, and human IgG1Fc protein with the substitution N297A as set forth in SEQ ID NO:2, wherein the fusion protein has the ability to selectively activate high affinity IL-2 receptors and thus selectively activate human regulatory T cells. Alternative proteins of the invention include: IL-2 variant proteins with amino acid substitutions N88R and C125S relative to human IL-2(SEQ ID NO:1-N88R), linker peptides as set forth in SEQ ID NO:15, and human IgG2Fc protein as set forth in SEQ ID NO: 3.

A more specific embodiment of the invention is a dimeric protein comprising two identical chains, wherein each chain comprises an N-terminal human IL-2 variant protein portion and a C-terminal IgG Fc protein portion, wherein the N-terminal human IL-2 variant protein portion has an N-terminus and a C-terminus, has at least one substitution variation compared to the wild type human IL-2 of SEQ ID NO:1 selected from the group consisting of N88R, N88G, D20H, Q126L and Q126F having at least 90 or 95 or 97% sequence identity to SEQ ID NO:1 and having the ability to activate those cells by binding IL2R αβ γ on Treg cells, the N-terminal human IL-2 variant protein is joined at its C-terminus to the N-terminus of an amino acid linker of 6-20 or 6-30 amino acid residues, wherein the linker also has a C-terminus, and the C-terminal Fc protein portion of the amino acid linker C-terminal IgG Fc protein portion of the amino acid linker is joined to the N-terminus of an amino acid linker of 6-20 or 6-30 amino acid residues, wherein the Fc protein portion of the amino acid linker is joined to the amino acid linker by a serine linker residue mixture of SEQ ID NO: 97, preferably comprising two amino acid residues of serine amino acid residues and serine linker 95, and 95, or 95, and 95, 95.

The present invention also provides the above composition in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

The invention also provides nucleic acids encoding the proteins described herein. The nucleic acid is preferably operably linked to an expression cassette which can be designed for recombination with the genome of a host cell or for introduction onto an independently replicating plasmid or extrachromosomal nucleic acid.

The present invention also provides a method of selectively activating human regulatory T cells in a patient in need thereof, the method comprising administering a pharmaceutical composition comprising administering the composition in a therapeutically effective dose until a desired level of human regulatory T cell concentration is achieved.

A method for measuring the number of Treg cells in a human blood sample by contacting human blood cells with the fusion protein of claim 1 at a concentration of 1nM-0.01nM and then detecting the protein-bound cells by flow cytometry.

Drawings

FIG. 1 is a graphical illustration of the relationship between circulating half-life, peak drug levels, biologically effective concentrations, and duration of time necessary to stimulate Treg cell proliferation following a single dose of IL-2 or IL2-Fc fusion protein with increased half-life the dotted lines represent IL-2 blood levels over time following subcutaneous injection, and the solid lines represent IL2-Fc fusion protein blood levels over time the dotted horizontal lines indicate the concentrations necessary to activate IL2R αβ γ and IL2R β γ expressing cells, respectively (EC50 values). double-headed arrows indicate the duration of exposure to IL-2 (5-6hr) under EC50 necessary to stimulate cell proliferation.

Figure 2 shows the design configuration of the Fc fusion protein. The fusion partner (X) may be fused at the N-terminus (X-Fc) or C-terminus (Fc-X) of the Fc protein. The linker peptide may be inserted between X and Fc.

Figures 3A-3C show the dose-response of IL-2 and N88RL9AG1 stimulated STAT5 phosphorylation in CD4+ T cells as measured by flow cytometry. Cells were treated with IL-2 or N88RIL2-Fc at the concentrations indicated above for 10 minutes at 37C, fixed, permeabilized, stained with antibody and then subjected to flow cytometry analysis as described in example 3. Cells sorted (gate) for CD4+ and further sorted for CD25 and pSTAT5 are shown, as shown in each of the 4 quadrants. Number in each quadrantThe percentage of CD4+ cells in each fraction is indicated. The cells in the upper quadrant represent the highest 1-2% CD25 expressing cells, an enriched population of Treg cells, and the cells in the right quadrant are pSTAT5 +. FIG. 3A. N88RL9AG1 stimulates only CD25 with high selectivity^{Height of}Cells, while IL-2 stimulates CD25 heavily^-/lowAnd CD25^{Height of}Both cells up to picomolar concentration. FIG. 3B, D20HL0G2 had no pSTAT5 stimulatory activity. No activation of pSTAT5 was observed in two independent experiments. FIG. 3℃ control shows D20H/IL2 stimulates CD25^{Height of}pSTAT5 but D20HL0G2 in cells were not stimulated. The graphics are displayed in a false color mode. The two proteins are expressed as 10^-8And (4) testing the concentration of M.

Figure 4 shows that CD4+ T cells treated with N88RL9AG1 showed stimulation of pSTAT5 levels in cells expressing high levels of FOXP 3. 4X 10 for cells^-9M IL-2 or N88RL9AG1 was treated and then analyzed as described in example 3. Most of the pSTAT5+ cells treated with N88RL9AG1 were also FOXP3+, while pSTAT5+ cells treated with IL-2 were also both FOXP 3-and FOXP3+, most of which were FOXP 3-.

Fig. 5A-5B show protein production of different Fc fusion constructs produced in HEK293 cells. The proteins were expressed in parallel in an optimized transient expression system and purified as described in example 1. The results are expressed as the final yield of purified protein from 30ml culture. FIG. 5A protein production of N88R/IL2-Fc fusion protein increased with increasing peptide linker length. Figure 5b. yield of wt IL2-Fc fusion protein only slightly increased with 15 residue peptide linker. Higher yields of D20H/IL2-Fc fusion protein were obtained in the X-Fc configuration but not in the Fc-X configuration.

FIGS. 6A-6B show the correlation of IL-2 bioactivity to peptide linker length in N88R/IL2-Fc fusion proteins. (FIG. 6A) CD25^{Height of}pSTAT5 signaling in CD4+ T cells (Tregs) increased with increasing peptide linker length. (FIG. 6B) in CD25^-/lowNo significant pSTAT5 signal was observed for any of the N88R/IL2-Fc proteins in the cells. 10^-8The pSTAT5 signal of the M IL-2 internal control is shown by black triangles in both figures.

FIG. 7 shows the biological activity of D20H/IL2-Fc fusion protein in human Tregs. The potency of D20HL15AG1 was significantly lower than that of N88RL15AG1, and D20HL15AG1(X-Fc configuration) and AG1L15D20H (Fc-X configuration) had similar potency. All 3 proteins have a 15 residue peptide linker.

FIGS. 8A-8B show the biological activity of wt IL-2-Fc pSTAT5 activity with or without a 15-residue peptide linker. IL-2 bioactivity was observed in Tregs cells (FIG. 8A) and CD25^-/lowOnly modest enhancement in cells (fig. 8B) was achieved by 15-residue peptide linker.

FIG. 9. selectivity of IL-2 and IL-2 selective agonist proteins on 7 different immune cell types in human PBMC. N88RL15AG1 was highly selective for tregs compared to wt IL-2 and WTL15AG1 and showed higher selectivity in various cell types than N88R/IL 2.

Detailed Description

Introduction to the design reside in

The present invention is a novel therapeutic fusion protein comprising three key protein elements: (1) an engineered IL-2 cytokine modified to be highly selective for Treg cells, (2) an effector function-deficient Fc protein that increases the circulating half-life of the protein and (3) a peptide linker between the two moieties, which is necessary for high biological activity of the fusion protein. The fusion protein is configured such that the IL-2 domain is linked to the N-terminus of the linker peptide via the C-terminus of the IL-2 domain. The Fc domain is linked via its N-terminus to the C-terminus of the linker. In previous studies with or without short peptide linkers, n-IL-2: c-Fc fusion proteins were reported to lack significant biological activity. This results in c-IL-2 focusing on the inverted configuration N-Fc, in which the Fc region connects the N-terminus of the linker and the IL-2 domain to form the carboxy terminus of the fusion protein.

Fc fusion proteins with a biologically active fusion partner domain at the N-terminus of the Fc are the preferred configuration because the biologically active domain replaces the Fab portion of IgG. Fc fusion proteins with a biologically active fusion partner domain on the C-terminus of the Fc are a less preferred configuration because the C-terminus of IgG Fc potentially compromises its ability to bind other molecules such as the Fc receptor FcRn, which is required for long circulating half-life of the Fc protein. IL2 fusion proteins fused to the C-terminus of Fc have been reported to have a much shorter circulating half-life than expected for Fc fusion proteins, indicating that the function or stability of Fc is compromised. Thus, the present invention with its long peptide linker necessary for IL-2 bioactivity represents a significant and unexpected advance in contrast to the teachings of prior art IL-2 fusion proteins. The molecules defined by the present invention will enable the safe and effective treatment of autoimmune diseases by stimulating new mechanisms that suppress the generation of small subpopulations of T cells of autoimmune and inflammatory pathologies. This paradigm-breaking therapeutic agent is expected to treat many different autoimmune diseases.

Definition of

As used herein, "at least a certain percentage (e.g., 90 or 95 or 97%) of sequence identity to sequence ID No. 1" refers to the degree to which the sequences of two or more nucleic acids or polypeptides are identical. The percent identity over the evaluation window (e.g., over the length of the target sequence) between the target sequence and the second sequence can be calculated by: aligning the sequences; determining the number of residues (nucleotides or amino acids) within the evaluation window relative to the same residues, allowing gaps to be introduced to maximize identity; divided by the total number of residues of the sequence of interest or of the second sequence (whichever is larger) that fall within the evaluation window; and multiplied by 100. When calculating the number of identical residues required to achieve a particular percent identity, the score will be rounded to the nearest integer. Percent identity may be calculated using a variety of computer programs. For example, computer programs such as BLAST2, BLASTN, BLASTP, GappedBLAST, and the like, generate alignments and provide percent identity between target sequences. The algorithms of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. ScL USA 87: 22264-. To obtain a gap-bearing alignment for comparison purposes, Gapped BLAST was utilized as described in Altschul et al (Altschul et al, Nucleic acids sres.253389-3402,1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs can be used. PAM250 or BLOSUM62 matrices may be used. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI). For these programs, see the Web site with the URL world-wide Web address "ncbi. In particular embodiments, percent identity is calculated using BLAST2 using the default parameters provided by NCBI.

"N-terminal" refers to the end of a peptide or polypeptide that carries an amino group, as opposed to a carboxyl end that carries a carboxylic acid group.

"C-terminal" refers to the end of a peptide or polypeptide bearing a carboxylic acid group, as opposed to the amino terminus bearing an amino group.

By "C-terminal IgG Fc protein portion" is meant the portion of a fusion protein derived from two identical protein fragments, each protein fragment having the hinge region, the second constant domain and the third constant domain of the two heavy chains of an IgG molecule and consisting of carboxy-terminal heavy chains disulfide-bonded to each other by the hinge region. It is functionally defined as the portion of an IgG molecule that interacts with complement protein C1q and an IgG-Fc receptor (fcyr), thereby mediating complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) effector functions. The sequences may be modified to reduce effector function, increase circulating half-life and eliminate glycosylation sites.

IL2 variants

The IL-2 variant proteins of the invention are IL-2 αβ γ selective agonists, functionally they selectively activate the IL2R αβ γ receptor complex relative to the IL2R β γ receptor complex, derived from a wild-type IL-2 protein, which is structurally defined as having at least 95% sequence identity to wild-type IL-2 of sequence ID No.1, and functionally defined by the ability to preferentially activate Treg cells, this protein can also be functionally defined by its ability to selectively activate IL-2 receptor signaling in tregs, as measured by the level of phosphorylated STAT5 protein in Treg cells compared to CD4+ CD 25-/low T cells or NK cells, or by the selective activation of phytohemagglutinin-stimulated T cells relative to NK cells.

By "N-terminal human IL-2 variant protein portion" is meant the N-terminal domain of the fusion protein, which is derived from the wild-type IL-2 protein as structurally and functionally defined above.

"C-terminal" refers to the end of a peptide or polypeptide bearing a carboxylic acid group, as opposed to the amino terminus bearing an amino group.

Treg

Regulatory T cells are a class of T cells that inhibit the activity of other immune cells and are defined by the cellular marker phenotypes CD4+ CD25+ FOXP3+ using flow cytometry since FOXP3 is an intracellular protein and requires cell fixation and permeabilization for staining, the cell surface phenotype CD4+ CD25+ CD 127-can be used to define live Treg. tregs also include various Treg subclasses such as tttregs (thymus-derived) and ptregs (derived, differentiated from the periphery of naive T cells in the periphery.) all tregs express the IL2R αβ gamma receptor, do not produce their own IL-2 and are dependent on IL-2 length to produce the same, and those skilled in the art will recognize that both classes will be selectively activated by IL2R αβ gamma selective agonists.

Peptide linker

A "peptide linker" is defined as an amino acid sequence located between two proteins comprising a fusion protein such that the linker peptide sequence is not derived from either partner protein. To promote proper protein folding and stability of the constituent protein parts, peptide linkers are incorporated into the fusion proteins as spacers to improve protein expression or to achieve better biological activity of the two fusion partners (Chen et al, 2013, Adv Drug Deliv Rev.65(10): 1357-69). Peptide linkers can be classified as unstructured flexible peptides or rigid structured peptides.

Fc fusion protein

An "Fc fusion protein" is a protein made by recombinant DNA technology in which the translational reading frame of the Fc domain of a mammalian IgG protein is fused to the translational reading frame of another protein ("Fc fusion partner") to produce a new single recombinant polypeptide. Fc fusion proteins are typically produced as disulfide-linked dimers joined together by disulfide bonds located in the hinge region.

Functional activation

"biological activity" refers to the measurement of biological activity in a quantitative cell-based in vitro assay.

"functional activation of Treg cells" is defined as an IL-2 mediated response in tregs. Analytical readings for functional activation of Treg cells included stimulation of pSTAT5, Treg cell proliferation, and stimulation of Treg effector protein levels.

Design and construction

There are a number of options for the design and construction of Fc fusion proteins, and the choice between these design options is given below to allow the generation of molecules with the desired biological activity and pharmacological profile. The key design options are: (1) the properties of selective agonists of IL2, (2) the choice of Fc protein moiety, (3) the configuration of the fusion partner in the fusion protein and (4) the amino acid sequence of the junction region between Fc and fusion partner.

General procedure

In general, the preparation of the fusion proteins of the invention can be accomplished by the procedures disclosed herein and by recognized recombinant DNA techniques including, for example, polymerase chain amplification reaction (PCR), preparation of plasmid DNA, DNA cleavage with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of DNA into an appropriate cell, transformation or transfection of a host, culture of a host. Alternatively, the fusion molecules can be isolated and purified using chaotropic agents and well-known electrophoretic, centrifugal and chromatographic methods. For disclosure of these methods see, generally, Sambrook et al, Molecular Cloning: Alaberration Manual (second edition) (1989); and Ausubel et al, Current Protocols in molecular biology, John Wiley & Sons, New York (1989).

The gene encoding the fusion protein of the present invention includes restriction enzyme digestion and ligation as essential steps for producing a DNA encoding the desired fusion. The ends of the DNA fragments may need to be modified prior to ligation, and this can be achieved by filling overhangs, deleting end portions of the fragments with nucleases (e.g., ExoIII), site-directed mutagenesis, or adding new base pairs by PCR. Polylinkers and adaptors can be used to facilitate ligation of selected fragments. Expression constructs are typically assembled at stages using rounds of restriction, ligation and E.coli transformation. Many cloning vectors suitable for constructing expression constructs are known in the art (cited in lambda. zap and pBLUESCRIPT SK-1, Stratagene, LaJolla, calif., pET, novagen inc., Madison, wis. - -Ausubel et al, 1999) and the particular choice is not critical to the present invention. The choice of cloning vector will be influenced by the gene transfer system chosen for introducing the expression construct into the host cell. At the end of each stage, the resulting constructs can be analyzed by restriction enzyme cleavage, DNA sequence, hybridization, and PCR analysis.

Site-directed mutagenesis is commonly used to introduce specific mutations into the gene encoding the fusion protein of the present invention by methods known in the art. See, e.g., U.S. patent application publication 2004/0171154; storici et al, 2001, Nature Biotechnology 19: 773-776; kren et al, 1998, nat. Med.4: 285-; and Calissano and Macino,1996, Fungal Genet.Newslett.43: 15-16. Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available for preparing variants of the invention.

Various promoters (transcription initiation regulatory regions) can be used according to the present invention. The choice of a suitable promoter depends on the proposed expression host. Promoters from heterologous sources may be used, provided they are functional in the host of choice.

Other suitable signal sequence/host cell pairs include a Bacillus subtilis sacB signal sequence for secretion in Bacillus subtilis and a Saccharomyces cerevisiae α -mating factor for secretion in Pichia pastoris (P.pastoris) or a Pichia pastoris acid phosphatase phoI signal sequence.

Elements for enhancing transcription and translation have been identified for use in eukaryotic protein expression systems. For example, placing the cauliflower mosaic virus (CaMV) promoter 1000bp on either side of a heterologous promoter can increase transcription levels 10 to 400 fold in plant cells. The expression construct should also include appropriate translation initiation sequences. Modifying the expression construct to include a Kozak consensus sequence for proper translation initiation can increase translation levels by 10-fold.

The expression cassette is ligated into a suitable vector compatible with the host being used. The vector must be able to adapt to the DNA sequence encoding the fusion protein to be expressed. Suitable host cells include eukaryotic and prokaryotic cells, preferably those that can be readily transformed and exhibit rapid growth in culture. Particularly preferred host cells include prokaryotic cells such as E.coli, Bacillus subtilis, and the like, and eukaryotic cells such as animal cells and yeast strains, e.g., Saccharomyces cerevisiae. Mammalian cells, in particular HEK, J558, NSO, SP2-O or CHO are generally preferred. Other suitable hosts include, for example, insect cells such as Sf 9. Conventional culture conditions were used. See Sambrook, supra. Stable transformed or transfected cell lines can then be selected. An in vitro transcription-translation system may also be used as an expression system.

The nucleic acid encoding the desired fusion protein can be introduced into the host cell by standard techniques for transfecting cells. The terms "transfection" or "transfection" are intended to include all conventional techniques for introducing nucleic acids into host cells, including calcium phosphate co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation, microinjection, viral transduction, and/or integration. Suitable methods for transfecting host cells can be found in Sambrook et al, supra and other laboratory texts.

Alternatively, one can use synthetic gene construction for the construction of all or part of the proteins described herein. This requires in vitro synthesis of the designed polynucleotide molecule to encode the polypeptide molecule of interest. Gene synthesis can be performed using a variety of techniques, such as the multiplex microchip-based technique described by Tian et al (Tian et al, Nature 432: 1050-.

The fusion protein of the invention is isolated from the harvested host cell or from the culture medium. The protein of interest is isolated from the culture medium or from the harvested cells using standard protein purification techniques. In particular, purification techniques can be used to express and purify the desired fusion protein on a large scale (i.e., in at least milligram quantities) from a variety of methods including roller bottles, spinner bottles, tissue culture plates, bioreactors, or fermentors.

IL2 selective agonist moieties

An exemplary case where IL-2 with the substitution of N88R is an IL2 selective agonist of the IL2R αβ gamma receptor (Shanafelt, A.B. et al, 2000, Nat Biotechnol.18: 1197-202.) IL2/N88R lacks binding to the IL2R β receptor subunit and IL2R β gamma receptor complex but is able to bind to the IL2R αβ gamma receptor complex and stimulate proliferation of PHA-activated T cells expressing IL2R αβ gamma as effectively as wt IL-2, although it appears that the ability to stimulate proliferation of truncated NK cells expressing IL2R β gamma is reduced by a factor of 3,000-fold other IL2R αβ gamma selective agonists with a similar spectrum of activity include IL-2 with the substitutions N88 and D20H, and other IL 585 gamma variants with the substitutions Q126L and Q126F (contact residues with IL2RG subunits) also have IL2 gamma agonist activity (Casl R αβ gamma selective agonists, cysteine residues), which are able to promote the activity of IL2 588 gamma receptor agonists of IL2 receptors by fusion with either of the above mentioned IL2, wt. 5, 23 gamma receptor antagonist, or cysteine substitution of the invention without the substitution of any of IL2, wt IL-35, cysteine, CD-35, CD.

Variants of the invention optionally include variants that apply to conservative substitutions of amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, when the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more (or all) of the selected codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-Buck 2608 (1985); Rossolini et al, mol.cell.Probes 8:91-98 (1994)). Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, that codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are silent variations, which are one type of conservatively modified variations. Each nucleic acid sequence herein that encodes a polypeptide also describes each possible silent variation of the nucleic acid. The skilled artisan will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each such sequence.

With respect to conservative substitutions of amino acid sequences, one of skill will recognize that a single substitution, deletion, or addition of a nucleic acid, peptide, polypeptide, or protein sequence that changes, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a conservatively modified variant, where the change results in the substitution of an amino acid to a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, but do not exclude, the polymorphic variants, interspecies homologs, and alleles of the invention.

The following groups each contain amino acids that are conservative substitutions for one another:

1) alanine (a), glycine (G);

2) serine (S), threonine (T);

3) aspartic acid (D), glutamic acid (E);

4) asparagine (N), glutamine (Q);

5) cysteine (C), methionine (M);

6) arginine (R), lysine (K), histidine (H);

7) isoleucine (I), leucine (L), valine (V); and

8) phenylalanine (F), tyrosine (Y), tryptophan (W).

FC protein moieties

The key design choice is the nature of the Fc protein portion. The major therapeutic applications of Fc fusion proteins are (1) conferring immunoglobulin Fc effector function to fusion partner proteins; or (2) increase the circulating half-life of the fusion partner protein (Czajkowsky et al, 2012, EMBO Mol Med.4: 1015-28). The major effector functions of IgG proteins are Complement Dependent Cytotoxicity (CDC) and Antibody Dependent Cellular Cytotoxicity (ADCC), which are functions mediated by Fc binding to the complement protein C1q and to IgG-Fc receptors (fcyr), respectively. These effector functions are important when therapeutic proteins are used to direct or enhance an immune response to a particular antigen or cell. The fusion proteins of the present invention are designed only to increase the circulating half-life of the IL2 selective agonist moiety, while effector function is not required and may even be toxic and therefore clearly undesirable. For example, an IL2 selective agonist-Fc fusion protein with Fc for effector functionality may potentially kill Treg cells that the fusion protein of the invention seeks to activate and expand, just as opposed to for the therapeutic purpose of autoimmune diseases. There are four human IgG subclasses that differ in effector function (CDC, ADCC), circulating half-life and stability (Salfeld, j.g.,2007, Nature Biotechnology 25: 1369-72). IgG1 has Fc effector function, is the most abundant IgG subclass, and is the most commonly used subclass in US FDA-approved therapeutic proteins. IgG2 is devoid of Fc effector functions, but undergoes dimerization with other IgG2 molecules, and also undergoes instability due to scrambling of disulfide bonds in the hinge region. IgG3 has Fc effector function and has an extremely long, rigid hinge region. IgG4 lacks Fc effector functions, has a shorter circulating half-life than other subclasses, and IgG4 dimer is biochemically unstable due to the exchange of H chains between different IgG4 molecules by a unique single disulfide bond in the hinge region. Those skilled in the art recognize that the Fc protein portions from IgG2 and IgG4 do not have effector function and can be used in the present invention. The skilled person will also recognise that Fc sequence modifications have been described in the art which allow the hinge region of IgG2Fc to be modified to prevent aggregation or the hinge region of IgG4Fc to be modified to stabilise the dimer. Alternatively, IgG1 variants deficient in effector function have been generated. One such variant has an amino acid substitution at position N297 (position of the N-linked glycosylation site). This substitution of the asparagine residue removes the glycosylation site and significantly reduces ADCC and CDC activity (Tao, M.H. et al, 1989, J Immunol.143: 2595-. This variation is used as an exemplary case in the present invention herein. Another effector function deficient IgG1 variant is IgG1(L234F/L235E/P331S /) (Oganesian et al, 2008, Acta Crystallogr D biol Crystallogr.64:700-4) which mutates amino acids in the C1q and Fc γ R binding sites, and one of skill in the art would consider using these or similar Fc variants to produce effector deficient and stable IL2SA-Fc fusion proteins. The skilled artisan also recognizes that forms of the Fc protein portion engineered to be stable monomers rather than dimers (Dumont, J.A. et al, 2006, BioDrugs 20: 151-60; Liu Z et al, J Biol chem.201520; 290:7535-62) may also be combined with the IL-2 selective agonists of the present invention. In addition, the skilled artisan will recognize that functional monomeric heterodimers consisting of an IL-2-Fc H chain polypeptide in combination with an Fc H chain polypeptide and assembled using bispecific antibody technology (Zhu Z et al, 1997Protein Sci.6:781-8) may also be combined with an IL-2 selective agonist of the invention. Some IL-2Fc fusion proteins were made with intact IgG antibody molecules, with (Penichet, M.L. et al 1997, Hum antibodies.8:106-18) or without (Bell et al 2015, J Autoimmun.56:66-80) antigen specificity in the IgG fraction. In addition, the skilled artisan will recognize that Fc variants lacking part or all of the hinge region may be used in the present invention.

Fc fusion proteins can be prepared in two configurations, denoted here as X-Fc and Fc-X (where X (fusion partner protein) is at the N-terminus and Fc is at the C-terminus) and Fc-X (where Fc is at the N-terminus and fusion partner protein is at the C-terminus) (FIG. 2). There are examples in the literature that indicate that different fusion partners can have different preferences for N-or C-terminal Fc fusions. For example, FGF21 has been shown to have a strong preference for the Fc-X configuration. Fc-FGF21 has essentially the same receptor-activating biological activity as FGF21 itself, whereas FGF21-Fc has a 1000-fold reduction in biological activity (Hecht et al, 2012, PLoS one.7(11): e 49345). A variety of IL-2Fc fusion proteins have been prepared for various applications, and these have been reported to have good IL-2 bioactivity when fused directly to Fc in both the Fc-X (Gillies et al, 1992, Proc Natl Acad Sci,89: 1428-32; Bell et al, 2015, JAutommun.56: 66-80) and X-Fc (Zheng, X.X. et al, 1999, J Immunol.163:4041-8) configurations. Gavin et al (US 20140286898A1) describe Fc fusion proteins comprising IL-2 and certain IL-2 variants in the Fc-X configuration, which have similar biological activity as free IL-2 cytokines, but in contrast to the results of Zheng et al (Zheng, X.X. et al, 1999, J Immunol.1999,163:4041-8), where IL-2 variant fusion proteins in the X-Fc configuration were found to have reduced or no biological activity. Therefore, Gavin et al generally teach against an N-terminal IL-2Fc fusion protein. Another factor that affects the choice of fusion protein configuration is the effect on circulating half-life. It is a repeated finding in the literature that the Fc-X configuration of the IL-2 fusion protein has a relatively low circulating half-life, much lower than the 21-day half-life of human IgG1 in humans or the half-life of current FDA approved Fc fusion proteins (table I). IgG-IL2 fusion proteins in the Fc-X configuration have been reported to be hour-scale in mice (Gillies S.D.,2002Clin Cancer Res.,8: 210-6; Gillies, S.D., US 2007/0036752A 2; Bell C.J.,2015J Autoimmun.56:66-80) and to have relatively short circulating half-lives in humans of about 3.3 hours (Ribas A., J2009 Transl Med.7:68) and 3.7 hours (KingD.M.,2004J Clin Oncol.,22:4463-73), and Fc-IL2 fusion proteins have been reported to have a circulating half-life in mice of 12.5 hours (Zhu E.F., Cancer. cell 2015., 13; 27 (489): 48501). Proteolysis between the C-terminal of the Fc portion and the IL-2 portion results in a short circulating half-life (Gillies S.D.,2002Clin Cancer Res.,8: 210-6; Zhu E.F.,2015Cancer cell.27: 489-501). Because of these relatively short half-lives, we focused on an IL2 selective agonist Fc fusion protein in the X-Fc configuration. Findings in this work indicate that IL2-Fc fusion proteins comprising an IgG1(N297A) substitution have high biological activity and are a particularly preferred class of the invention. Variants of the IgG1Fc fusion protein that eliminate O-linked sugars are also a particularly preferred class because it is highly biologically active and offers advantages for the manufacture of pure and homogeneous drug products. The findings in this patent further indicate that the IgG1 variant with defective effector function and the IL2-Fc fusion protein of IgG4Fc, although slightly less active, are also preferred species. Fusions with IgG2 and serum albumin (HSA) have lower biological activity and are a less preferred species, although they may be suitable for therapeutic use if they have other advantageous properties.

Joint

Fc andthe amino acid sequence of the junction region between the fusion partner proteins may be (1) a direct fusion of two protein sequences or (2) a fusion with an intervening linker peptide of the 10 Fc fusion proteins currently approved by the US FDA for clinical use (Table I), 8 are fusion partner proteins directly fused to Fc and 2 have linker peptides, so many Fc fusion proteins may be functional without linker peptides the linker peptide includes as a spacer between the two protein parts the linker peptide may promote correct protein folding and stability of the constituent protein parts, improve protein expression and achieve better biological activity of the constituent protein parts (Chen et al, 2013, Adv Dr Deliv Rev.65: 1357-69.) the peptide linker used in many fusion proteins is designed as a non-structured flexible peptide the length, sequence and configuration of the linker peptide between the independent domains in the native protein has provided a theoretical basis for the design of a flexible peptide linker (Argos, J. MolJ. 1990: 353. the length, sequence and configuration of the linker peptide between the independent domains in the native protein are designed as a non-structured flexible peptide linker peptide with a number of amino acid residues such as Argos, a serine amino acid sequence providing a high affinity for the design of a hydrophobic amino acid sequence, or a hydrophobic amino acid sequence such as a polypeptide with a low affinity, a hydrophobic amino acid sequence, such as a hydrophobic amino acid sequence, a hydrophobic linker peptide, a hydrophobic linkerEnd-to-end length of. Thus, linker peptides of 5, 10, 15, 20, 25 or 30 residues, respectively, haveOrIs extended to the maximum fully extended length. The maximum end-to-end length of a peptide linker may also be a guide for defining the characteristics of the peptide linker of the invention.

The skilled artisan will also recognize that non-peptide flexible chemical linkers may also be substituted for polypeptide linkers having the lengths shown above, e.g.OrThe purpose of the linker peptide of the invention is to enable the appropriate conformation and orientation of the individual fusion protein moieties to allow binding of the IL-2 selective agonist moiety to its cognate receptor and to allow binding of the Fc moiety to FcRn, thereby achieving fusion protein recycling and prolonged circulating half-life. Many Fc fusion proteins do not require linker peptides as demonstrated by 8 of the 10 US FDA-approved Fc fusion proteins listed in table I that lack such peptides. In contrast, dulaglutide, a fusion of GLP-1 and Fc, contains a 15-residue peptide linker that has a strong effect on biological activity (glaesener, US patent 7,452,966B 2). Previous work performed in the art on IL-2-Fc fusion proteins has shown that linker peptides are not necessary for biological activity. Fc-X directed IL-2 fusion proteins comprising either wt IL-2 or IL-2 with the substitution C125S have been reported to have IL-2 bioactivity similar to free IL-2 cytokine, either without (Gillies et al, 1992, Proc Natl Acad Sci,89: 1428-32; Gavin et al, US patent application 20140286898A1) or with (Bell et al, 2015, J Autoimmun.56:66-80) peptide linkers. In the X-Fc targeting, Zheng et al reported the IL-2 bioactivity of IL-2 fusion proteins in the X-Fc configuration, which is essentially indistinguishable from IL-2 itself (Zheng, X.X. et al, 1999, J Immunol.1999,163: 4041-8). This broad prior art teaches that fusion of an IL-2 protein to an Fc does not require a linker peptide to have high IL-2 bioactivity. However, the Gavin et al report, includes the presence of changesHas reduced or no biological activity without a peptide linker or with a 5-residue peptide linker (Gavin et al, US patent application 20140286898a 1). The work reported in this patent demonstrates that a peptide linker of at least 6 and preferably at least 9 amino acids is necessary to stabilize IL-2 bioactivity on tregs, and further shows that the improvement in bioactivity reaches a plateau at 15 amino acids and is maintained for linkers up to 30 amino acids in length.

Biological assay

Robust and quantitative bioassays are essential for the characterization of the biological activity of candidate proteins. These assays should measure activation of the IL2 receptor, measure downstream functional consequences of activation in tregs, and measure treatment-related consequences and function of activated tregs. These assays can be used to measure the therapeutic activity and potency of IL2 selective agonist molecules, and can also be used to measure the pharmacodynamics of IL2 selective agonists in animals or humans. One assay measures phosphorylation of the signal transducer STAT5, measured by flow cytometry using an antibody specific for phosphorylated protein (pSTAT 5). Phosphorylation of STAT5 is an essential step in the IL-2 signaling pathway. STAT5 is essential for Treg development, and a constitutively active form of STAT5 expressed in CD4+ CD25+ cells is sufficient to produce Treg cells in the absence of IL-2 (mahmudd, s.a. et al, 2013, JAKSTAT 2: e 23154). Thus, measurement of phosphorylated STAT5(pSTAT5) in Treg cells is well recognized by those skilled in the art as a reflection of IL-2 activation in these cells and would predict other biological consequences of IL-2 treatment given appropriate exposure times and conditions. Another assay for functional activation measures IL-2 stimulated Treg cell proliferation. One skilled in the art will recognize that Treg proliferation can be measured by: incorporation of tritiated thymidine into purified Treg cells, flow cytometry measurement of the frequency of the increase in the number of Treg cells and CD4+ CD25+ FOXP3+ or CD4+ CD25+ CD 127-marker phenotype in mixed cell populations, increased expression of proliferation-associated cyclins (such as Ki-67) in Treg cells, or measurement of cell division-related dilution of live fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) by flow cytometry in Treg cells. Another assay for functional activation of tregs using IL-2 is the increased stability of tregs. pTreg cells are considered by some to be unstable and have the potential to differentiate into Th1 and Th17 effector T cells. IL-2 activation of Tregs stabilizes Tregs and prevents this differentiation (Chen, Q. et al, 2011, J Immunol.186: 6329-37). Another consequence of IL-2 stimulation of tregs is stimulation of the levels of Treg functional effector molecules such as CTLA4, GITR, LAG3, TIGIT, IL-10, CD39 and CD73, which contribute to the immunosuppressive activity of tregs.

To develop an IL2 selective agonist Fc protein, we initially focused on proteins in the X-Fc configuration because of the short circulating half-life that has been reported for Fc-X configured IL-2 fusion proteins. The first two proteins, one with and the other without a linker peptide, produced and tested, unexpectedly showed that the protein with the peptide linker had IL-2 bioactivity and the protein without the peptide linker had no detectable bioactivity. Both proteins showed high binding affinity for IL2RA, indicating that both proteins fold correctly. These results indicate that linker peptides are essential for IL-2 receptor activation and bioactivity. A series of additional analogs were then generated to eliminate other variables and to test this hypothesis. The results from these studies led to the discovery of key structure-activity relationships for this therapeutic protein and the generation of new molecules with desirable activity and pharmacological attributes.

The following table provides a list of preferred categories.

Preparation

The pharmaceutical compositions of the fusion proteins of the present invention are defined as formulated for parenteral (in particular intravenous or subcutaneous) delivery according to conventional methods. Typically, a pharmaceutical formulation will include a fusion protein of the invention in combination with a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% aqueous dextrose, and the like. The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent loss of protein on the vial surface, and the like. Formulation methods are well known in The art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa.,19th ed., 1995.

By way of illustration, the pharmaceutical formulation may be provided as a kit comprising a container containing the fusion protein of the invention. The therapeutic protein may be provided in the form of an injectable solution for single or multiple doses, as a sterile powder for reconstitution prior to injection, or as a pre-filled syringe. Such kits may also include written information regarding the indications and the usage of the pharmaceutical composition. Furthermore, such information may include statements that the fusion protein of the invention is contraindicated in patients with known allergies to the fusion protein of the invention.

The IL-2 selective agonist fusion proteins of the invention can be incorporated into compositions, including pharmaceutical compositions. Such compositions typically comprise the protein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the compositions.

The pharmaceutical composition is formulated to be compatible with its intended route of administration. The IL-2 selective agonist fusion proteins of the present invention may be administered by parenteral routes. Examples of parenteral routes of administration include, for example, intravenous, intradermal, and subcutaneous. Solutions or suspensions for parenteral administration may include the following components: sterile diluents such as water for injection, saline solution, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for regulating tonicity such as sodium chloride or dextrose. The pH may be adjusted (e.g., to a pH of about 7.2-7.8, e.g., 7.5) with an acid or base such as sodium dihydrogen phosphate and/or disodium hydrogen phosphate, hydrochloric acid, or sodium hydroxide. Parenteral formulations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or Phosphate Buffered Saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that ready syringability exists. It should be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In the case of dispersions, maintenance of the desired particle size may be facilitated by the use of surfactants, such as polysorbates or tweens. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.

Sterile injectable solutions can be prepared by: the desired amount of active compound is incorporated in a suitable solvent with one or a combination of the above ingredients as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a base dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the previously active ingredient plus any additional desired ingredient from a sterile-filtered solution thereof.

In one embodiment, the IL-2 selective agonist fusion protein is prepared with a carrier that protects the IL-2 selective agonist fusion protein from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations may be prepared using standard techniques.

The pharmaceutical composition may be included in a container, package, or dispenser with instructions for administration.

Administration of

The fusion protein of the invention will preferably be administered by parenteral route. The subcutaneous route is the preferred route, but intravenous, intramuscular, and subcutaneous administration may also be used. For the subcutaneous or intramuscular routes, depot (depot) and depot formulations can be used. For certain diseases, a specific route of administration may be used. For example, for inflammatory eye diseases, intraocular injection may be used. The fusion protein may be used at a concentration of about 0.1 to 10mcg/ml total volume, although concentrations in the range of 0.01mcg/ml to 100mcg/ml may be used.

Determination of dosage is within the level of ordinary skill in the art. Daily or weekly administration during the treatment period, or may be administered at another intermittent frequency. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations may also be used. In general, a therapeutically effective amount of a fusion protein of the invention is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant change in circulating Treg cells, a clinically significant change in Treg cells present within diseased tissue, or a clinically significant change in disease symptoms.

Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dose of such compounds is preferably within a range of circulating concentrations that have little or no toxicity, including the half maximal effective concentration (EC 50; i.e., the concentration of the test compound that achieves half maximal stimulation of Treg cells). The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, a therapeutically effective dose can be initially estimated from cell culture assays. The dose may be formulated in animal models to achieve a circulating plasma concentration range that includes EC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by enzyme-linked immunosorbent assay.

As defined herein, a therapeutically effective amount (i.e., an effective dose) of an IL-2 selective agonist fusion protein depends on the polypeptide selected and the frequency of administration. For example, a single dose amount in the range of about 0.001 to 0.1mg/kg of patient body weight may be administered; in some embodiments, about 0.005, 0.01, 0.05mg/kg may be administered. The composition may be administered from once a day to once or more times a week, or once or more times a month; including once every other day. One skilled in the art will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, the level of Treg cells present in the patient, and other diseases present. Furthermore, treatment of a subject with a therapeutically effective amount of an IL-2 selective agonist fusion protein of the invention is likely to be a series of treatments.

Autoimmune diseases

Some diseases that may benefit from the treatment of the present invention have been noted. However, the role of Treg cells in autoimmune diseases is a very active area of research and additional diseases would likely be identified as treatable by the present invention. Autoimmune diseases are defined as human diseases in which the immune system attacks its own proteins, cells and tissues. A comprehensive list and overview of Autoimmune Diseases can be found in The Autoimmune Diseases (Rose and Mackay,2014, academic Press). Diseases for which evidence exists for benefit from Treg enhancement include graft versus host disease, pemphigus vulgaris, systemic lupus erythematosus, scleroderma, ulcerative colitis, crohn's disease, psoriasis, type 1 diabetes, multiple sclerosis, amyotrophic lateral sclerosis, alopecia areata, uveitis, neuromyelitis optica, and duchenne's muscular atrophy.

Other fusion proteins

Because the purpose of the Fc protein portion of the present invention is only to increase the circulatory half-life, one skilled in the art, using the structure-activity relationships found in the present invention, will recognize that the IL-2 selective agonist portion can be fused to the N-terminus of other proteins to achieve the same purpose of increasing molecular size and reducing renal clearance. An IL2 selective agonist may be fused to the N-terminus of serum albumin (Sleep, D. et al, 2013, Biochim Biophys acta.1830:5526-34), which increases the hydrodynamic radius of the fusion protein relative to the IL-2 moiety and is also recycled through the FcRN. The skilled artisan will also recognize that the IL2 selective agonist moiety of the invention may also be fused to the N-terminus of a recombinant non-immunogenic amino acid polypeptide. Two examples of non-immunogenic amino acid polypeptides developed for this purpose are the XTEN polymer, the chain of A, E, G, P, S and T amino acids (Schellenberger, v. et al, 2009, Nat biotechnol.27:1186-90)), and the PAS polymer, the chain of P, A and S amino acid residues (Schlapschy, m. et al, 2007, Protein Eng Des sel.20: 273-84).

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Examples

The following examples are offered by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize that a variety of non-critical parameters may be altered or modified to produce substantially similar results.

Example 1 cloning, expression and purification of IL-2 Selective agonist-IgG Fc fusion proteins

The cDNA encoding N88RL9AG1(SEQ ID NO:4) was constructed by DNA synthesis and PCR assembly N88RL9AG1 construct consisting of mouse IgG1 signal sequence, mature human IL-2(SEQ ID NO:1) sequence with substitutions N88R and C125S, 9 amino acid linker peptide sequence (SEQ ID NO:15) and Fc region of human IgG1 containing the substitution N297A N88R/IL2 is an IL2 selective agonist with reduced binding to IL2RB and selective agonist activity on IL2R αβ γ receptor-expressing cells (Shanafelt, A.B. et al 2000, Nat Biohntecol.18: 1197-202), the elimination of the N297 site of IgG linked glycosylation at N297 on IL 5961 Fc reduces effector functions (Tao, M.H. et al 1989, JImmtecol.143: 25143-202), SEQ ID NO: 358, SEQ ID NO: 31-99, SEQ ID NO: 31 J. 11-11, SEQ ID NO: 35 J. 11, 35, SEQ ID NO: 11-11, 35 J. 11, 35, III) and mouse IgG 3520 variants with substitutions IgG 23, IgG-19, IgG-expressing the Fc 2 selective agonist activity.

These cDNAs were cloned into pcDNA3.1(+) (Life technologies, Carlsbad, Calif.) using restriction sites HindIII and NotI. The purified expression vector plasmid containing this construct was transiently transfected into HEK293 cells. HEK293 cells were seeded in shake flasks 24 hours prior to transfection and grown using serum-free chemically defined media. The DNA expression constructs were transiently transfected into 0.1 liter suspension HEK293 cells. After 24 hours, cells were counted to obtain viability and viable cell count. Cultures were harvested on day 5 and conditioned medium supernatant was passed at 3000 fX g clarified by centrifugation for 15 minutes. The protein was purified by running the supernatant on a protein A column (GE Healthcare), eluting with 0.25% acetic acid (pH 3.5), neutralizing the eluted protein with 1M Tris (pH8.0) and dialyzing against 30mM HEPES, pH 7,150mM NaCI. The samples were then sterile filtered through 0.2 μm membrane filters and analyzed by SDS PAGE under reducing and non-reducing conditions. Proteins migrate as disulfide-linked dimers. Protein concentration 1.11mg/ml cm calculated for use^-1The extinction coefficient of (a) was determined by absorbance, and aliquots were stored at-80C.

Cytokines N88R/IL2 and D20H/IL2 are variants of SEQ ID NO 1 and were produced in e.coli essentially as described in US patent 6,955,807B1, except that an additional mutation C125S was added for improved stability.

Example 2 assay of the receptor binding activity of N88RL9AG1 and D20HL0G 2.

To determine whether N88RL9AG1 and D20HL0G2 folded correctly, their affinities for IL-receptor subunits IL2RA and IL2RB were determined by Surface Plasmon Resonance (SPR) using a Biacore T-200 instrument (GEHealthcare). IL2RA and IL2RB extracellular domain proteins and IL-2 protein (R & D Systems, Minneapolis, MN) were immobilized on a CM-5Biacore chip via NHS/EDC coupling to final RU (resonance units) values of 30 and 484, respectively. The binding kinetics with IL2RA were measured at a flow rate of 50 ul/min at five IL2 and N88RL9AG1 concentrations in the range of 0.6nM to 45 nM. The binding kinetics with IL2RB were measured at five concentrations in the range of 16.7nM-450nM for IL2 and 14nM-372nM for Fc fusion protein at a flow rate of 10 ul/min. Dissociation constants (Kd) were calculated from kinetic constants using Biacore evaluation software version 2.0, assuming a 1:1 fit for IL-2 and a bivalent fit for N88RL9AG1 and D20HL0G 2. Equilibrium Kd values were calculated by Biacore evaluation software using steady state binding values.

Binding to IL2RA was detected against both IL-2 and N88RL9AG 1. Rmax values for N88RL9AG1 (14.43) were 5.5 times higher than Rmax values for IL2 (2.62), which is in contrast to N88RL9AG1 (82)916g/M) has a higher molecular weight than IL-2(15,444 g/M). The kon, koff and Kd values for IL-2 were within the range predicted from published SPR values (Table II). The affinity of N88RL9AG1 was approximately 2-fold higher than that of IL2, as determined by both kinetic and equilibrium methods. Binding of IL2 to IL2RB was detected as having an Rmax of 6.19. The values determined for kon, koff and Kd are within the ranges reported in the literature. Reported values are 3.1X 10^-8M (IL2RA) and 5.0X 10^-7M (IL2RB) (Myszka, D.G. et al, 1996, Protein Sci.5: 2468-78); 5.4X 10^-8M (IL2RA) and 4.5X10^-7(IL2RB) (Shanafelt, A.B. et al 2000, Nat Biotechnol.18: 1197-202); and 6.6X 10^-9M (IL2RA) and 2.8X 10^-7M (IL2RB) (Ring, A.M. et al, 2012, Nat Immunol.13: 1187-95). Essentially no binding of N88RL9AG1 to IL2RB was detected, slight binding (Rmax 0.06) was detected at the highest concentration tested, much lower than expected based on the molecular weight difference between IL2 and N88RL9AG1 and based on IL2RA binding results. The D20HL0G2 protein was also tested for binding to IL2RA and was found to have 8.3X 10^-9The Kd of M is similar to that of N88RL9AG 1. Thus, SPR binding studies showed that both N88RL9AG1 and D20HL0G2 proteins bound IL2RA, indicating that the proteins were correctly folded.

Example 3 biological activity of N88RL9AG1 and D20HL0G2 on T cells.

The biological activity of N88RL9AG1 and D20HL0G2 on T cells was determined by measuring the level of phosphorylated STAT5(pSTAT5) in a specific subpopulation of T cells. Levels of pSTAT5 were measured by flow cytometry in fixed and permeabilized cells using antibodies against phosphorylated STAT5 peptide. Treg cells constitutively express CD25, and cells in the first 1% of CD25 expression levels are highly enriched for Treg cells (Jailwala, p. et al, 2009, PLoS one.2009; 4: e 6527; Long, s.a. et al, 2010, Diabetes 59: 407-15). Thus, flow cytometry data were sorted for CD25 for Treg and CD4 effector T cell subsets, respectively^{Height of}(first 1-2% of CD 25-expressing cells) and CD25^-/lowAnd (4) grouping.

Cryopreserved CD4+ T cells (Astarte Biologics, Seattle, WA) were thawed, washed in X-VIVO15 (Lonza, Allendale, NJ) medium containing 1% human AB serum (Mediatech, Manassas, VA), and allowed to recover at 37C for 2 hours. Cells were then plated at 5X10 in 0.1ml⁶The concentration of cells/ml was distributed into 15X75mm tubes. Cells were treated with varying concentrations of IL-2 or Fc fusion protein at 37C for 10 minutes. The cells were then fixed with Cytofix fixation Buffer for 10 minutes at 37C, permeabilized with Perm Buffer III (BD Biosciences, Santa Clara, CA) for 30 minutes on ice, and then washed. Cells were then stained with a mixture of anti-CD 4-Pacific Blue (BD Biosciences, santa clara, CA), anti-CD 25-AF488(eBioscience, San Diego, CA), and anti-pSTAT 5-AF547(BDBiosciences) antibodies at the manufacturer's recommended concentrations for 30 minutes at 20C, washed, and flow cytometry data were obtained on a LSRII instrument (BD Biosciences). Data were analyzed using Flowjo analysis software (Flowjo, Ashland, OR).

The results of N88RL9AG1 in this assay showed that N88RL9AG1 had significant selectivity for the Treg population compared to IL-2 (fig. 3A). N88RL9AG1 activated less than 1% of CD4+ cells, for CD25^{Height of}The cells have very strong selectivity. In contrast, IL-2 activated more than 80% of CD4+ T cells at a concentration of 40nM, with a high proportion of pSTAT5+ cells expressing low or no CD 25. IL-2 was found to be in CD25 even at 4pM (lowest concentration tested)^-/lowCells and CD25^{Height of}Significant levels of pSTAT5 were still stimulated in both cells.

D20HL0G2 was then tested for activity in the CD4+ T cell pSTAT5 assay. Surprisingly, D20HL0G2 was not active in this assay (fig. 3B). Use 10^-8M D20 additional control of 20H/IL2 cytokines (variant IL-2 cytokines not fused to Fc) showed CD25^{Height of}Stable and selective activation of pSTAT5 in cells (fig. 3C). Given that D20HL0G2 binds IL2RA with a similar Kd to IL-2 and N88RL9AG1 (thereby indicating that it is correctly folded), the lack of activity of D20HL0G2 is particularly surprising.

To confirm CD25 selectively activated by N88RL9AG1^{Height of}The cells were tregs, and activated cells were co-stained for pSTAT5 and FOXP3 (another molecular marker of Treg cells). CD4+ cells were treated with 4nM IL-2 or N88RL9AG1, fixed, and permeabilized as described above for pSTAT5 staining, and then treated with 1ml FOXP3Perm Buffer (BioLegend, San Diego, CA) for 30min at room temperature, and then washed and resuspended in FOXP3Perm Buffer. The permeabilized cells were stained with a mixture of anti-FOXP 3-eFluor450, anti-CD 25-AF488(eBioscience, San Diego, Calif.) and anti-pSTAT 5-AF547(BD Biosciences) antibodies at 20C for 30 minutes, washed, and analyzed by flow cytometry. The results of this experiment indicate that a high proportion of N88RL9AG 1-treated cells with activated STAT5(pSTAT 5+ cells) also expressed high levels of FOXP 3. This result provides further evidence that activated cells are highly enriched for Treg cells. In contrast, IL-2 treated pSTAT5+ cells were both FOXP3+ and FOXP 3-most were FOXP 3-cells.

Example 4 determination of structure-activity relationship important for biological activity.

The unexpected results described in example 3 indicate that the IL2 bioactivity measured with N88RL9AG1, but not D20HL0G2, is due to the presence of a linker peptide. To verify this finding and to eliminate the effect of other variables such as isotype of Fc portion and selective mutations in IL-2 portion, a panel of analogs (all using IgG1N297A Fc) was designed and generated (table III).

cDNA was constructed and proteins were expressed and purified as described in example 1, except that the C-terminal lysine residue of Fc was detected in all constructs and the producer cell culture was in a volume of 30ml instead of 100 ml. All proteins were recovered in good yield. Indeed, comparison of the yields of the N88R/IL2 series of molecules showed a clear trend of increasing protein yield with increasing peptide linker length, with N88RL20AG1 (with the longest peptide linker) recovery 6.8-fold higher than N88RL0AG1 (without peptide linker) (fig. 5A). The basis for improved yield of the linker peptide-containing protein is not clear, but may be due to increased expression levels, increased secretion rates, increased protein stability or increased purification efficiency. Interestingly, the yield of WTL15AG1 was only marginally (1.8-fold) higher than that of WTL0AG1, in contrast to the 4.5-fold higher yield of N88RL15AG1 compared to N88RL0AG 1. D20HL15AG1 production was similar to N88RL15AG1 production, indicating that IL-2 selective mutation had no significant effect on production and that both proteins had significantly higher production (4.3-fold and 3.4-fold, respectively) than AG1L15D20H (fig. 5B). Overall, these results indicate that increased peptide linker length correlates with higher protein production of Fc fusion proteins comprising N88R/IL2, that production of Fc fusion proteins comprising wt IL-2 is much less sensitive to the presence of linker peptide, and that IL-2-Fc fusion proteins of the X-Fc configuration are produced.

These purified proteins were tested in the human T cell pSTAT5 bioassay essentially as described in example 3, except that human CD3+ T cells (negatively selected) were used instead of CD4+ cells, and the cells were incubated with the test protein for 20min instead of 10 min.

Results from the N88R/IL2 series of molecules demonstrated that biological activity in the Treg-enriched population was significantly affected by the length of the peptide linker (fig. 6A). The pSTAT5 signal (% pSTAT5+ cells) in the Treg population increased stepwise with increasing peptide linker length. This increased bioactivity is at 10^-8The maximal pSTAT5 signal neutralization at M test proteins is reflected by both EC50 values (table IV). N88RL20AG1, protein with the longest peptide linker, showed a 4.2-fold increase in maximal pSTAT5 signal over N88RL0AG 1. Since the N88RL0AG1pSTAT5 signal was at its highest concentration (10)^-8M) did not reach 50% IL-2 activation, it was not possible to determine the fold increase in EC50 relative to N88RL0AG1 for the protein comprising the linker peptide. However, based on the highest concentrations of N88RL20AG1EC50 and N88RL0AG1 tested, it can be estimated that N88RL20AG1 exhibits compared to N88RL0AG1>100 times lower EC 50.

As expected, at CD25^-/lowSubstantially free of any detectable activity of the N88R/IL2 molecule on the population, and 10^{- 8}M IL-2 at 54.2% CD25^-/lowCellsMiddle stimulation of pSTAT5 activity (fig. 6B).

Comparison of WTL0AG1 with WTL15AG1 demonstrated that the linker peptide had a much lower significant effect on the wt IL-2-Fc fusion protein than the N88R/IL2-Fc fusion protein (fig. 7). In the Treg subpopulations, both WTL0AG1 and WTL15AG1 had significant biological activity and in fact stimulated the highest pSTAT5 phosphorylation level, approximately 2-fold higher than IL-2. However, WTL0AG1 and WTL15AG1 are also in CD25^-/lowLarge pSTAT5 signals were stimulated in cells at approximately 10-fold higher concentrations. WTL15AG1 and WTL0AG1 on Treg and CD25^-/lowBoth cell populations showed approximately 10-fold difference in EC50 values.

The maximal pSTAT5 signal for D20HL15AG1 in Treg was significantly lower than N88RL15AG1 (fig. 8). This indicates that the lack of any detectable activity in example 3 using D20HL0G2 is due in part to the lower activity of the D20H/IL2 moiety compared to the N88R/IL2 moiety in the context of Fc fusion proteins. The activity of AG1L15D20H was slightly higher than D20HL15AG1, indicating that the configuration of the IL-2 moiety in the Fc fusion protein (i.e., X-Fc vs Fc-X) had no major effect on Treg biological activity.

Overall, these results define the key features of the N88R/IL2-Fc fusion protein necessary for optimal biological activity. The N88R/IL2-Fc protein requires a linker peptide for optimal Treg bioactivity, with a tendency for bioactivity to increase with increasing linker peptide length. Second, consistent with other human work, the linker peptide had a milder effect on the biological activity of the wt IL-2 containing Fc fusion protein. These different requirements for linker peptides may be the result of the fact that N88R/IL2 lacks binding to IL2RB, which may lead to more stringent requirements for receptor binding and increased sensitivity to steric hindrance caused by the Fc fusion protein partner. These results also define the most potent IL2 selective agonist-Fc fusion protein.

Example 5 Selectivity of IL2 Selective agonist-Fc fusion proteins in human PBMCs

To determine that the N88R/IL2-Fc fusion protein is in a broader organismSelectivity in the scientific background, an assay was developed that measures STAT5 activation of all key immune cell types in crude unfractionated human PBMC. Human PBMC were isolated from normal volunteers by Ficoll-Hypaque centrifugation. Will 10⁶PBMCs were suspended in X-VIVO15 medium with glucose (Lonza) and 10% FBS (omega) and incubated at 37 ℃ with 10^-8M test protein treatment for 20 min. Cells were then treated with Foxp 3/transcription factor staining buffer set (EBIO) according to the manufacturer's instructions. The cells were then fixed with Cytofix buffer and permeabilized with Perm buffer III as described in example 3. The fixed and permeabilized cells were then washed with 1% FBS/PBS and stained with the antibody mixture for 60 minutes at room temperature in the dark. The stained cells were then washed in 1% FBS/PBS, resuspended in PBS, and analyzed on a Fortessa flow cytometer (BD Biosciences). The antibody mixture consisted of: anti-CD 4-PerCP-Cy5.5(BD, #560650), anti-pSTAT 5-AF-488(BD, #612598), anti-CD 25-PE (BD, #560989), anti-CD 56-PE-CF594(BD, #562328), anti-FOXP 3-AF647(BD, #560889), anti-CD 3-V450(BD, 560366) and anti-CD 8-BV650 (Biolegged, # 301041). This staining procedure enabled monitoring of pSTAT5 levels in 7 key immune cell types.

The cell phenotype is defined as follows: treg cells: CD3+, CD4+, Foxp3+, CD25^{Height of}CD8-, CD 56-; activated CD4Teff cells: CD3+, CD4+, Foxp3-, CD25^{Height of}CD8-, CD 56-; CD4Teff cells: CD3+, CD4+, Foxp3-, CD25^{Is low in}CD8-, CD 56-; NKT cells: CD3+, CD4-, Foxp3-, CD25^{Is low in}CD8-, CD56 +; NK cells: CD3-, CD4-, Foxp3-, CD25^{Is low in}CD8-, CD56 +; b cell: CD3-, CD4-, Foxp3-, CD25^{Is low in}、CD8-、CD56-。

The protein was analyzed at 10^-8And (4) testing the concentration of M. The results shown in figure 9 and summarized in table V demonstrate that N88RL15AG1 shows significant selectivity compared to wt IL2 and WTL15AG1, both of which activate pSTAT5 in a large fraction of all cell populations. N88RL15AG1 stimulated pSTAT5 signaling in Treg populations at levels close to wt IL-2, with other than NK cellsInsignificant activation of cell types. Additional analysis (not shown) demonstrated that pSTAT5+ NK cells were CD25^{Height of}It is NK-CD56^{Bright Light (LIGHT)}Characteristics of cells (NK cell subsets also having immunomodulatory activity) (Poli, A et al, 2009immunology.126(4): 458-65). Several cell types with low levels of pSTAT5 signaling for N88R/IL2 (activated CD4Teff cells, NK T cells, and NK cells) showed no or low pSTAT5 signaling for N88RL15AG 1. These results show the activity and high selectivity of N88RL15AG1 for Tregs in a complex biological environment.

Example 6 investigation of additional structure-function relationships important for biological activity.

The results presented in example 5 indicate a strong demand for stable biological activity of the N88R/IL-2-Fc fusion protein for linker peptides of 6-20 amino acids in length, wherein an increased linker peptide length correlates with an increased biological activity. To determine whether even longer linker peptides promote increased biological activity, additional protein constructs with peptide linker lengths of 25-30 amino acids were prepared as described in example 1 and tested in the T cell pSTAT5 biological activity assay as described in example 3, together with separate preparations of N88RL15AG1 and N88RL20AG 1. The results of this experiment showed that adding a peptide linker to 25(N88RL25AG1) or 30(N88RL30AG1) amino acids did not result in higher biological activity on CD25hi cells compared to proteins with a 20 amino acid linker (table VII). These results, together with the results given in example 4, indicate the ability of the peptide linker to promote the IL-2 bioactivity platform at a length of 15-20 amino acids and that longer linker peptides do not further promote an increase in bioactivity.

Alternative Fc fusion partners that can increase circulating half-life and lack Fc effector function were evaluated. IgG1Fc (SEQ ID NO.22), IgG2Fc (SEQ ID NO.23) with mutations E233P/L234A/L235A/G236del and IgG4Fc (SEQ ID NO.24) with hinge region mutation S228P that stabilizes the Fc dimer were prepared and tested similarly. In addition, fusion proteins in which IL2/N88R was fused to human albumin (fused to the N-terminus (N88RL15HSA, SEQ ID NO.25) or C-terminus (HSAL15N88R, SEQ ID NO.26)) of human albumin were prepared and tested. Although all proteins were biologically active on CD25hi cells, none of these fusion proteins showed higher biological activity than that observed for N88RL15AG1 (table VIII).

The effect of different IL-2 selective mutations was examined on the backbone of an IgG1N297A Fc fusion (Table IX). Protein with selective mutations N88G and Q126F at 10^-8M (highest concentration tested) had lower biological activity on CD25hi cells compared to N88R, while no biological activity was shown on CD 25-/low cells (data not shown). The protein with the substitution N88I was not active on any cell type. This may indicate that the original report of selective agonist activity for this variant was wrong, or alternatively this may indicate that it is not active in the case of an Fc fusion protein. The protein with the substitution Q126L had higher biological activity than N88R, as reflected by a greater pSTAT5 response at the highest tested concentration on CD25hi cells, although this was accompanied by 10^-8Expression of CD 25-/low in M moderate increase in cell activation (data not shown). These results indicate that Q126L/IL2 is a more potent selective agonist with higher biological activity on both CD25hi and CD 25-/low cells.

Finally, the effect of eliminating the O-linked glycosylation site at threonine 3 of the IL2/N88R moiety was evaluated by making the variant N88RT3AL15AG 1. The O-linked glycosylation site in IL-2 is not required for biological activity (Robb, R.J. et al, 1984, Proc Natl Acad Sci U S A.81:6486-90), and elimination of this glycosylation site should result in a fully deglycosylated protein with fewer post-translational modifications, thereby contributing to product heterogeneity. A variant N88RT3AL15AG1 was prepared and tested and was shown to have biological activity similar to N88RL15AG1 in CD25hi cells (table IX).

Table form

US FDA-approved Fc fusion proteins and features thereof

TABLE I

Affinity of IL-2Fc fusion proteins for IL2RA and IL2RB subunits

TABLE II

nd binding not detected

TABLE III

TABLE IV

TABLE V

Percentage of pSTAT5+ cells among 7 immune cells of human PBMC. Cells were treated with the proteins shown in the column headings and analyzed as described in example 6.

TABLE VI

TABLE VII

TABLE VIII

TABLE IX

Sequence listing

SEQ ID NO.1

Human IL-2(N88R)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

SEQ ID NO.2

Human IgG1(N297A) Fc

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.3

>human IgG2 Fc

VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

SEQ ID NO.4

>N88RL9AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

SEQ ID NO.5

>D20HL0G2

APTSSSTKKTQLQLEHLLLHLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

SEQ ID NO.6

>N88RL0AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.7

>N88RL5AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.8

>N88RL10AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.9

>N88RL15AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.10

>N88RL20AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.11

>WTL0AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.12

>WTL15AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.13

>D20HL15AG1

APTSSSTKKTQLQLEHLLLHLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.14

>AG1L15D20H

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLHLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT*

SEQ ID NO.15

>L9

GGGGAGGGG

SEQ ID NO.16

>L5

GGGGS

SEQ ID NO.17

>L10

GGGGSGGGGS

SEQ ID NO.18

>L15

GGGGSGGGGSGGGGS

SEQ ID NO.19

>L20

GGGGSGGGGSGGGGSGGGGS

SEQ ID NO.20

>N88RL25AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.21

>N88RL30AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.22

>N88RL3G1ED(E233P/L234A/L235A/G236del)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPPAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.23

>N88RL3G2

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.24

>N88RL3G4(S228P)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*

SEQ ID NO.25

>N88RL15HSA

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDY

LSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL*

SEQ ID NO.26

>HSAL15N88R

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGS

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT*

SEQ ID NO.27

>N88IL15AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.28

>N88GL15AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.29

>Q126FL15AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSFSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.30

>Q126LL15AG1

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

SEQ ID NO.31

>N88RT3AL15AG1

APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

Claims (19)

1. A fusion protein comprising a human IL-2 variant protein domain, a 6-35 amino acid peptide linker fragment domain, and an IgG Fc domain, wherein each domain has an amino terminus (N-terminus) and a carboxy terminus (C-terminus); and wherein the fusion protein is configured such that the C-terminus of the human IL-2 variant protein domain is fused by a peptide bond to the N-terminus of the peptide linker and the N-terminus of the IgG Fc protein moiety is fused by a peptide bond to the C-terminus of the peptide linker;

wherein the IL-2 fusion protein has the ability to selectively activate a high affinity IL-2 receptor and thereby selectively activate human regulatory T cells.

2. The fusion protein of claim 1, wherein the IL-2 variant protein comprises a human IL-2 having a substitution relative to a human IL2 protein (SEQ id no:1) selected from the group consisting of: N88R, N88G, D20H, Q126L and Q126F.

3. The fusion protein of claim 1, wherein the IL-2 variant protein comprises human IL-2 having the substitution C125S.

4. The fusion protein of claim 1, wherein the IL-2 variant protein is N88R provided in SEQ ID No. 1.

5. The fusion protein of claim 1, wherein the linker comprises glycine and serine residues and wherein the linker is 10-30 amino acids.

6. The fusion protein of claim 1, wherein the IgG Fc protein comprises one or more amino acid substitutions that reduce effector function of the Fc portion of the fusion protein.

7. A fusion protein comprising:

an IL-2 variant protein having the amino acid substitutions N88R and C125S relative to human IL-2(SEQ ID NO: 1);

b. a linker peptide as shown in SEQ ID NO. 15; and

c. the human IgG1Fc variant protein as shown in SEQ ID NO.2,

wherein the fusion protein has the ability to selectively activate the high affinity IL-2 receptor and thereby selectively activate human regulatory T cells.

8. A fusion protein comprising:

an IL-2 variant protein having the amino acid substitutions N88R and C125S relative to human IL-2(SEQ ID NO: 1);

b. a linker peptide as shown in SEQ ID NO. 15; and

c. human IgG2Fc protein as shown in SEQ ID NO 3.

9. A pharmaceutical composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier.

10. A method of selectively activating human regulatory T cells, the method comprising administering a pharmaceutical composition comprising: an IL-2 variant protein having the amino acid substitutions N88R and C125S relative to human IL-2(SEQ ID NO:1), a linker peptide as set forth in SEQ ID NO:15 and a human IgG1Fc protein as set forth in SEQ ID NO:2, wherein the pharmaceutical composition is administered in a therapeutically effective dose until the human regulatory T cell concentration reaches a desired level.

11. A method of selectively activating human regulatory T cells, the method comprising administering a pharmaceutical composition comprising: the IL-2 variant protein of claim 2 and a human IgG Fc protein selected from the group consisting of:

a. human IgG1Fc protein as shown in SEQ ID NO 2;

b. human IgG2Fc protein as shown in SEQ ID NO 3; and

c. the structural domain of human IgG4Fc protein as shown in SEQ ID NO:24,

wherein the pharmaceutical composition is administered in a therapeutically effective dose until the human regulatory T cell concentration reaches a desired level.

12. A method of measuring the number of Treg cells in a human blood sample by contacting human blood cells with the fusion protein of claim 1 at a concentration of 1nM to 0.01nM, and then detecting cells bound to the protein by flow cytometry.

13. A dimeric protein comprising two identical chains, wherein each chain comprises an N-terminal human IL-2 variant protein portion and a C-terminal IgG Fc protein portion, wherein:

the N-terminal human IL-2 variant protein portion

a. Having an N-terminus and a C-terminus;

b. a change in at least one substitution compared to the wild type human IL-2 in SEQ ID NO 1 selected from: N88R, N88G, D20H, Q126L and Q126F;

c. (ii) has at least 97% sequence identity to SEQ ID NO 1; and

d. has the ability to activate Treg cells by binding to IL2R αβ γ on those cells;

the N-terminal human IL-2 variant protein is joined at its C-terminus to the N-terminus of an amino acid linker of 6-30 amino acid residues, wherein the linker also has a C-terminus; and is

The C-terminus of the amino acid linker is joined to the N-terminus of an IgG Fc protein portion having 95% sequence identity to seq id No.2 and comprising a cysteine residue; and wherein the two chains are linked to each other via a cysteine residue of the IgG Fc protein moiety.

14. The dimeric protein of claim 13, wherein the IL-2 variant protein further comprises human IL-2 having the substitution C125S.

15. The protein of claim 13, wherein the amino acid linker consists of a linker selected from the group consisting of glycine residues, serine residues, and mixtures of glycine and serine residues.

16. The protein of claim 13, wherein the IL-2 variant protein portion has the substitution N88R.

17. The protein of claim 13, wherein the linker comprises a mixture of 12-17 serine and glycine residues.

18. The fusion protein of claim 13, wherein the linker comprises a glycine residue to serine residue ratio of 4: 1.

19. A nucleic acid encoding the fusion protein of claim 1.