TW201531482A - Modified interleukin 21 receptor proteins - Google Patents

Abstract

Disclosed herein are modified interleukin 21 receptor extracellular domain (IL21R ECD) proteins comprising a modification selected from: one or more amino acid substitutions at or within 4 amino acid residues of the tryptophan at position 148 of the amino acid sequence of SEQ ID NO:1, an insertion of one or more amino acids at position 148 of the amino acid sequence of SEQ ID NO:1, or deletion of the tryptophan at position 148 of the amino acid sequence of SEQ ID NO:1. Additional substitutions are also contemplated. Also disclosed are fusion proteins comprising the modified IL21R ECD proteins, pharmaceutical compositions comprising the modified IL21R ECD proteins and fusion proteins, and methods of treatment and uses relating to the modified IL21R ECD proteins and fusion proteins.

Description

Modified interleukin 21 receptor protein

The present invention relates to novel modified IL21R ECD proteins and fusion proteins thereof having improved stability, activity and increased performance as well as other properties. The invention further relates to the use of such proteins for the treatment of a disease or condition mediated by the interaction of IL21 with IL21R.

Human interleukin 21 (IL21) is a cytokine that shares sequence homology with IL2, IL4 and IL15 (Parrish-Novak et al. (2000) Nature 408: 57-63). The human IL21 receptor (IL21R) is a class I cytokine receptor that is expressed in lymphoid tissues, especially in T cells, B cells, natural killer (NK) cells, dendritic cells (DC), and macrophages. (Parrish-Novak et al. (2000) supra), such that these cells can react with IL21 (Leonard and Spolski (2005) Nat. Rev. Immunol. 5:688-98). The broad lymphatic distribution of IL21R indicates that IL21 plays an important role in immune regulation. In vitro studies have demonstrated that IL21 significantly regulates the function of B cells, CD4 ⁺ and CD8 ⁺ T cells and NK cells (Parrish-Novak et al. (2000) supra; Kasaian et al. (2002) Immunity 16: 559-69). Recent evidence suggests that IL21-mediated signaling can have anti-tumor activity (Sivakumar et al. (2004) Immunology 112: 177-82), and IL21 prevents antigen-induced asthma in mice (Shang et al. (2006) Cell. Immunol .241:66-74).

In autoimmunity, disruption of the IL21 gene and injection of recombinant IL21 have been shown to regulate the process of experimental autoimmune myasthenia gravis (EAMG) and experimental autoimmune encephalomyelitis (EAE), respectively (King et al. (2004) ) Cell 117: 265-77; Ozaki et al. (2004) Immunol. 173: 5361-71; Vollmer et al. (2005) Immunol. 174: 2696-2701; Liu et al. (2006) Immunol. 176: 5247-54) . In these experimental systems, it has been shown that manipulation of IL21-mediated signaling directly alters the function of CD8 ⁺ cells, B cells, T helper cells, and NK cells. Animal studies using a replacement murine IL21RFc fusion protein in a mouse model susceptible to lupus have demonstrated the pathogenic role of IL21 in disease models and can slow disease progression by administering a replacement fusion protein. (Herber et al. (2011) J. Immunol. 178: 3822-3830).

The amino acid sequences of IL21 and IL21R and the nucleotide sequences encoding the same are described in WO00/57761, WO01/85792 and WO2003/028630. IL21 and IL21R antagonists, including antibodies and fusion proteins, are also described in these applications. Despite these prior advances, there is a long-felt need to improve IL21R proteins, including IL21R fusion proteins with improved therapeutic features. The present invention satisfies this need.

The present application discloses modified IL21R extracellular domain (ECD) proteins or polypeptides and related reagents, compositions and methods.

Example 1 (E1). According to a first aspect of the invention there is provided a modified IL21R ECD comprising a modification selected from the group consisting of: four amine groups at position 148 of the amino acid sequence set forth as SEQ ID NO: 1. Substituting one or more amino acids at or within the acid residue; insertion of one or more amino acids at position 148 of the amino acid set forth as the amino acid sequence of SEQ ID NO: 1; Deletion of the tryptophan residue (W148) at position 148 of the amino acid sequence of SEQ ID NO: 1 is illustrated.

A plurality of embodiments (E) of this first aspect of the invention are described below, wherein E1 is identical thereto for the sake of convenience.

E.2. The modified IL21R ECD of E1, wherein one or more substituted or inserted amino acids are naturally occurring amino acids.

E3. The modified IL21R ECD of E2, wherein the substituted or inserted amino acid is selected from the group consisting of glycine, alanine, leucine, methionine, phenylalanine, lysine, glutamic acid , glutamic acid, serine, valine, valine, isoleucine, cysteine, tyrosine, histidine, arginine, aspartic acid, aspartic acid and A group consisting of threonine.

E4. The modified IL21R ECD of E1, wherein the substituted amino acid at position 148 is selected from the group consisting of serine or aspartic acid.

E5. The modified IL21R ECD of E1, further comprising at least one amino acid substitution at amino acid positions 147, 149 and 150 of the amino acid sequence set forth as SEQ ID NO: 1.

E6. The modified IL21R ECD of E5, wherein the amino acid substitution is selected from the group consisting of: a. glycine at position 147, serine at position 148, and glycine at position 149 ( SEQ ID NO: 2); b. Aspartic acid at position 148 and serine at position 150 (SEQ ID NO: 3); c. Aspartic acid at position 148, at position 149 Glycine and serine at position 150 (SEQ ID NO: 4); and d. serine at position 148 and glycine at position 149 (SEQ ID NO: 7).

E7. A modified IL21R ECD of E1 comprising a serine at position 148 (SEQ ID NO: 6).

E8. The modified IL21R ECD of E5 comprising aspartic acid at position 148, a serine at position 150, and further comprising aspartic acid at position 49 (SEQ ID NO: 5) .

E9. The modified IL21R ECD of claim 5, which is comprised at position 147 Glyceric acid, a serine at position 148, a glycine at position 149, and further comprising an amino acid substitution at position 122, relative to the amino acid sequence set forth as SEQ ID NO: 1.

E10. The modified IL21R ECD of E9 comprising alanine at position 122 (SEQ ID NO: 8).

E11. The modified IL21R ECD of E9 comprising the isoleucine at position 122 (SEQ ID NO: 9).

E12. The modified IL21R ECD of E9 comprising tryptophanic acid at position 122 (SEQ ID NO: 10).

E13. The modified IL21R ECD of E9 comprising amphetamine at position 122 (SEQ ID NO: 11).

E14. The modified IL21R ECD of E9 comprising tyrosine at position 122 (SEQ ID NO: 12).

E15. A fusion protein comprising a modified IL21R ECD of any one of E1 to E14 fused to a heterologous amino acid sequence.

E16. The fusion protein of E15, wherein the IL21R ECD is fused to a heterologous amino acid sequence via a linker.

E17. The fusion protein of E16, wherein the linker is a peptidyl linker comprising a sequence selected from the group consisting of GSGEGEGSEGSG (SEQ ID NO: 13); GGSEGEGSEGGS (SEQ ID NO: 14); and GGGGS (SEQ ID NO: 15).

E18. The fusion protein of E17, wherein the linker comprises the amino acid sequence GSGEGEGSEGSG (SEQ ID NO: 13).

E19. The fusion protein of E17, wherein the linker comprises the amino acid sequence GGSEGEGSEGGS (SEQ ID NO: 14).

E20. The fusion protein of E15, wherein the heterologous amino acid sequence comprises a human IgG1 Fc domain.

E21. The fusion protein of E20, wherein the Fc domain comprises an amino acid sequence set forth as SEQ ID NO: 16.

E22. A fusion protein according to E20, wherein the Fc domain is modified to alter the effector function of the Fc domain.

E23. The fusion protein of E22, wherein the Fc domain comprises an amino acid sequence set forth as SEQ ID NO: 19.

E24. A fusion protein according to E20, wherein the Fc domain is modified to increase the half-life of the fusion protein.

E25. The fusion protein of E24, wherein the Fc domain comprises an amino acid sequence set forth as SEQ ID NO: 17.

E26. The fusion protein of E24, wherein the Fc domain comprises an amino acid sequence set forth as SEQ ID NO: 18.

E27. A fusion protein according to E15, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

E28. A fusion protein comprising the amino acid sequence SEQ ID NO:20.

E29. A pharmaceutical composition comprising a modified IL21R ECD according to any one of E1 to E14, or a fusion protein according to any one of E15 to E28, and a pharmaceutically acceptable agent.

E30. The pharmaceutical composition according to E29, wherein the composition comprises 0.5-200 mg/mL of the modified IL21R ECD according to any one of E1 to E17 or the fusion protein of any one of E18 to E31, 10-50 mM slow-blooding Acid amine, 0.01-0.10 mg/mL EDTA, 20-120 mg/mL sucrose, and 0.01-0.4 mg/mL polysorbate 80.

E31. The pharmaceutical composition of E30, wherein the composition comprises 100 mg/ml of a fusion protein comprising the amino acid sequence set forth as SEQ ID NO: 20, and further comprising 40 mM. Serum amine Tris, 0.05 mg/mL EDTA, 100 mg/ml sucrose, and 0.2 mg/ml polysorbate 80.

E32. An isolated nucleic acid encoding a modified IL21R ECD of any one of E1 to E14 or a fusion protein of any one of E15 to E28.

E33. An isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO:31.

E34. A vector comprising a nucleic acid as E33.

E35. A host cell comprising the nucleic acid of claim 33.

E36. A host cell comprising the vector of claim 34.

E37. A method of making a modified IL21R protein comprising growing a host cell, such as E34 to E35, under conditions which exhibit a protein encoded by the nucleic acid.

E38. The method of E36, which further comprises isolating the protein.

E39. A modified IL21RFc fusion protein encoded by a nucleic acid insert contained in a vector of ATCC Accession No. PTA-120480.

E40. A modified IL21R-Fc fusion protein comprising an amino acid sequence encoded by a nucleic acid insert comprised by a vector deposited under ATCC Accession No. PTA-120480.

E41. A modified IL21R Fc fusion protein comprising an amino acid sequence consisting of the amino acid sequence SEQ ID NO: 20.

E42. A modified IL21R Fc fusion protein comprising an amino acid sequence consisting of the amino acid sequence SEQ ID NO:21.

E43. A modified IL21R Fc fusion protein comprising an amino acid sequence consisting of the amino acid sequence SEQ ID NO: 22.

E44. A modified IL21R Fc fusion protein comprising an amino acid sequence consisting of the amino acid sequence SEQ ID NO: 23.

E45. A modified IL21R Fc fusion protein comprising an amino acid sequence consisting of the amino acid sequence SEQ ID NO: 24.

E46. A modified IL21R Fc fusion protein comprising an amino acid sequence The amino acid sequence consisting of SEQ ID NO:25.

E47. A modified IL21R Fc fusion protein comprising an amino acid sequence consisting of the amino acid sequence SEQ ID NO:26.

E48. A modified IL21R Fc fusion protein comprising an amino acid sequence consisting of the amino acid sequence SEQ ID NO:27.

E49. A modified IL21R Fc fusion protein comprising an amino acid sequence consisting of the amino acid sequence SEQ ID NO:28.

E50. A modified IL21R Fc fusion protein comprising an amino acid sequence consisting of the amino acid sequence SEQ ID NO:29.

E51. A method of treating a disease or condition mediated by the interaction of IL21 and IL21R, comprising the IL21R ECD protein according to any one of claims E1 to E14, a pharmaceutical combination according to any one of claims E29 to E31 The fusion protein according to any one of claims E15 to E28 or E39 to E50 is administered to a patient in need thereof.

E52. The method of E51, wherein the disease or condition is an inflammatory or autoimmune disease or condition.

E53. The method of E51, wherein the disease or condition is selected from the group consisting of: transplant rejection, graft versus host disease (GVHD), multiple sclerosis, allergy, atopic allergy, diabetes, arthritic condition, class Rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, spondyloarthropathy, multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, skin Lupus erythematosus, autoimmune thyroiditis, dermatitis, atopic dermatitis, eczema dermatitis, psoriasis, Sjogren's syndrome, IBD, Crohn's disease Ulcerative colitis, asthma, endogenous asthma, allergic asthma, scleroderma, vasculitis, and Behcet's Disease.

E54. Use of a modified IL21R ECD according to any one of E1 to E14, or a fusion protein according to any one of claims E15 to E28 or E39 to E50, for use in the manufacture of A medicament for treating a disease or condition mediated by the interaction of IL21 with IL21R.

E55. The use of E54, wherein the disease or condition is an inflammatory or autoimmune disease or condition.

E56. The use of E54, wherein the disease or condition is selected from the group consisting of: transplant rejection, graft versus host disease (GVHD), multiple sclerosis, allergy, atopic allergy, diabetes, arthritic condition, rheumatoid Arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, spondyloarthropathy, multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, skin erythema Lupus, autoimmune thyroiditis, dermatitis, atopic dermatitis, eczema dermatitis, psoriasis, Hugh's syndrome, IBD, Crohn's disease, ulcerative colitis, asthma, internal Source asthma, allergic asthma, scleroderma, vasculitis, and Behcet's disease.

The use of the IL21R ECD protein of any one of E1 to E14, the pharmaceutical composition of any one of the aspects of E29 to E31, or the fusion protein of any one of claims E15 to E28 or E39 to E50, It is used to treat diseases or conditions mediated by the interaction of IL21 and IL21R in a patient in need thereof.

E58. The use of E57, wherein the disease or condition is an inflammatory or autoimmune disease or condition.

E59. The use of E57, wherein the disease or condition is selected from the group consisting of: transplant rejection, graft versus host disease (GVHD), multiple sclerosis, allergy, atopic allergy, diabetes, arthritic condition, class Rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, spondyloarthropathy, multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, skin Lupus erythematosus, autoimmune thyroiditis, dermatitis, atopic dermatitis, eczema dermatitis, psoriasis, Hugh's syndrome, IBD, Crohn's disease, ulcerative colitis, asthma, Endogenous asthma, allergic asthma, scleroderma, vasculitis, and Behcet's disease.

E60. The IL21R ECD protein according to any one of claims E1 to E14, such as E29 The pharmaceutical composition of any one of E31 or the fusion protein of any one of E15 to E28 or E39 to E50 for use in the treatment of a disease or condition mediated by IL21 and IL21R interaction.

E61. The pharmaceutical composition of E60, wherein the disease or condition is an inflammatory or autoimmune disorder.

E62. The pharmaceutical composition according to E60, wherein the inflammatory condition is selected from the group consisting of transplant rejection, graft versus host disease (GVHD), multiple sclerosis, allergy, atopic allergy, diabetes, arthritic condition, Rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, spondyloarthropathy, multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, Skin erythematosus, autoimmune thyroiditis, dermatitis, atopic dermatitis, eczema dermatitis, psoriasis, Hugh's syndrome, IBD, Crohn's disease, ulcerative colitis, asthma , endogenous asthma, allergic asthma, scleroderma, vasculitis, and Behcet's disease.

The foregoing summary of the invention, as well as the following embodiments, may be The presently preferred embodiments are shown in the drawings for the purpose of illustrating the invention. However, it should be understood that the invention is not limited to the precise arrangements and means shown.

In the drawings: Figure 1 depicts a graph showing the X-ray crystal structure of two chains of IL21R ECD at a resolution of 2.8 Angstrom, which is illustrated in shades of gray (chain A) and black (chain B). The individual IL21 cytokines complexed to the acceptor chain are represented by a surface density model. The insert map provides a close-up view of the flexible loop of the C-terminal domain of the receptor. The tryptophan acid (Trp or W) at position 148 of each chain is represented by a bar and the two tryptophan acids are shown to be linked via a pi-stacking interaction, which helps to make IL21 without any particular theoretical limitations. The homodimer of the receptor is stable.

2 depicts a graph illustrating data from a differential scanning calorimetry thermogram showing that the aspartate at position 122 is replaced with p-ethyl phenylalanine (paF) relative to other positions within the IL21R ECD. Modified IL21R ECD produced by (Asp or D) (D122) The thermal stability is enhanced.

Figure 3 depicts Western blots showing various transient expressions of modified IL21R ECD proteins containing His tags from HEK 293 cells. The cells were transiently transfected with each construct indicated at the bottom of the gel, and the supernatant was collected after five days of culture. Western blot analysis was performed on the culture medium of various mutants. More specifically, after transfer, mouse anti-histidine IgG (Qiagen, catalog number: 34670) as a primary antibody, rabbit anti-mouse IgG as a secondary antibody in combination with horseradish peroxidase ( Pierce, catalog number: 32430) to detect nitrocellulose membranes. Each lane was loaded with an aliquot of conditioned medium from the sample containing an equal number of cells (hereinafter referred to as "equal cell counts"). Wild-type ECD expression was directly compared to three different IL21R ECD variants ECD1, ECD2 and ECD3 containing mutations at and around Trp 148 (see Table 1 below). This Western blot analysis showed an increased expression of the modified IL21R ECD protein relative to the wild-type IL21R ECD protein.

Figure 4 is a Western blot image showing the performance of various modified IL21R ECD constructs from transiently transfected HEK 293 cells. The experimental method is as described for Figure 3 above.

Figure 5 shows a graph depicting a thermogram of differential scanning calorimetry showing enhanced thermostability of modified IL21R ECD constructs ECD1, ECD2 and ECD4 compared to wild-type IL21R ECD (WTECD).

Figure 6 depicts a graph showing the results from analytical size exclusion chromatography demonstrating a reduced level of aggregation of the modified IL21R construct ECD1 compared to wild-type ECD after nickel NTA initial affinity capture purification.

Figure 7, comprising Panels A and B, depicts a graph showing the results of analytical size exclusion chromatography showing enhanced resistance to cohesion of modified IL21R constructs ECD1 and ECD2 compared to wild-type ECD. The protein sample was subjected to overnight incubation at 4 ° C (Figure 7A shows that none of the proteins were agglomerated) and then subjected to a stress temperature of 40 ° C (Figure 7B), Set the chromatogram. After overnight incubation at 40 °C, peaks showing 16% protein aggregation for wild-type ECD were not present in the chromatogram of ECD1 or ECD2.

Figure 8 is a diagram depicting the structure of an IL21RFc fusion protein as disclosed herein. One IL21R-Fc fusion protein is shown in black and the other IL21R-Fc is shown in light gray. The two fusion proteins are displayed in the form of homodimers stabilized by two disulfide bridges formed by two cysteine acids in each hinge region of the Fc of each fusion peptide.

Figure 9, comprising Figures A through D, shows the results of an isothermal titration calorimetry (ITC) experiment that titrates the IL21 cytokine to the monomeric form of IL21R (ECD1) and to the homodimer comprising the linker and the Fc domain. The ECD1 fusion construct (ECD1-L3-FC3) was titrated against the IL21 cytokine. Figure 9A presents an experimental thermogram of IL-21 titration to monomeric ECD1. Figure 9B shows the fitted curve of the data in Figure 9A for the molar ratio (N) determination. Figure 9C presents an experimental thermogram of IL-21 titration to the homodimeric ECD1 Fc fusion construct ECD1-L3-FC3. Figure 9D shows the fitted curve of the data in Figure 9C for the molar ratio (N) determination. For monomeric ECD1, the resulting molar ratio N = 0.829 (Fig. 9B) approximates the expected binding stoichiometry N = 1 for one cytokine binding to one receptor. In the case of an Fc fusion molecule comprising linker L3, the measured molar ratio N = 0.888 (Fig. 9D) unexpectedly remained at approximately the same level of N = 1. The Fc fusion molecule contains two receptors and the expected result is closer to N=2.

Figure 10, comprising Panels A and B, depicts the results of a BIAcore IL21 titration study demonstrating that the linker L1 design allows each IL21RFc fusion protein homodimer to bind two IL21 cytokines. Figure 10A depicts an experimental sensor map showing the saturation of the reaction signal when IL-21 is titrated at a higher concentration. ECD1-L1-FC1 was coated on a BIAcore wafer to react the theoretical Rmax to 50.83 RU. The IL-21 cytokine was gradually titrated to saturation, and as seen in the fit curve of Figure 10B, Rmax was recorded as 49.5 RU. This observation indicates that each IL-21RFc homodimer on the wafer binds 97.4% of the complete stoichiometry of the two cytokines.

Figure 11 shows the amino acid sequence compared to linker L1 (top sequence: GSGEGEGSEGSG), description of changes in linker L2 (bottom sequence: GGSEGEGSEGGS).

Figure 12 is a Western blot image showing the transient expression of various modified IL21RFc constructs ECD1-L1-FC1, ECD1-L1-FC2, ECD7-L2-FC1, and ECD1-L2-FC1 in the cell. After the gel was transferred to a nitrocellulose membrane (NCM), NCM was visualized with a horseradish peroxidase-conjugated goat anti-human IgG (Sigma, catalog number: A0170) antibody to detect human Fc. Each sample identified at the bottom of the gel was loaded in duplicate and each lane was loaded with an aliquot of conditioned medium normalized to an equal number of cells. The rightmost lane contains molecular weight standards, where the weight is in kD. The migration position of the IL21Fc fusion construct is approximately 150 kD. Immediately above the fusion structure is a stain associated with the product of the higher molecular weight material. Smudges are produced by heterogeneous O-linked glycosylated materials. These post-translational modifications have been characterized and are located on specific serine residues in the linker L1 sequence. Comparison of the lanes of ECD1-L1-FC1 with ECD1-L1-FC2 shows that the two molecules have the same band pattern. This demonstrates the SDS-PAGE characteristics of the unmodified molecule of the R445K mutation in FC of FC1 to FC2. The comparison of the lanes of ECD1-L1-FC1 with ECD1-L2-FC1 was highlighted by a clef to show a significant reduction in stained material. This shows that the linker L2 design with two serine position substitutions is not effectively supported even if glycosylation supporting the O-linkage of the linker. Comparison of the lanes of ECD1-L2-FC1 with ECD7-L2-FC1 shows that the strip pattern of the ECD7 construct has an extremely significant improvement. Almost all high molecular weight stains above the 150kD strip have been removed. This demonstrates that the D122 mutation contained in the ECD7 design, by virtue of its stabilizing effect and in combination with the L2 linker design, allows for the production of more homogeneous molecules while reducing high molecular weight species.

Figure 13 including Figure A and Figure B shows an image of an isoelectric focusing (IEF) gel (Figure 13A) and an image of an SDS PAGE gel (Figure 13B) of the modified IL21RFc fusion construct ECD1-L1-FC1, Show the isoelectric point (pI). The empirical pI of the IEF gel was observed by a tight band group around the 4.2 pI standard. The pI sequence of this construct was calculated to be approximately 5.44. The difference is due to the fact that the six N-linked glycosylation sites are occupied by sialic acid and covered, allowing the molecule to The amino acid sequence is expected to be significantly more acidic than the amino acid sequence. The SDS PAGE gel exhibits non-reducing and reducing conditions and highlights the purity of the tested material and the dimer nature of the disulfide bond of the molecule, in contrast to disulfide in the denaturing conditions of the gel. Sugar alcohol (DTT) treatment may be completely reduced to monomer.

Figure 14 shows a graph depicting the results of a differential scanning calorimetry thermogram showing modified IL21RFc fusion proteins ECD7-L1-FC1, ECD8-L1-FC1, ECD9-L1 compared to wild-type IL21RFc (ECD1) The thermal stability of -FC1, ECD10-L1-FC1 and ECD11-L1-FC1 is enhanced. All constructs have the same design and comprise the same linker (L1) and Fc construct (FC1), except for the amino acid at position 122 of the IL21R ECD component of the fusion protein. The wild type IL21R ECD has Asp (D122) at position 122. ECD7, ECD8, ECD9, ECD10, and ECD11 have Ala, Ile, Trp, Phe, and Tyr at position 122, respectively. The CH2 and CH3 domain transition midpoint (Tm) did not change due to mutations. The wild-type ECD (having D122) has a lower Tm of 50.22 degrees, while the hydrophobic mutants at position 122 overlap, with an average increase of more than 4 degrees and a higher Tm of about 54 degrees. This display enhances thermal stability by mutating D122 to a hydrophobic residue.

Figure 15 shows a graph depicting the results of a BIAcore study of the binding of various modified IL21RFc fusion proteins to FcRn. The solid line refers to ECD1-L1-FC3. This FC3 molecule has a triple mutation (3m) and a reduced effector function (ie, LLGL has been mutated to AAGA). The dotted line indicates ECD1-L1-FC1. This FC1 molecule was designed to extend in vivo half-life and has a 3m mutation and LS double mutant to enhance binding to FcRn (see Table 4). The BIAcore experimental sensing map of this figure shows that the in vitro affinity of the FC1 construct for human FcRn is more than 10 times higher than that of the FC3 construct (ECD1-L1-FC3 mean K _D (nM) = 2114.5, sd 102.5 compared to ECD1 -L1-FC1 average K _D (nM) = 194.1, sd 17.1).

Figure 16 shows a graph depicting the pharmacokinetic (PK) profiles of various modified IL21 RFc fusion constructs in non-human primates administered intravenously (IV) or subcutaneously (SC) at a dose of 2 mg/kg. Solid circles showing intravenous ECD1-L1-FC3; solid squares showing intravenous ECD1-L1-FC1; open squares show subcutaneous ECD1-L1-FC1; and open diamonds show intravenous WT ECD-L3-Fc3. By measuring the serum concentration of molecules over time, this figure illustrates the progressive improvement of PK characteristics in vivo obtained with different IL21RFc constructs. Comparison of WTECD-L3-FC3 to ECD1-L1-FC3 demonstrates the combined advantages of the ECD1 mutation at position 148 and the L1 linker compared to wild-type ECD and linker L3. This design resulted in a 18-fold improvement in clearance rate (CL) from 15.9 mL/h/Kg to 0.882 mL/h/Kg. Comparison of ECD1-L1-FC3 with ECD1-L1-FC1 also demonstrates the direct beneficial effects of the LS mutations contained in FC1. Without wishing to be bound by any particular theory, FcRn binding enhancement significantly mediated a 3.5-fold improvement in terminal half-life for up to 247 hours and a half-cutting rate of 0.241 mL/h/Kg. In addition, the intravenous (IV) and subcutaneous (SC) data of ECD1-L1-FC1 showed a good bioavailability of about 84% for this molecule.

Figure 17 shows a graph depicting the results of primary B cell proliferation assays and the ability of different IL21 RFc constructs to inhibit proliferation. Neutralizing activity was not shown for the control human IgGl molecule in the range tested up to 50 nM. At slightly below 10 nM, both ECD1-L1-FC1 and WTECD-L1-FC3 exhibited equal neutralization performance. This display stabilizes the mutation introduced in the design of ECD1 and the use of LS mutations in FC1 and FC3 does not affect the in vitro biological function of IL-21 receptor.

Figure 18 shows a graph depicting the results of an IL21-dependent primary T cell assay using modified IL-21RFc constructs ECD7-L1-FC1, ECD8-L1-FC1, ECD9-L1-FC1, ECD10-L1 - FC1 and ECD11-L1-FC1. Cell proliferation was measured in counts per minute. The control human IgG1 with a solid black line did not exhibit neutralization activity in the range tested up to 1000 nM. All other molecules (wild-type ECD and D122 ECD mutants) exhibited equal neutralization potency at approximately 25 nM. This mutation at position D122 indicates that the hydrophobic residue sufficiently retains the biological function of the IL-21 receptor protein.

This application discloses modified IL21R ECD protein with unmodified wild type Compared to the IL21R protein, it has improved properties including higher in vitro performance, improved stability, reduced aggregation, and increased half-life. The half-life of the modified IL21R ECD protein can be further improved by fusing a heterologous protein such as the Fc region of the antibody to the IL21R ECD protein. The fusion protein can be further modified by substituting an amino acid of the Fc region of the fusion protein.

Definition and general technology

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meaning as commonly understood by one of ordinary skill. In addition, singular terms shall include the plural and plural terms shall include the singular unless the context requires otherwise. In general, nomenclature and techniques for cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly employed in the art. Their nomenclature and technology.

The methods and techniques of the present invention are generally described in accordance with the <RTIgt;conventional</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt; See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al, Current Protocols in Molecular Biology , Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which is incorporated herein by reference. Enzymatic reactions and purification techniques are typically accomplished in such techniques or as described herein according to the manufacturer's instructions. The nomenclature used in the analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmaceutical chemistry described herein, as well as their laboratory procedures and techniques, are well known and commonly used in the art, and their laboratory procedures and techniques. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

Unless otherwise indicated, the following terms are understood to have the following meaning: The term "polypeptide" encompasses natural or artificial proteins, protein fragments, and polypeptide analogs of protein sequences. The polypeptide can be a monomer or a polymer.

The term "isolated protein", "isolated polypeptide" or "isolated antibody" is a protein, polypeptide or antibody having one or four of the following origins or sources by virtue of its derivation: (1) not in its natural The state is associated with its natural association component, (2) does not contain other proteins from the same species, (3) is expressed by cells from different species, or (4) does not exist in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from a cell of natural origin will be "isolated" from the component with which it is naturally associated. Proteins can also be substantially free of natural association components by separation using protein purification techniques well known in the art.

A protein or polypeptide is "substantially pure", "substantially homogeneous" or "substantially purified" when at least about 60% to 75% of the sample exhibits a single species of polypeptide. The polypeptide or protein can be a monomer or a multimer. Substantially pure polypeptides or proteins typically comprise about 50%, 60%, 70%, 80% or 90% W/W of the protein sample, more typically about 95% and preferably more than 99% pure. Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of protein samples, followed by staining the gel with a dye well known in the art to observe a single polypeptide strip. band. For some purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

The term "polypeptide fragment" as used herein refers to a polypeptide having a deletion of an amino terminus and/or a carboxy terminus, but wherein the remaining amino acid sequence is identical to the corresponding position in the naturally occurring full length sequence. Furthermore, fragments according to the invention can be made by truncation, for example by removal of one or more amino acids from the N-terminus and/or C-terminus of the polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids can be removed from the N-terminus and/or C-terminus in this manner. Fragments can also be generated by one or more internal deletions. In some embodiments Where the fragment is at least 5, 6, 8 or 10 amino acids in length, in other embodiments, the fragment is at least 14, at least 20, at least 50 or at least 70, 80, 90, 100, 150 or 200 amines in length. Base acid.

In certain embodiments, the amino acid substitution of the protein or portion thereof is substituted with an amino acid: (1) reduced sensitivity to proteolysis, (2) reduced sensitivity to oxidation, (3) Altering the binding affinity used to form the protein complex, or (4) imparting or altering other physicochemical or functional properties. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be carried out in the sequences normally present.

Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence. Examples of recognized secondary and tertiary structures of the polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze) Edited by Garland Publishing, New York, NY (1991); and Thornton et al, Nature 354: 105 (1991), each of which is incorporated herein by reference.

As used herein, the term "binding affinity (K _D)" is intended to refer to a specific antigen - antibody off-rate of the solution interaction. K _D is the rate of dissociation (also known as "off-rate (k _off )") and the association rate (or "association rate" (on-rate) (k _on The ratio of ))). Therefore, K _D is equal to k _off /k _on and expressed as the molar concentration (M). Therefore, the smaller the K _D is, the stronger the binding affinity is. Therefore, compared with the K _D 1nM, K _D of the [mu] M indicates weak binding affinity. The K _D values for antibodies can be determined using techniques already improve the method. Method for determining the K _D of an antibody is generally used by the system such as the BIAcore biosensor system of such systems using surface plasmon resonance.

The term "surface plasmon resonance" (SPR) as used herein refers to an optical phenomenon that is allowed by, for example, using the BIACORE ^(TM) system (formerly Pharmacia Biosensor AB, Uppsala, Sweden, GE Healthcare, Little Chalfont, UK) Detection of changes in protein concentration in the biosensor matrix to analyze immediate biospecific interactions. For further description, see Johnsson U. et al., Ann. Biol. Clin. 51: 19-26 (1993); Jonsson U. et al., Biotechniques 11: 620-627 (1991); Jonsson B. et al., J. Mol. Recognit. 8: 125-131 (1995); and Johnsson B. et al, Anal. Biochem. 198:268-277 (1991).

The twenty naturally occurring amino acids and their abbreviations as used herein follow conventional usage. See Immunology-A Synthesis (Second Edition, ES Golub and DR Gren ed., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

The term "polynucleotide" as used herein, means a nucleotide of at least 10 bases in length (ribonucleotide or deoxyribonucleotide or a modified form of any type of nucleotide). The form of aggregation. The term includes both single and double shares.

The term "isolated polynucleotide" as used herein, means a genomic, cDNA or synthetically derived polynucleotide or a combination thereof, by virtue of its origin or source, "isolated polynucleotide" Having one or three of the following: (1) does not associate with all or a portion of the polynucleotides, wherein the polynucleotides are present in nature together with the "isolated polynucleotides", (2 ) operably linked to a polynucleotide in which the polynucleotide is not linked in nature, or (3) does not exist in nature as part of a larger sequence.

The term "oligonucleotide" as used herein includes naturally occurring and modified nucleotides joined together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subpopulation of polynucleotides that generally comprise a length of 200 bases or less. Preferred oligonucleotides are 10 to 60 bases in length and have an optimal length of 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 bases. Oligonucleotides are typically single stranded, for example, for primers and probes; but oligonucleotides can also be double stranded, for example, for use in constructing genetic mutants. The oligonucleotide of the invention may be a sense or antisense oligonucleotide.

The term "naturally occurring nucleotide" as used herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" as used herein includes a nucleotide having a modified or substituted sugar group and the like. The term "oligonucleotide linkage" as used herein includes, for example, phosphorothioate, dithiophosphate, selenophosphate, diselenyl phosphate, aniline phosphorothioate, aniline phosphate, phosphoniumamine. Oligonucleotide linkages of acid esters and the like. See, for example, LaPlanche et al, Nucl. Acids Res. 14:9081 (1986); Stec et al, J. Am. Chem. Soc. 106: 6077 (1984); Stein et al, Nucl. Acias Res. 16:3209 ( 1988); Zon et al, Anti-Cancer Drug Design 6:539 (1991); Zon et al, Oligonucleotides and Analogues: A Practical Approach , pp. 87-108 (edited by F. Eckstein, Oxford University Press, Oxford England (1991) U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990), the disclosure of which is incorporated herein by reference. Oligonucleotides can include detection labels as needed.

Sequences that are "operably linked" include expression control sequences adjacent to the relevant gene and expression control sequences that act in trans or at a distance to control the associated gene. The term "expression control sequence" as used herein means a polynucleotide sequence necessary for the performance and processing of the coding sequence to which it is ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie, Kozak common Sequence); sequences that enhance protein stability; and sequences that increase protein secretion if desired. The nature of the control sequences will vary depending on the host organism; in prokaryotes, such control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences generally include initiation. And transcription termination sequences. The term "control sequences" is intended to include at least all of the components necessary for expression and processing, and may also include other components that are preferred, such as leader sequences and fusion partner sequences.

The term "carrier" as used herein means capable of transporting another core to which it is connected. Acidic nucleic acid molecule. In some embodiments, the vector is a plastid, meaning a circular double stranded DNA loop into which other DNA segments can be joined. In some embodiments, the vector is a viral vector in which other DNA segments can be ligated into the viral genome. In some embodiments, the vector is capable of autonomous replication in a host cell into which it is introduced (eg, a bacterial vector having a bacterial origin of replication and a free mammalian vector). In other embodiments, a vector (eg, a non-episomal mammalian vector) can be integrated into the genome of the host cell after introduction into the host cell for replication with the host genome. In addition, certain vectors are capable of directing the performance of their operably linked genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The term "recombinant host cell" (or simply "host cell") as used herein means a cell into which an exogenous nucleic acid and/or a recombinant vector has been introduced. It should be understood that "recombinant host cells" and "host cells" are not only meant to refer to a particular individual cell, but also to the progeny of that cell. Since some changes may occur in the offspring due to mutation or environmental influences, in fact the progeny may differ from the parental cells, but are still included within the scope of the term "host cell" as used herein.

The term "selective hybridization" as used herein means detectable and specific binding. The polynucleotides, oligonucleotides and fragments thereof of the invention are selectively hybridized to the nucleic acid strand under hybridization and wash conditions that minimize the significant amount of detectable binding to the non-specific nucleic acid. As is known in the art and discussed herein, "high stringency" or "highly stringent" conditions can be used to achieve selective hybridization conditions. An example of a "high stringency" or "highly stringent" condition is at 6X SSPE or SSC, 50% formamide, 5 x Denhardt's reagent, 0.5% at a hybridization temperature of 42 °C. The SDS, 100 μg/ml of the disrupted squid sperm DNA in the hybridization buffer, the polynucleotide is incubated with another polynucleotide for 12-16 hours, wherein one of the polynucleotides can be immobilized to a solid surface such as a membrane It was then washed twice with a washing buffer of 1 x SSC, 0.5% SDS at 55 °C. See also Sambrook et al., supra, pages 9.50-9.55.

In the context of a nucleic acid sequence, the term "percent sequence identity" means the percentage of residues when comparing and aligning the first adjacent sequence with the second adjacent sequence to obtain maximum correspondence. The sequence identity comparison can be an extension of more than at least about 9 nucleotides, typically an extension of at least about 18 nucleotides, more typically an extension of at least about 24 nucleotides, usually at least An extension of about 28 nucleotides, more typically an extension of at least about 32 nucleotides, and preferably an extension of at least about 36, 48 or more nucleotides. A variety of different algorithms known in the art can be used to measure nucleotide sequence identity. For example, FASTA, Gap, or Bestfit can be used to compare polynucleotide sequences, such as the Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, including, for example, the programs FASTA2 and FASTA3, provides an alignment of the optimal overlap region between the query and the search sequence and a percent sequence identity (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84 (1998); herein incorporated by reference) . Preset parameters for a particular program or algorithm are used unless otherwise specified. For example, FASTA as provided in GCG version 6.1 (which is incorporated herein by reference) may be used with its preset parameters (word length 6 and NOPAM coefficients for the scoring matrix) or use thereof The provided Gap uses its preset parameters to determine the percent sequence identity between nucleic acid sequences.

Unless otherwise specified, reference to a nucleotide sequence encompasses its complement. Thus, reference to a nucleic acid having a particular sequence is to be understood as encompassing its complementary strand and its complement.

The term "percent sequence identity" means a ratio expressed as a percentage of the number of identical residues compared to the number of residues compared.

When referring to a nucleic acid or a fragment thereof, the terms "substantial similarity" or "substantial sequence similarity" means that the nucleic acid having the appropriate nucleotide insertion or deletion is optimally aligned with another nucleic acid (or its complementary strand). Time, such as by any well-known algorithm of sequence identity (such as above) As measured by FASTA, BLAST or Gap), there is at least about 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96% in the nucleotide base. , 97%, 98% or 99% nucleotide sequence identity.

The term "substantially consistent" as applied to a polypeptide means that the two peptide sequences have at least 70%, 75%, when optimally aligned, such as by program GAP or BESTFIT using a preset gap weight supplied by the program. 80% or 85% sequence identity, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In certain embodiments, inconsistent residue positions differ due to conservative amino acid substitutions.

A "conservative amino acid substitution" is the replacement of an amino acid residue with an amino acid substituted with another amino acid residue having a side chain R group of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Where two or more amino acid sequences differ from each other due to conservative substitutions, the percent sequence identity can be adjusted up to correct for the conservative nature of the substitution. The method of making this adjustment is well known to those skilled in the art. See, for example, Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of groups of amino acids having side chains having similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic hydroxyl side Chain: serine and threonine; 3) guanamine containing side chain: aspartic acid and glutamic acid; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) alkali Side chains: aminic acid, arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyramine, lysine-arginine, alanine-proline, glutamate-day Aspartate and aspartic acid-glutamic acid.

Alternatively, the terms conservatively substituted or permuted as used herein have any variation of positive values in the PAM250 log-like matrix, which is disclosed in Gonnet et al., Science 256:1443-, herein incorporated by reference. 45 (1992). The "moderately conservative" substitution is any change in the PAM250 log-like matrix that has a non-negative value.

Sequence analysis software is commonly used to measure the sequence identity of a polypeptide. Protein analysis software performs sequence matching using a measure that confers similarity to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG contains programs such as "Gap" and "Bestfit" that use the preset parameters specified by the program to determine closely related polypeptides (such as homologous polypeptides from organisms of different species) or in the wild. Sequence homology or sequence identity between a protein and its mutant protein. See, for example, GCG version 6.1. FASTA can also be used to compare polypeptide sequences using preset or recommended parameters, see GCG version 6.1. (Wisconsin WI University) FASTA (e.g., FASTA2 and FASTA3) provides an alignment of the optimal overlap regions between the query and search sequences and a percent sequence identity (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000)). When comparing the sequences of the present invention with databases containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, especially blastp or tblastn, which uses preset parameters as supplied by such programs. See, for example, Altschul et al, J. Mol. Biol. 215:403-410 (1990); Altschul et al, Nucleic Acids Res. 25:3389-402 (1997).

The polypeptides of comparative homology are typically at least about 16 amino acid residues, typically at least about 20 residues, more typically at least about 24 residues, usually at least about 28 residues, and preferably greater than about. 35 residues. When searching for a library containing sequences from a large number of different organisms, it is preferred to compare the amino acid sequences.

The term "performance" is a measure of biological activity, and can be expressed as IC _50, or effective concentration of a desired activity of the proteins that regulate cell 50% inhibition of the biological activity of the protein.

The term "effective amount" or "therapeutically effective amount" as used herein refers to the amount necessary to achieve the desired therapeutic result (dose and duration and mode of administration). The effective amount is at least the minimum amount of active agent necessary to impart a therapeutic benefit to the individual, but less than the toxic amount.

The term "inhibiting" as used herein with respect to the biological activity of a polypeptide of the invention or By "neutralizing" is meant that the polypeptide is capable of substantially antagonizing, inhibiting, preventing, limiting, slowing, destroying, eliminating, terminating, attenuating or reversing the progression or severity of, for example, but not limited to, a biologically active inhibitor.

The term "competition" as used herein with respect to a polypeptide means that the first polypeptide binds to the epitope in a manner sufficiently similar to the binding of the second polypeptide, thereby being compared to the second polypeptide in comparison to the first polypeptide. Binding in the presence of a peptide, the binding of the first polypeptide to its cognate epitope is detectably reduced in the presence of the second polypeptide. Alternatively, the binding of the second polypeptide to its epitope in the presence of the first polypeptide may also be detectably reduced, but this is not necessarily the case. That is, the first polypeptide inhibits binding of the second polypeptide to its epitope, and the second polypeptide does not inhibit binding of the first polypeptide to its respective epitope. However, if each polypeptide detects, in the same, greater or lesser extent, the other polypeptide is inhibited from binding to its cognate epitope or ligand, the polypeptides are said to "cross-competitively" bind to each other. Individual epitopes. The invention encompasses competition and cross-competing polypeptides. Regardless of the mechanism by which competition or cross-competition occurs (eg, steric hindrance, conformational changes, or binding to a common epitope or a portion thereof), those skilled in the art will understand that such competition and/or based on the teachings provided herein. Or cross-competing polypeptides are encompassed by and applicable to the methods disclosed herein.

"IgG" as used herein means a polypeptide that is substantially of the class of antibodies encoded by the identified immunoglobulin gamma gene. In humans, this category contains IgG1, IgG2, IgG3, and IgG4. In mice, this class contains IgG1, IgG2a, IgG2b, IgG3. As used herein, "immunoglobulin (Ig)" means a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have a number of structural forms including, but not limited to, full length antibodies, antibody fragments, and individual immunoglobulin domains. As used herein, "immunoglobulin (Ig) domain" means an immunoglobulin region that exists as a unique structural entity as recognized by those skilled in the art of protein structure. Ig domains typically have a characteristic folding topology. The known Ig domain in IgG class antibodies is a variable heavy chain domain (VH); the heavy chain constant domains Cγ1, Cγ2, Cγ3, which together comprise Cγ1 and Cγ2 The Cγ domain between the hinge regions; the light chain variable domain (VL); and the light chain constant domain (CL), which in humans contain a kappa (Cκ) or lambda (Cλ) light chain constant domain.

The term "Fc region" as known in the art is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" (also referred to as "fragment crystallizable" region or "tail" region) may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as an extension sequence from position Cys226 or from an amino acid residue at Pro230 to its carboxy terminus. For all heavy chain constant region amino acid positions discussed in the present invention, the numbers are based on the Eu index, which was first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1): 78-85. In it, it describes the amino acid sequence of the myeloma protein Eu (the first sequenced human IgG1). The EU index of Edelman et al. is also described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991. Thus, "such as the EU index as set forth in Kabat" or "the EU index of Kabat" refers to the amino acid residue numbering system based on the human IgGl EU antibody of Edelman et al. as set forth in Kabat 1991.

The Fc region of an immunoglobulin typically contains two constant domains: CH2 and CH3. Generally, the term "Fc polypeptide" as used herein encompasses the CH2 and CH3 domains and may include at least a portion of the hinge domain, but typically does not include the entire CH1 domain. As is known in the art, the Fc region may exist in a dimeric or monomeric form.

"Fc receptors" and "FcR" as used in the art describe receptors that bind to the Fc region of an antibody. Preferably, the FcR is a native sequence human FcR. Furthermore, preferred FcRs are FcRs (gamma receptors) that bind to IgG antibodies and include receptors of the FcyRI, FcyRII and FcyRIII subclasses, including dual gene variants and alternatively spliced forms of such receptors. Fc[gamma]RII receptors include FcyRIIA ("Activated Receptor") and FcyRIIB ("Inhibition Receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domain. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al; 1994, Immunomethods, 4:25-34; and de Haas et al; 1995, J. Lab. Clin. Med., 126: 330-41. "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, 1976, J. Immunol., 117: 587; and Kim et al, 1994, J. Immunol., 24: 249). ).

"Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein, includes any ingredient that, when combined with the active ingredient, allows the ingredient to retain biological activity and does not react with the individual's immune system. substance. Compositions comprising such carriers are by well known methods (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice) Of Pharmacy 20th Edition Mack Publishing, 2000) to deploy.

The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression or delay of the condition or condition to which the term applies or delaying the progression of one or more symptoms of the condition or condition. Attack or prevent it. The term "treatment" as used herein, unless otherwise indicated, refers to a therapeutic act as "treatment" as just defined above. The term "treatment" also includes adjuvant therapy and neoadjuvant therapy for an individual. For the avoidance of doubt, reference to "treatment" in this context includes reference to curative, palliative and prophylactic treatment.

In the present specification and claims, the word "comprising" is to be understood to include the integer or integer group, but does not exclude any other integer or group of integers.

Modified IL21R ECD protein

The present application discloses novel modified IL21R ECD proteins having substantially improved useful characteristics. Such proteins and fusion proteins thereof can be used, for example, as antagonists of IL21 cytokine activity.

By examining the X-ray crystal structure of the human wild-type IL21R ECD protein complexed to the IL21 cytokine, two of the dimeric configurations of the IL21R-IL21 cytokine complex The tryptophan residue (also known as Trp148 or W148) at position 148 of the wild type IL21R protein was identified at the interface of the IL21R monomers. The modified IL21R protein is designed by mutating the amino acid at or around W148 (ie, within the four amino acid residues of W148) using SEQ ID NO: 1 as a reference, yielding at least one of the following characteristics Constructs: greater stability, in vitro performance, reduced aggregation, and increased half-life, in order to develop therapeutic proteins with improved characteristics over wild-type proteins, thereby improving therapeutic efficacy and reducing commodity costs It also meets commercial needs.

Thus, the modified IL21R ECD protein is disclosed herein, which has one or both of the four amino acid residues on either side of W148 (ie, "lateral" W148) relative to SEQ ID NO: Multiple amino acid substitutions. The substitution in W 148 or in the four amino acid residues on either side of W148 may be any naturally occurring amino acid, including glycine, alanine, leucine, methionine, phenylalanine, Amino acid, glutamic acid, glutamic acid, serine, valine, valine, isoleucine, cysteine, tyrosine, histidine, arginine, aspartate Amino acid, aspartic acid and threonine. In a particular embodiment, W148 is substituted with serine or aspartate.

In other embodiments, the amino acid added to the four amino acid residues surrounding W148 or W148 may also be substituted. In some embodiments, the substitution can be at position 49 relative to SEQ ID NO: 1. In other embodiments, the substitution can be made at one or more of position 49, position 147, position 148, position 149, or position 150. In an exemplary embodiment, the modified IL21R ECD may comprise a substitution selected from the group consisting of: a. glycine at position 147, serine at position 148, glycine at position 149 (SEQ) ID NO: 2); b. Aspartic acid at position 148, serine at position 150 (SEQ ID NO: 3); c. Aspartic acid at position 148, at position 149 Aminic acid, a serine at position 150 (SEQ ID NO: 4); d. a serine at position 148, a glycine at position 149 (SEQ ID NO: 7); and e. an aspartic acid at position 49, an aspartic acid at position 148, and in position 150 of serine (SEQ ID NO: 5).

In another embodiment, the modified IL21R ECD protein can further comprise a substitution of an aspartic acid residue (D122) at position 122. In a particular embodiment, D122 can be substituted with alanine, isoleucine, tryptophan, amphetamine or tyrosine.

Table 1 provides an overview of the various exemplary IL21R ECD mutations disclosed herein. Table 2 provides the complete sequences of the various IL21R ECD proteins.

Modified IL21R fusion protein

The present application also discloses fusion proteins of the modified IL21R ECD proteins disclosed herein.

The term "IL21R fusion polypeptide" or "IL21R fusion protein" as used herein, refers to the fusion of one or more amino acid residues at the N-terminus or C-terminus of any of the modified IL21R ECD proteins described herein (such as Heterologous protein or peptide). Thus, the term "fusion protein" refers to a protein or polypeptide having an amino acid sequence derived from two or more proteins. The fusion protein may also include a linking region derived from an amino acid between the amino acid moieties of the respective proteins.

Heterologous peptides and polypeptides include, but are not limited to, epitopes (eg, FLAG) or tag sequences (eg, His ₆ and analogs thereof) such that IL21R polypeptide mutants can be detected and/or isolated; transmembrane receptor proteins Or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof, which binds to a transmembrane receptor protein; an enzyme or a portion thereof, which has catalytic activity; a polypeptide or peptide that promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide that increases stability, such as an immunoglobulin constant region (eg, an Fc domain); a half-life extension sequence that comprises two or more (eg, 2, 5, 10, 15) , 20, 25, etc. combinations of naturally occurring or non-naturally occurring charged and/or uncharged amino acids (eg, serine, glycine, glutamic acid or aspartic acid) designed To form a fusion partner for significant hydrophilicity or significant hydrophobicity of a modified IL21R ECD protein; a functional or non-functional antibody or a heavy or light chain thereof; and a polypeptide having activity such as therapeutic activity, The modified IL21RECD protein of the present invention is not .

The IL21R fusion protein can be prepared by fusing a heterologous sequence at the N-terminus or C-terminus of the modified IL21R ECD protein. As described herein, the heterologous sequence can be an amino acid sequence or a polymer containing a non-amino acid. The heterologous sequence can be fused directly to the modified IL21R ECD protein either chemically or by recombinant expression from a single polynucleotide, or it can be joined via a linker or adaptor molecule. The peptidyl linker or adaptor molecule can be one or more amino acid residues (or polymers), such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 residues (or polymers). Preferably 10 to 50 amino acid residues (or polymers), such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 residues (or polymer); and more preferably 15 to 35 amino acid residues (or polymers). The linker or adaptor molecule can also be designed to have a cleavage site for a DNA restriction endonuclease or protease such that the fusion moiety can be isolated.

a) linker

When forming a fusion protein of the invention, a linker can be used, but not necessarily. The linker may be composed of an amino acid linked together by a peptide bond, that is, a peptidyl linker. In some embodiments of the invention, the linker consists of from 1 to 20 or more amino acids joined by a peptide bond, wherein the amino acid is selected from the group consisting of 20 naturally occurring amino acids. In some embodiments, the amino acid is selected from the group consisting of amino acids, glycine, serine, and glutamate. In some embodiments, suitable linkers include: GSGEGEGSEGSG (SEQ ID NO: 13); GGSEGEGSEGGS (SEQ ID NO: 14); and GGGS (SEQ ID NO: 15). Although a linker of 12 amino acid residues has been found to be useful for the modified IL21 RFc fusion proteins disclosed herein, the present invention contemplates linkers of any length or composition. Exemplary linkers are shown in Table 3.

The linkers described herein are exemplary, and the invention encompasses linkers that are much longer and include other residues.

b) Fc protein

In some embodiments of the invention, the modified IL21R ECD protein is fused to an Fc domain, such as one or more domains of the Fc region of human IgG. The antibody comprises two functionally independent moieties: a variable domain called a "Fab" binding antigen; and a constant domain termed "Fc" which is particularly involved in effector functions such as complement activation and by phagocytic invasion. Fc has a long serum half-life, while Fab is short-lived (Capon et al., 1989, Nature 337: 525-31), so when conjugated to a therapeutic protein, the Fc domain provides a longer half-life or in a therapeutic protein. Effect functions such as Fc receptor binding, protein A binding, complement binding, and other desirable features are incorporated.

In vivo pharmacokinetic analysis indicated that wild-type human IL21R has a short half-life. Thus, to extend the half-life of IL21RFc, various Fc sequences were fused to the modified IL21R ECD proteins disclosed herein. Fusion of the Fc sequence to wild-type IL21R ECD did not extend the half-life to the desired value, however this observation allowed the identification of the modified IL21R ECD (as disclosed herein and illustrated in Table 2 above) and as illustrated in Example 16. A modified IL21RFc fusion protein with an increased half-life. These and other IL21RFc fusion proteins form embodiments of the invention. Table 4 below illustrates some of the Fc modifications exemplified in the present application, and Table 5 below illustrates some of the modified IL21RFc fusion protein constructs. In one embodiment, the modified ECD Fc fusion protein comprises an amino acid sequence encoded by a polynucleotide insert of the vector deposited on the ATCC with ECD1-L1-FC1 on July 17, 2013, wherein the ATCC registration number For PTA-120480.

The resulting modified IL21RFc fusion protein can be purified, for example, by using a Protein A affinity column. Peptides and proteins fused to the Fc region have been found to exhibit substantially greater in vivo half-lives than unfused counterparts. Fusion to the Fc region also allows the fusion polypeptide to dimerize/multimerize. The Fc region can be a naturally occurring Fc region, such as IgGl, IgG2, IgG3 or IgG4 Fc. In a In one example, the Fc region is a human IgGl Fc, such as SEQ ID NO: 16. The Fc region can also be altered to modify certain qualities, such as modifications that reduce effector function, such as SEQ ID NO: 19; or modified to improve therapeutic quality, such as increased cycle time (half-life), such as SEQ ID NO: 17.

Pharmaceutical composition

Pharmaceutical compositions comprising a modified IL21R ECD protein or fusion protein thereof are within the scope of the invention and are specifically encompassed by the present invention in light of the identification of several modified IL21R ECD sequences and fusion proteins exhibiting enhanced properties. Such pharmaceutical compositions may comprise a therapeutically effective amount of a modified IL21R ECD protein or a fusion protein thereof, in admixture with a pharmaceutically or physiologically acceptable formulation selected for the mode of administration. Acceptable formulations are preferably non-toxic to the recipient at the dosages and concentrations employed.

The pharmaceutical composition may contain, for example, altering, maintaining or maintaining, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or Penetrating formulation. Suitable formulating agents include, but are not limited to, amino acids (such as glycine, glutamic acid, aspartic acid, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite) , methionine or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, histidine, citrate, phosphate or other organic acids); bulking agents (such as mannitol or Glycine; chelating agent (such as ethylenediaminetetraacetic acid (EDTA)); complexing agent (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); filler Monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrin), proteins (such as serum albumin, gelatin or immunoglobulin); stains, flavorings and diluents; emulsifiers; hydrophilic polymerization (such as polyvinylpyrrolidone); low molecular weight polypeptide; salt-forming relative ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, p-hydroxyl Methyl benzoate, propyl p-hydroxybenzoate, chlorhexidine, sorbus Or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene Glycols; sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or humectants (such as pluronic; PEG; sorbitan esters; polysorbates, such as polysorbates) 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal; stability enhancers (such as sucrose or sorbitol); Such as alkali metal halides (preferably sodium chloride or potassium chloride) or mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see, for example, Remington's Pharmaceutical Sciences (18th Edition) , ARGennaro ed., Mack Publishing Company 1990 and subsequent versions of this document, which are incorporated herein by reference for all purposes.

The optimal pharmaceutical composition will be determined by those skilled in the art, for example, by the intended route of administration, the mode of delivery, and the desired dosage (see, for example, Remington's Pharmaceutical Sciences, supra). Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the modified IL21R ECD protein or fusion protein thereof.

The primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection may be water, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials commonly used in compositions for parenteral administration. Neutral buffered saline or a mixture with saline and serum albumin is another exemplary vehicle. Other exemplary pharmaceutical compositions comprise a histidine or Tris buffer of about pH 6.0-8.5, which may further comprise sorbitol or a suitable substitute. In one embodiment of the invention, the IL21RFc fusion protein composition can be prepared by mixing a selected composition of the desired purity with an optionally formulated formulation (Remington's Pharmaceutical Sciences, supra) in the form of an aqueous solution. For storage.

The pharmaceutical composition is optionally used for parenteral delivery. Alternatively, the composition can be optionally delivered for inhalation or via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation component is present at a concentration that is acceptable for the site of administration. For example Buffering agents are used to maintain the composition at a physiological pH or a slightly lower pH, typically in the range of from about 6 to about 8 pH.

When parenteral administration is desired, the therapeutic composition for use in the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired modified IL21R ECD protein or fusion protein thereof in medicine. A solution in an acceptable vehicle. Particularly suitable for parenteral injection is sterile distilled water, and the modified IL21R ECD protein or its fusion protein is formulated into a sterile, isotonic solution for proper preservation. Another preparation may include formulating the desired molecule with an agent such as an injectable microsphere, a bioerodible particle, a polymeric compound such as polylactic acid or polyglycolic acid, a bead or a liposome such that the subsequent injection via a reservoir The delivered product can be controlled for release or sustained release. Hyaluronic acid can also be used and it can have the effect of prolonging the duration of the cycle. Other suitable methods for introducing the desired molecule include implantable drug delivery devices.

In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the pharmaceutical composition can be formulated as a dry powder for inhalation. The inhalation solution can also be formulated with a propellant for aerosol delivery. In another embodiment, the solution can be sprayed. Transpulmonary administration is further described in International Publication No. WO 94/20069, which describes the transpulmonary delivery of chemically modified proteins.

Certain formulations are also expected to be administered orally. In one embodiment of the invention, the formulations administered in this manner may or may not be formulated with carriers that are typically used to compound solid dosage forms such as tablets and capsules. For example, the capsules can be designed to release the active portion of the formulation when the bioavailability in the gastrointestinal tract is greatest and the pre-system degradation is minimal. Other agents may be included to promote absorption. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders can also be used.

Another pharmaceutical composition can include a mixture of an effective amount of a modified IL21R ECD protein or a fusion protein thereof and a non-toxic excipient suitable for use in the manufacture of a tablet. The solution can be prepared in unit dosage form by dissolving the tablet in sterile water or another suitable vehicle. Suitable for excipient package Including (but not limited to): an inert diluent such as calcium carbonate, sodium or sodium bicarbonate, lactose or calcium phosphate; or a binder such as starch, gelatin or gum arabic; or a lubricant such as magnesium stearate, hard Fatty acid or talc.

Other pharmaceutical compositions will be readily apparent to those skilled in the art, including formulations comprising a modified IL21R ECD protein or fusion protein thereof in a sustained delivery or control delivery delivery formulation. Techniques for formulating a variety of other sustained or controlled delivery modes such as liposome carriers, bioerodible microparticles or porous beads and reservoir injections are also known to those skilled in the art (see, for example, International Publications). WO 93/15722, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions; and Wischke and Schwendeman, 2008, Int. J. Pharm. 364: 298-327, and Freiberg and Zhu, 2004, Int. J .Pharm. 282: 1-18, which discusses microsphere/particle preparation and use). As described herein, a hydrogel is an example of a sustained delivery or controlled delivery formulation.

Other examples of sustained release formulations include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. The sustained release matrix may include polyester, hydrogel, polylactide (U.S. Patent No. 3,773,919 and European Patent No. 0 058 481), copolymerization of L-glutamic acid and γ-ethyl-L-glutamate. (Sidman et al., 1983, Biopolymers 22: 547-56), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al., supra) or poly-D (-)-3-hydroxybutyric acid (European Patent No. 0 133 988) . Sustained release compositions can also include liposomes that can be prepared by any of a number of methods known in the art. See, for example, Epstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-92; and European Patent No. 0 036 676, No. 0 0 046 and No. 0 143 949.

Pharmaceutical compositions to be used for in vivo administration should generally be sterile. This can be achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, it can be sterilized using this method either before or after lyophilization and reconstitution. The composition for parenteral administration can be in a lyophilized form Or store in the form of a solution. Additionally, parenteral compositions are typically placed in a container having a sterile access port, for example, in an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle. The parenteral composition can be diluted in a parenterally acceptable diluent such as saline and 5% dextrose.

Once the pharmaceutical composition has been formulated, it may be stored in a sterile vial as a solution, suspension, gel, emulsion, solid form or as a dehydrated or lyophilized powder. Such formulations may be stored in ready-to-use form or in a form to be reconstituted prior to administration (eg, lyophilized form).

In one embodiment, the invention is directed to a kit for producing a single dose administration unit. The kits can each comprise a first container having dried protein and a second container having an aqueous formulation. Also included within the scope of the invention are kits comprising single chamber and multi-chamber pre-filled syringes (e.g., liquid injectors and dual chamber injectors).

In one embodiment, the invention is directed to a pharmaceutical composition comprising a modified IL21RFc fusion protein formulated into a powder for reconstitution into a solution for injection followed by injection. The formulation component can be in the range of concentrations: 0.5-200 mg/mL modified IL21R ECD or fusion protein disclosed herein, 10-50 mM tromethamine, 0.01-0.10 mg/mL EDTA, 20-120 mg/mL sucrose And 0.01-0.4 mg/mL polysorbate 80. In a specific embodiment, after reconstitution, the composition will contain 100 mg/ml IL21 RFc fusion protein, 40 mM tromethamine (Tris base), 0.05 mg/ml EDTA, 100 mg/ml sucrose, 0.2 mg/ml polysorbate. Ester 80.

The choice of treatment regimen for treatment will depend on a number of factors, including the serum or tissue turnover rate of the entity, the degree of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix. In certain embodiments, the dosing regimen maximizes the amount of treatment delivered to the patient while leaving the side effects at an acceptable level. Thus, the amount of biological agent delivered will depend, in part, on the severity of the particular entity and the condition being treated. Guidance for the selection of appropriate antibodies, Fc fusion therapeutic proteins, cytokines, and small molecule doses can be obtained (see, for example, Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxford.shire, UK; Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.), 1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al., 2003, New Engl. J. Med. 348: 601-608; Milgrom et al., 1999, New Engl. J. Med. 341: 1966-1973; Slamon et al., 2001, New Engl. .Med. 344: 783-792; Beniaminovitz et al, 2000, New Engl. J. Med. 342: 613-619; Ghosh et al, 2003, New Engl. J. Med. 348: 24-32; Lipsky et al. , 2000, New Engl. J. Med. 343: 1594-1602).

The appropriate dosage is determined by the clinician, for example, using parameters or factors known or suspected to affect treatment or are expected to affect treatment in the art. In general, the dose begins with an amount that is slightly less than the optimal dose and then increases in small increments until the desired or optimal effect is achieved with respect to any negative side effects. Important diagnostic measures include, for example, the symptoms of inflammation or the amount of inflammatory cytokines produced.

The actual dosage of the active ingredient in the pharmaceutical compositions of the present invention can be varied to achieve an amount effective to achieve the desired therapeutic response to a particular patient, composition, and mode of administration without being toxic to the patient. The selected dose will depend on a number of pharmacokinetic factors, including the activity of the particular composition of the invention or its ester, salt or guanamine; the route of administration; the time of administration; the rate of excretion of the particular compound employed; the duration of treatment Other drugs, compounds and/or substances used in combination with the particular compositions used; the age, sex, weight, condition, general health and prior medical history of the patient being treated; and similar factors well known in the medical arts.

Compositions comprising the modified IL21R ECD of the invention may be provided by continuous infusion or by administration at intervals of, for example, one day, one week, or one to seven times per week. The dose can be administered intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracranically or by inhalation. A specific dosage regimen is the maximum dose or frequency of administration that involves avoiding a large number of adverse side effects The program. The total weekly dose may be at least 0.05 μg/kg body weight, at least 0.2 μg/kg, at least 0.5 μg/kg, at least 1 μg/kg, at least 10 μg/kg, at least 100 μg/kg, at least 0.2 mg/kg, at least 1.0 mg/kg. At least 2.0 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg or at least 50 mg/kg (see, eg, Yang et al., 2003, New Engl. J. Med. 349:427 -434; Herold et al, 2002, New Engl. J. Med. 346: 1692-1698; Liu et al, 1999, J. Neurol. Neurosurg. Psych. 67: 451-456; Portielji et al, 2003, Cancer. Immunol. Immunother. 52: 133-144). The dose may be at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg. At least 95 μg or at least 100 μg. The dose administered to the individual may total at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 times, or more than 12 times.

For the therapeutically modified IL21R protein of the invention, the dosage to the patient to be administered may range from 0.0001 mg to 100 mg per kg of patient body weight. The dose may be 0.0001 mg to 20 mg, 0.0001 mg to 10 mg, 0.0001 mg to 5 mg, 0.0001 mg to 2 mg, 0.0001 mg to 1 mg, 0.0001 mg to 0.75 mg, 0.0001 mg to 0.5 mg, 0.0001 mg to 0.25 mg per kg of patient body weight, 0.0001 mg to 0.15 mg, 0.0001 mg to 0.10 mg, 0.001 mg to 0.5 mg, 0.01 mg to 0.25 mg or 0.01 mg to 0.10 mg.

The therapeutic protein dose of the present invention can be calculated by multiplying the patient's body weight in kilograms (kg) by the dose to be administered in mg/kg. The protein dosage of the present invention may be 150 μg/kg or 150 μg/kg or less, 125 μg/kg or 125 μg/kg or less, 100 μg/kg or less, 95 μg/kg or 95 μg/kg or less, 90 μg/kg or less. 90 μg/kg or less, 85 μ/kg or 85 μ/kg or less, 80 μ/kg or 80 μ/kg or less, 75 μ/kg or 75 μ/kg or less, 70 μ/kg or 70 μ/kg or less, 65 μ/kg or 65 μ/kg or less, 60μ/kg or 60μ/kg or less, 55μ/kg or 55μ/kg or less, 50μ/kg or 50μ/kg or less, 45μ/kg or 45μ/kg or less, 40μ/kg or 40μ/kg or less, 35μ/kg or 35μ /kg or less, 30μ/kg or 30μ/kg or less, 25μ/kg or 25μ/kg or less, 20μ/kg or 20μ/kg or less, 15μ/kg or 15μ/kg or less, 10μ/kg or 10μ/kg or less, 5μ/kg or 5μ/kg or less, 2.5μ/kg or 2.5μ/kg or less, 2μ/kg or 2μ/kg or less, 1.5μ/kg or 1.5gkgk or less, 1μ/kg or 1/kgk or less, 0.5μ/kg or 0.5μ/kg or less or 0.1 //kg or less than 0.1μ/kg.

The unit dose of the therapeutic protein of the present invention may be 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg To 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg or 1 mg to 2.5 mg.

The therapeutic protein dose of the invention may be at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least 15 μg in the individual. /ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml, at least 250 μg Serum titers of /ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml or at least 400 μg/ml. Alternatively, the antibody dose of the invention may be at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ml, at least in the individual. 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ml, at least Serum titers of 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml or at least 400 μg/ml.

The dosage of the therapeutic protein of the present invention may be repeated and the administration may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or at least 6 month.

The effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the route of administration, and the severity of the dosage and side effects (see, for example, Maynard et al., 1996, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FIa.; Dent, 2001, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK).

The route of administration can be via, for example, topical or dermal administration, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracranial, intralesional injection or infusion or by sustained release system or implant (see For example, Sidman et al, 1983, Biopolymers 22: 547-556; Langer et al, 1981, J. Biomed. Mater. Res. 15: 167-277; Langer, 1982, Chem. Tech. 12: 98-105; Epstein et al. USA, 1985, Proc. Natl. Acad. Sci. USA 82: 3688-3692; Hwang et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4030-4034; U.S. Patent No. 6,350,466 and 6,316,024 number). If desired, the compositions may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Alternatively, pulmonary administration can be employed, for example, by using an inhaler or nebulizer and by aerosol formulation. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication No. WO 92/19244, WO 97 /32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference in its entirety. In one embodiment, an engineered antibody or engineered antibody conjugate, combination therapy or composition of the invention is administered using Alkermes AIR ^(TM) via pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).

The frequency of administration will depend on the pharmacokinetic parameters of the modified IL21R ECD protein or its fusion protein in the formulation used. Typically, the clinician will administer the composition until a dose that achieves the desired effect is achieved. Therefore, it can be in a single dose form, twice or two times over time The above dosage forms (which may or may not contain the same amount of the desired molecule) or administration of the composition in a continuous infusion via an implant device or catheter. Further improvements in the appropriate dosages are routinely performed by the average skilled person and within the scope of their routine work. The appropriate dose can be confirmed by using appropriate dose-response data.

The pharmaceutical composition is administered according to known methods, such as oral; via subcutaneous, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal or intralesional routes. Injection; by means of a sustained release system (which may also be injected) or by implantation of a device. If desired, the composition can be administered by rapid injection or by continuous infusion or by implantation.

Alternatively or additionally, the composition can be administered topically via implantation of a membrane, sponge or another suitable substance that has absorbed or encapsulated the desired molecule. When an implant device is used, the device can be implanted into any suitable tissue or organ and the delivery of the desired molecule can be effected via diffusion, time release of the bolus or continuous administration. To deliver a drug such as the modified IL21R ECD protein or fusion protein thereof disclosed herein at a predetermined rate such that the drug concentration can be maintained at the desired therapeutic level for a prolonged period of time, a variety of different methods can be used. In one example, a hydrogel comprising a polymer such as gelatin (eg, bovine gelatin, human gelatin or gelatin from another source) or a naturally occurring or synthetically produced polymer can be used. Any percentage of polymer (e.g., gelatin) can be used in the hydrogel, such as 5%, 10%, 15%, or 20%. The choice of the appropriate concentration can depend on a number of factors, such as the desired therapeutic profile and the pharmacokinetic profile of the therapeutic molecule.

Examples of polymers that can be incorporated into the hydrogel include polyethylene glycol ("PEG"), polyethylene oxide, polyethylene oxide-co-polyoxypropylene, co-polyoxyethylene block or random copolymer, poly Vinyl alcohol, poly(vinylpyrrolidone), poly(amino acid), polydextrose, heparin, polysaccharides, polyethers and the like.

Another factor that can be considered when producing a hydrogel formulation is the degree of crosslinking in the hydrogel and crosslinker. In one embodiment, the crosslinking can be via methyl propyl methacrylate The acylate reaction is achieved. In some cases, a high degree of crosslinking may be required, while in other cases, a lower degree of crosslinking is preferred. In some cases, a higher degree of crosslinking provides a longer sustained release. A higher degree of crosslinking provides a stronger hydrogel and a longer drug delivery time. Any ratio of polymer to crosslinker (e.g., methacrylic anhydride) can be used to produce a hydrogel having the desired characteristics. For example, the ratio of polymer to crosslinker can be, for example, 8:1, 16:1, 24:1, or 32:1. For example, when the hydrogel polymer is gelatin and the crosslinker is methacrylate, a methacrylic anhydride: gelatin ratio of 8:1, 16:1, 24:1 or 32:1 can be used.

treatment method

A modified IL21R ECD protein, a fusion protein thereof, and a pharmaceutical composition comprising the modified IL21R ECD protein or fusion protein thereof can be used to modulate at least one IL21-mediated or IL21R-mediated immune response, such as T cells, B cells, One or more of cell proliferation, cytokine expression or secretion, chemokine secretion, and cytolytic activity of NK cells, macrophages, or synoviocytes. Thus, the proteins of the present invention can be used to inhibit the activity (e.g., proliferation, differentiation, and/or survival) of immune or hematopoietic cells (e.g., cells of the bone marrow, lymph, or erythroid cells or their precursor cells), and thus can be used to treat each other by IL21 and IL21R. Roles mediated by various diseases or conditions. Thus, a modified IL21R ECD protein of the invention, or a fusion protein thereof, or a pharmaceutical composition thereof, can be used to treat or prevent an IL21 or IL21R mediated disorder. Further, the present invention provides the use of the modified IL21R ECD protein of the present invention or a fusion protein thereof or a pharmaceutical composition thereof for the manufacture of a medicament for the treatment or prevention of an IL21 or IL21R-mediated disorder. In another embodiment, the application discloses a modified IL21R ECD protein or fusion protein thereof, or a pharmaceutical composition thereof, for use in the treatment of a condition mediated by IL21 or IL21R. In another embodiment, the application discloses a pharmaceutical composition comprising a modified IL21R ECD protein or fusion protein thereof of the invention for use in the treatment or prevention of an IL21 or IL21R mediated disease.

Examples of treatable immune disorders include, but are not limited to, transplant rejection; graft versus lodging Primary disease (GVHD); allergies (eg atopic allergy); and autoimmune diseases, including diabetes, arthritic conditions (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis) And ankylosing spondylitis), spondyloarthropathy, multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, erythematous lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic Dermatitis and eczema dermatitis), psoriasis, Hugh's syndrome, IBD (including Crohn's disease and ulcerative colitis), asthma (including endogenous asthma and allergic asthma), scleroderma, Vasculitis and Behcet's disease.

In use, the treatment of IL21 and IL21R by treatment of a modified IL21R ECD protein, or a fusion protein thereof, or a pharmaceutical composition thereof, as described herein, can be administered to a patient in need thereof in a therapeutically effective amount. A condition or condition mediated by a role. Administration can be carried out as described herein, such as by intravenous injection, intraperitoneal injection, intramuscular injection or orally in the form of a lozenge or liquid formulation. In most cases, the desired dose can be determined by a clinician as described herein and can represent a therapeutically effective dose of a modified IL21R ECD or fusion protein. It will be readily apparent to those skilled in the art that the therapeutically effective dose will depend, inter alia, on the time course of administration, the unit dosage of the agent administered (whether or not the composition is administered in combination with other therapeutic agents), the immune status, and the health of the recipient. And set. The term "therapeutically effective dose" as used herein, refers to a biological or medical response (which includes alleviating the symptoms of a disease or condition being treated) sought by a researcher, doctor or other clinician in a tissue system, animal or human. The amount of the modified IL21R ECD protein or its fusion protein.

Biological deposit

A representative substance of the present invention was deposited on July 17, 2013 at the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209, USA). The vector ECC1-L1-FC1 with ATCC accession number PTA-120480 contains a DNA insert encoding the modified IL21R ECD1, and the linkage Sub-1 and Fc variants designated as FC1. Deposited in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and its regulations (Budapest treaty). This guarantee maintains the vitality of the deposit for 30 years from the date of deposit. The deposit will be made by the ATCC under the terms of the Budapest Treaty and subject to the agreement between Pfizer Inc. and the ATCC, which guarantees that after the publication of the relevant US patent or after the publication of any US or foreign patent application to the public (in no particular order) The public can use the children of the culture of the deposit permanently and without restriction, and is guaranteed by the authorized US Patent and Trademarks (USCommissioner of Patents and Trademarks) in accordance with 35 USC Part 122 and its membership rules (including 37 CFR Part 1.14, especially with reference to 886 OG 638) Determinants can use the progeny.

The assignee of the present application has agreed that if the culture of the substance at the time of storage is killed or lost or damaged when cultured under suitable conditions, the substance is replaced with another identical substance immediately after notification. The availability of a hosted material is not to be construed as a license to practice the invention in a manner that violates the rights granted by any government agency under its patent laws.

Illustrative embodiment

The invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should not be construed as being limited to the following examples, but should be construed as including any and all variations that are obvious as the teachings provided herein.

Instance Example 1. X-ray crystal structure of wild-type human IL21R ECD

The X-ray structure of the complex of human IL21R protein and its cytokines was resolved. More specifically, IL21 and IL21R ECD were expressed in CHO cells and purified independently. The cytokine and the receptor are then mixed in a one-to-one ratio and applied to the SourceQ anion. Converter chromatography to further purify the complex. The selected fractions were concentrated to 7.3 mg/ml and subjected to hanging drop crystallization screening. After optimization, good quality crystals were obtained from 1720 mM ammonium sulfate, 10 mM cobalt chloride, and 100 mM MES buffer at pH 6.43. The crystals were frozen and collected at 2.6 Å resolution under diffraction generated by a National Synchrotron Light Source (NSLS, Brookhaven, NY). The symmetric space group is I4122 and the structure is resolved via molecular replacement techniques using the IL4/IL4R complex structure purchased from the protein library.

The crystal structure identified the tryptophanic acid (W148) at position 148 of the IL21R ECD as a key contributor to the dimer interface between the two monomers of the receptor (Figure 1).

Example 2. Production of modified IL21R ECD protein

Wild-type human IL21R ECD has a relatively low thermal stability measured by DSC to be Tm = 51 ° C, which begins to melt at 40 ° C. When formatted as an Fc fusion, the midpoint of the wild-type IL21R ECD specific transition was further reduced to 48 °C. Various mutants of human IL21R ECD having a C-terminal hexahistidine tag were designed by studying the X-ray structure of the IL21 receptor (Fig. 1). In particular, the tryptophan acid at position 148 is structurally in a flexible loop exposed to solvent and can result in an undesired configuration due to its highly hydrophobic nature. Surprisingly, mutations in this amino acid and, where appropriate, flanking residues to different residues (including more hydrophilic residues) stabilize and reduce the flexible loop of IL21R ECD with native tryptamine The negative entropy effect associated with the high solvation loss of acid.

In addition, the aspartate residue (D122) at position 122 was identified as a residue that caused significant and unexpected stability of the modified IL21R ECD construct upon mutation. D122 is located in the middle of the hydrophobic structural environment. As shown in Figure 2, the substitution of D122 with paF significantly enhanced the thermal stability of the modified IL21RFc ECD relative to other p-acetylphenylalanine (paF) mutants at other solvent exposure sites on the protein. Table 6 illustrates the Tm values for the various modified IL21R ECDs from Figure 2.

Table 6: Tm values of variant IL21R ECD

Other D122 position mutants substituted with hydrophobic residues were designed to mimic the stabilizing effects observed in the substitution of the non-natural amino acid paF. Table 2 lists various IL21R ECD mutants and their amino acid sequences. In each modified IL21R ECD construct, the bolded and underlined residues were mutated relative to wild-type IL21R ECD (SEQ ID NO: 1).

Example 3: Performance of Modified IL21R ECD Constructs

The various modified IL21R ECD constructs as listed in Table 2 were transiently expressed as IL21R-ECD-His6 constructs in HEK293 cells.

HEK293F cells obtained from the [US Type Culture Preservation Center (ATCC, Manassas, VA)] were cultured in freestyle 293 medium (Invitrogen, Grand Island, NY). The cells were grown and maintained in a moisture-containing incubator with 7% CO ₂ at 37 °C. Conditioned media for constructs were generated by standard large-scale transient HEK293 transfection methods. The medium was filtered at 0.22 μm prior to purification.

Secreted proteins from conditioned medium were analyzed by Western blotting as follows. Serum-free conditioned medium samples were resolved by SDS PAGE (Invitrogen) on a pre-cast NuPAGE Bis-Tris microgel on a 4-12% gradient according to the manufacturer's protocol. In addition, the same aliquot of the conditioned medium obtained from the sample was loaded into each well, which contained an equal number of cells compared to the other lanes of the gel. The gel was stained for protein observation using Coomassie Brilliant Blue R-250, and the gel was transferred to a nitrocellulose membrane at 0.5 °C for 0.5 hour at 4 °C. In the case of a monomer-His-tagged protein, mouse anti-histidine IgG (Qiagen, catalog number: 34670) as a primary antibody, rabbit anti-small in combination with horseradish peroxidase as a secondary antibody Mouse IgG (Pierce, catalog number: 32430) was used to detect Western membranes. For Fc fusion eggs The Western blotting method was used to develop a nitrocellulose membrane using a single goat anti-human IgG (Sigma, catalog number: A0170) antibody that binds horseradish peroxidase.

Figures 3 and 4 depict Western blot analysis showing that the performance of the modified IL21R ECD constructs ECD1, ECD2, ECD3, and ECD4 is greater than that of wild-type IL21R ECD. Thus, each modified construct has improved performance in HEK293 cells compared to wild-type proteins.

Example 4: Purification of a modified IL21R ECD construct containing a His tag

Conditioned medium from transient HEK293 or CHO stable cultures is typically diafiltered against PBS (pH 7.2) and then loaded onto a suitably sized nickel NTA superfluid (Qiagen) chromatogram equilibrated in 300 mM NaCl, 50 mM NaHPO (pH 8). On the pipe column. The column was washed with 10 CV of 300 mM NaCl, 50 mM NaHPO, 15 mM imidazole (pH 8), followed by resolution by a 5 CV linear gradient to 300 mM NaCl, 50 mM NaHPO, 250 mM imidazole (pH 8). The protein peak pool eluted from the nickel NTA step was loaded onto a Superdex 200 size exclusion chromatography column (GE Healthcare) equilibrated in PBS and developed. Finally, the SEC fractions with high purity were pooled, concentrated and frozen for long-term storage at -80 °C. The biophysical characteristics of the purified modified IL21R ECD construct were further investigated as discussed below.

Example 5: Differential Scanning Calorimetry

Thermal denaturation experiments were performed on a capillary-DSC instrument (MicroCal, Northampton, MA). Protein samples were dialyzed against PBS buffer (pH 7.2) at 4 °C, normalized to a concentration of 1 mg/ml and fully degassed prior to analysis. The samples were scanned at a scan rate of 1 ° C min ^-1 from 20 ° C to 90 ° C against the dialysis buffer contained in the reference cells. The thermograms generated from the protein samples were analyzed using Origin 7.0 software (MicroCal Software). The data was converted to waste heat capacity, the reference traces were subtracted, the concentration was normalized and analyzed for Tm determination.

Figure 5 depicts a differential scanning calorimetry thermogram showing thermal stability enhancement of modified IL21R ECD constructs ECD1, ECD2 and ECD4 compared to wild-type IL21R ECD. IL21R ECD monomer by mutations of T _m increased by about 6-8 ℃, clearly show compared to the wild-type sequence mutations positive stabilizing effect. The IL21R ECD1 formatted as an Fc fusion retained an increase in thermal stability at 4 °C (data not shown). Similarly, the monomer IL21R ECD construct mutations of T _m increased as compared with the wild-type protein, thereby showing a GSG W148 substituted at a position 147 and 149 and the side of flanking residues (respectively PWA) to impart the modified IL21R ECD1 Greater stability.

Example 6: Analytical Size Exclusion Chromatography

The purity of the various modified IL21R ECD constructs was analyzed by analytical size exclusion chromatography using a Superdex 200 10/300 GL (GE Healthcare) chromatography column on an Agilent 1200 system. Typically 20 micrograms to 100 micrograms of sample were loaded via an autosampler and injected onto a column deployed in PBS (pH 6.8) at a flow rate of 0.75 ml/min.

Figure 6 depicts an analytical size exclusion chromatography demonstrating a reduced level of aggregation of the modified IL21R construct ECD1 compared to wild-type IL21R after nickel NTA initial affinity capture purification. Molecules were analyzed by analytical SEC immediately after IMAC capture and imidazole elution. The IL21R wild-type molecule exhibited approximately 30% high molecular weight (HMW) material which was dissolved before the relevant main peak visible at 20 minutes. Under the same conditions, the amount of HMW material in the ECD1 construct was significantly reduced to 10%, and about 20% higher molecular weight aggregates were reduced compared to the wild type. This not only improves the overall production process yield, but also contributes to the final formulation and increases the stability of the product.

Figure 7 depicts the results of analytical size exclusion chromatography showing reduced levels of aggregation of modified IL21R constructs using ECD1, ECD2 and ECD4 compared to wild type. Each construct was purified to homogeneity and free of high molecular weight species by nickel affinity capture and size exclusion chromatography. Wild type, ECD1 and ECD2 molecules were incubated overnight in PBS buffer at two different temperatures of 4 °C and 40 °C. Protein analysis by SEC immediately after incubation sample. The 4 ° C samples were all consistent with the quality of the starting material and did not have visible HMW species detectable by SEC. In contrast, the 40 °C sample exhibited a differential profile between WT and ECD1/ECD2. That is, high temperature incubation only caused agglomeration of the wild type sample, which represents 16% of the total integrated OD280nm signal. Compared with wild-type IL21R, the modified IL21R constructs ECD1 and ECD2 showed resistance to aggregation under temperature stress conditions.

Example 7: Production of modified IL21RFc constructs

Various modified IL21RFc fusion proteins were designed based on the modified IL21R ECD constructs described in Table 2 above. The linker and human IgG1 Fc protein were selected as described below. Figure 8 shows a schematic representation of the structure of a modified IL21RFc fusion protein.

a) connector design

By exploring the linker structure selection, it is not easy to determine the stoichiometry of the two cytokine molecules over the one Fc dimer fusion molecule. That is, the classical (Gly4Ser) _n motif commonly used for protein engineering with gene-linked protein domains is not sufficient to support the desired target binding stoichiometry. Isothermal titration calorimetry was used to study the stoichiometry of IL21 cytokine binding in IL21RFc fusion protein constructs. As shown in the graph of Figure 9, the IL21R monomer molecule exhibits an appropriate binding stoichiometry N = 1 that approximates the binding of one cytokine to one monomeric receptor. In the case of a homodimeric Fc fusion molecule, attachment of the IL21 receptor to the Fc domain using G4S motifs (four glycine acids and one serine) does not allow for the expected N=2 binding stoichiometry. In fact, only the stoichiometry of approximately N = 1 was measured. It should be noted that longer lengths of linkages in multiple G4S motifs do not help to improve stoichiometry back to the expected N=2.

As shown in Table 3, the modified linker sequence L1 allowed for complete engagement of IL21, wherein one IL21RFc homodimeric molecule was able to capture two free IL21 cytokine molecules. The results of the BIAcore study depicted in Figure 10 demonstrate a complete 2:1 stoichiometry of IL21 binding to IL21RFc. Briefly, IL21R Fc was coated on a BIAcore wafer to give a theoretical Rmax value of 50.83 RU. The IL21 cytokine was gradually titrated to saturation and the Rmax was recorded as 49.5RU. This observation represents 97.4% of the complete stoichiometry of the binding of two cytokines per dimer of each IL21RFc on the wafer.

In addition, as illustrated in Figure 11, linker L1 is modified by exchanging SG residues at the ends of the linker to remove glycosylation of the O-linkage. As shown in Table 3, the resulting linker was designated as linker L2. After fine characterization by mass spectrometry and sugar analysis, it was concluded that the linker L1 sequence supports post-translational modification of two serines by O-linked glycosylation. The heterogeneity of the substance is highlighted by the O-linked glycan possessing a portion of its variable size. In a particular linker sequence L1 GSGEGEGSEGSG (SEQ ID NO: 13), the first and last serine acids have a modification of the O linkage. Since the SG motif has been associated with the potential of a higher O-linkage (Mann et al., J Biol Chem. March 25, 1990; 265(9): 5317-23), the sequence is changed from SG to GS to destroy This O-linked glycosylation motif. As discussed in Example 8 below, the resulting new linker sequence (GGSEGEGSEGGS) exhibited better homogeneity by Western blotting.

b) Human IgG1 Fc design

Since one of the desirable properties of the IL21RFc fusion protein is the capture and clearance of plasma and tissue circulating IL21 cytokines, natural Fc functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) Generally not preferred. One of the group mutations as described in U.S. Patent Nos. 5,624,821 and 5,648,260, by the mutating of the positions Leu242, Leu243 and Gly245 to alanine (each Kabat antibody number 234, 235 and 237, respectively). (See Table 4)) to engineer the loss of effector function in human IgGl Fc. These mutations from LLGL to AAGA are expected to reduce Fc effector function including Fc-mediated cell removal.

Serum retention time is also increased by engineering the two residues at the interface between CH2 and CH3 in the Fc domain. That is, as described in International Patent Publication No. WO 2009/086320, it has been shown that the mutations M436L and N442S (Eu antibody Kabat numbers 428 and 434, respectively (see Table 4)) increase the binding affinity to FcRn and in vivo half. decline period.

As disclosed in Example 11 below, the BIAcore study demonstrates that these mutations are administered to the modified IL21RFc fusion proteins disclosed herein (such as ECD1-L1-FC1) such that they are directly human compared to their Fc wild-type homolog molecules The affinity of FcRn is increased by a factor of ten. As further revealed in Example 12, the actual in vivo NHP PK profile significantly benefited from both mutations, which doubling the drug exposure and positively affecting the terminal half-life (making it increase compared to the wild-type protein WTECD-L1-FC1) Triple) and the rate of removal (to make it halfway).

A variety of modified IL21RFc fusion proteins were designed and several of these modified IL21RFc fusion proteins are listed in Table 5. As is well known to those skilled in the art, many other combinations of modified IL21RFc fusion proteins can be made by combining any of the modified IL21R ECDs disclosed above with Linker 1 or Linker 2 or any other suitable linker. Any one of the human IgG1 Fc proteins disclosed above or any other suitable Fc protein combination is produced.

Example 8: Performance and Purification of Modified IL21RFc Fusion Protein

Figure 12 depicts the performance of modified IL21RFc constructs ECD1-L1-FC1, ECD1-L1-FC2, ECD-L2-FC1, and ECD7-L2-FC1 as analyzed in Table 5 using Western blot analysis. The culture supernatant was manipulated by non-reducing SDS gel electrophoresis. After the protein was electrotransferred from the gel to a nitrocellulose membrane (NCM), NCM was visualized with a goat anti-human IgG (Sigma, catalog number: A0170) antibody conjugated with horseradish peroxidase. Each lane was loaded with an equal cell count. ECD1-L1-FC1 and ECD1-L1-FC2 exhibit the same strip pattern by Western blotting, and the main migration strip of non-reducing IL-21RFc is indicated by the arrow and presents a distinct stain on it. This unique stain pattern may be associated with a glycosylation post-translational modification of the heterologous O-ligation of the L1 linker sequence. The banding pattern of ECD1-L2-FC1 shows a significant reduction in this stain, thereby demonstrating the beneficial effect of the optimized L2 linker sequence, which removes the O-linked glycosylation motif of the L1 linker. ECD7-L2-FC1 is very To a further improved and cleaner strip pattern, it does not have high molecular weight stains above the IL-21RFc strip. This demonstrates other beneficial effects on coacervation obtained via ECD7 engineering comprising substitutions of GSG at amino acid residues 147, 148 and 149 and another substitution of D122A, respectively. To further purify the modified IL21RFc fusion protein, conditioned medium from transient or stable CHO cultures is typically loaded in neat form on a properly sized Protein A chromatography column (rmpProtein A Sepharose Fast Flow) equilibrated in PBS (pH 7.2). , GE Healthcare). The column was then washed with 5 column volumes (CV) of PBS followed by a 10 CV linear gradient to 20 mM citric acid, 150 mM NaCl (pH 2.5). The Protein A peak pool was diluted 1 to 3 with 20 mM Tris (pH 8.6) for binding to a Source 15Q column (GE Healthcare) and was eluted with a 15 CV linear gradient to 20 mM Tris 1 M NaCl (pH 8.6). The Source 15Q peak containing IL21R Fc was then pooled and applied to a Superdex 200 size exclusion chromatography column (GE Healthcare) equilibrated and expanded in PBS. The final fraction was filtered at 0.2 microns and concentrated to a variable concentration, after which it was frozen for long term storage at -80 °C. The protein was further characterized as described below.

Example 9: SDS PAGE and IEF gel electrophoresis

The purity of the modified IL21RFc fusion protein construct ECD1-L1-FC1 was analyzed by SDS PAGE as described above and by isoelectric focusing (IEF) to determine pI. IEF gel electrophoresis was performed on pre-cast vertical Novex pH 3-10 IEF 5% polyacrylamide gel (Invitrogen) according to the manufacturer's protocol. The gel was stained with Coomassie Brilliant Blue R-250 for observation of the heart protein. Figure 13 depicts SDS and IEF gels.

As illustrated by the diffusion band that migrates around the 4.2 pI marker of the IEF gel, the IL21R ECD has five occupied N-linked glycosylation sites, resulting in molecular charge heterogeneity.

SDS PAGE reveals the high purity of the material in which no other bands are detected under reducing conditions. Non-reducing conditions primarily exhibit a covalent IL21RFc homodimeric fusion protein bridged by a disulfide bond expected to migrate at about 140 kD, and its reduced at about 70 kD migration. A minor component of the monomeric form of the non-covalently bound equivalent.

Example 10: Differential Scanning Calorimetry (DSC) of Modified IL21RFc Constructs

DSCs were performed on the modified IL21RFc fusion proteins ECD7-L1-FC1, ECD8-L1-FC1, ECD9-L1-FC1, ECD10-L1-FC1 and ECD11-L1-FC1 listed in Table 5 as described above, and Compared to the ECD1-L1-FC1 fusion protein. A DSC thermogram is depicted in FIG. Table 7 is an overview of the Tm temperatures of the various constructs from the thermograms of Figure 14.

For each modified IL21RFc construct, the midpoint of the DSC transition of the mutated IL21R domain was increased from 3.2 °C to 4.6 °C relative to the native D122 native molecule. Compared to the increase in monomer by 6 degrees (see Figure 2), these lower values are attributed to the different structural contributions of the native amino acid and the destabilization of the Fc format compared to paF.

Example 11: BIAcore study of modified IL21RFc fusion constructs conjugated to FcRn

BIAcore analysis was performed to determine the steady-state affinity (KD) of different IL21RFc engineered molecules for binding to human FcRn. The BIAcore technique utilizes the IL21RFc molecule to bind to a human FcRn protein immobilized on the surface of the sensor, and the refractive index at that layer changes. The bonding is detected by surface plasmon resonance (SPR) of the laser light refracted from the surface. Human FcRn was labeled with an engineered Avi-tag using BirA reagent (catalog number: BIRA500, Avidity, LLC, Aurora, Colorado) specific biotin and immobilized on a streptavidin (SA) sensor wafer In order to achieve a uniform orientation of the FcRn protein on the sensor. Subsequently, various concentrations of IL21RFc molecules were applied to 20 mM MES (2-(N- A solution of N-morpholinyl)ethanesulfonic acid (pH 6.0) was injected onto the surface of the wafer with 150 mM NaCl, 3 mM EDTA (ethylenediaminetetraacetic acid), and 0.5% surfactant P20 (MES-EP). The surface was regenerated using an HBS-EP + 0.05% surfactant P20 (GE Healthcare, Piscataway, NJ) (pH 7.4) between injection cycles. The steady state binding affinity of the modified IL21RFc constructs was determined and compared to each other. The data is the average of at least two experiments for at least two different surfaces. The BIAcore results shown in Figure 15 illustrate the enhanced kinetic effects of engineered FcRn binding in the Fc domain. The FC1-containing IL21RFc fusion protein containing Fc component mutations to enhance FcRn binding and increase in vivo half-life exhibited a 10-fold improvement in the steady-state KD for FcRn compared to molecules containing unengineered FC3.

Example 12: Overview of non-human primate pharmacokinetics (PK) of various modified IL21RFc fusion proteins

In this assay, test articles were captured by recombinant human IL-21-FLAG (DYKDDDDK) tag and detected using biotinylated anti-human IgG antibodies. Use of streptavidin poly-80 horseradish peroxidase (HRP) conjugate and substrate ABTS (2,2'-azo-di(3-ethyl-benzylthiazoline-6-sulfonate) To produce a colored final product, and to measure the optical density by spectrophotometry at a wavelength of 405 nm. The sample concentration is determined by interpolation from a standard curve fitted using a 4-parameter logistic equation. The lower limit of quantification is 15-40 ng/ mL.

A single 2 mg/kg dose of each modified IL21 RFc fusion protein was administered intravenously or subcutaneously to untreated female cynomolgus macaques. Figure 16 depicts the PK profile of non-human primates injected with modified IL21RFc fusion protein constructs ECD1-L1-FC1, ECD1-L1-FC3, and wild-type IL21RFc. Table 8 is an overview of the PK profile depicted in Figure 16.

Formatted as a linker containing four glycine acids and one serine (G4S; Wild-type ECD, which is an L3) linked Fc fusion, exhibits a poor PK profile and low drug exposure, which is believed to be caused by low bioavailability, low average terminal half-life, and unexpectedly high clearance rates. The first round of architecture, introduced with ECD1 and linker L1 optimization, engineered the PK profile back to the expected value of a typical Fc fusion molecule with a clearance rate below 1 ml/h/kg. Subsequent introduction of FC1 engineering with FcRn binding enhancing mutations as discussed in Examples 6 and 11 above above further improved the pharmacokinetic properties with twice the overall exposure increase. In addition, ECD1-L1-FC1 exhibited an increase in bioavailability of 83.6%.

Example 13: Analysis of human primary B cell proliferation using modified IL21RFc fusion constructs

Leukocyte cells from healthy human donors were incubated with B cell enrichment cocktail (RosetteSep; StemCell Technologies, Vancouver, British Columbia, Canada) and isolated by the negative selection method described in the manufacturer's instructions. After staining with anti-CD19, the purity of the isolated B cells was measured by flow cytometry. The enriched cells are about 60-80% CD19+ B cells.

Enriched humans in 1×10 ⁵ cells/well in 0.2 mL RPMI containing 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin and 2 mM L-glutamic acid in a 96-well flat-bottom plate B cells. B cells were incubated with serially diluted anti-human IL21 receptor antibody, 1 μg/mL anti-CD40 mAb (BD Bisosciences) and 20 ng/mL IL21 cytokine for 3 days in a 37 °C incubator adjusted to 5% CO ₂ . On day 3, cultures were pulsed with 0.5 [mu]Ci/well [3H] thymidine (Perkin-Elmer NEN, Boston, MA) and collected on glass fiber filter mats after 5 hours. Incorporation of [3H] thymidine was determined by liquid scintillation counting. The original data was plotted in Excel/XLFIT4.

Figure 17 is a graph depicting the results of a primary IL21 dependent B cell proliferation assay using modified IL21RFc constructs ECD1-L1-FC1 and wild-type IL21RFc positive control WTECD-L1-FC3. The human IgGl control exhibited no effect on B cell proliferation. In this assay, both the mutant and the wild-type IL21RFc protein exhibited an IC _{50 with} an equivalent potency of about 10 nM, thereby demonstrating that the GSG mutation of ECD1 did not affect the biological function of the IL21 receptor in recognizing cytokines.

Example 14: Analysis of human T cell proliferation using modified IL21RFc fusion constructs

Human leukocyte layer was separated from the CD4 + T cells with ROSETTESEP ^TM CD4 + T cells were enriched mixture (Stem Cell Technologies, Vancouver, BC , Canada) according to the manufacturer instructions. Coating anti-CD3/anti-CD28 in RPMI (37 ° C, 5% CO ₂ ) containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamic acid and HEPES Microspheres (internal preparation) activated cells (80-90% CD4+/CD3+ T cells) for 3 days. Microspheres then removed, and the cells were washed once, followed by about 1 x 10 ⁶ cells / ml in medium was allowed to stand overnight, and the cells were washed prior to addition to the assay plate at once. Test articles were diluted with medium in a flat bottom 96-well plate, followed by sequential addition of human IL-21 (25 ng/ml final concentration) and 10 ⁵ cells/well. After 72 hours, cells were pulsed for 6 hours with 1 [mu]Ci/well 3H-thymidine (Perkin Elmer (NEN)) and collected on glass fiber filter mats for liquid scintillation counting.

Figure 18 shows a graph depicting the results of an IL21-dependent primary T cell proliferation assay using modified IL21RFc constructs ECD7-L1-FC1, ECD8-L1-FC1, ECD9-L1-FC1, ECD10-L1- FC1 and ECD11-L1-FC1. Table 9 is an overview of the IC ₅₀ for each building.

In this assay, IL21 cytokines at a concentration of 25 ng/ml maintained proliferation of T cells. All 5 mutants demonstrated good potency in neutralizing T cell proliferation compared to the human IgG1 anti-tetanus toxin negative control antibody and the monomeric IL21R as a positive control. The IC ₅₀ difference was in the range of 19.6 nM to 28.3 nM and the difference was not significant.

Example 15: Characterization of a modified IL21RFc construct containing the Fc variant T445K

The modified IL21RFc constructs ECD1-L1-FC1 and ECD1-L1-FC2 were characterized and compared in various side-by-side experiments. Both proteins exhibited similar amounts and were purified identically following the same protocol. Analytical characterization by SDS PAGE, IEF, size exclusion, ion exchange, and hydrophobic interaction chromatography revealed that ECD1-L1-FC1 is similar to the ECD1-L1-FC2 molecule. Differential scanning calorimetry results highlight a slight destabilization of the Fc CH2 and CH3 domains of ECD1-L1-FC2, but this is considered to be within the variation criteria observed with molecules containing the same human IgG1 Fc fragment. In addition, in the functional analysis of IL21 neutralization, IL21 binding, and FcRn binding, no significant difference in activity between ECD1-L1-FC1 and ECD1-L1-FC2 was observed.

Example 16: Preparation of IL21RFc Constructs

The modified IL21RFc construct ECD1-L1-FC1 was formulated into a powder for injection at a dose concentration of 100 mg/mL after recovery. The total protein content of each reconstituted vial was 140 mg (1.4 mL) of ECD1-L1-FC1, but 100 mg (1.0 mL) was withdrawn from the vial. That is, to ensure that 1.0 mL can be withdrawn from the vial, 0.4 mL is overfilled. The components of one embodiment of the formulation are listed in Table 10.

Although the teachings disclosed in the various applications, methods, kits, and composition descriptions have been described, it should be understood that the teachings of the present invention and the invention as claimed hereinafter may be Make various changes and modifications. The foregoing examples are provided to fully illustrate the teachings of the present invention and are not intended to limit the scope of the teachings presented herein. Although the teachings of the present invention have been described in terms of these exemplary embodiments, those skilled in the art will readily appreciate that many variations and modifications may be made to the exemplary embodiments without departing from the practice. All such changes and modifications are within the scope of this teaching.

All references, including patents, patent applications, essays, textbooks, and the like, and references cited therein, are hereby incorporated by reference in their entirety to the extent of the entireties. In the event that one or more of the incorporated documents and similar materials are different from or inconsistent with the present application (including but not limited to, defined terms, term usage, the techniques, or the like) ), subject to this application.

The above description and examples are illustrative of certain specific embodiments of the invention and are described in the preferred embodiments. It should be understood, however, that the present invention may be practiced in various ways, and the invention should be construed as being consistent with the scope of the appended claims and any equivalents thereof.

<110> American Pfizer Pharmaceutical Factory Stephanie Olam Lonno William Kleiz William Stewart Somos Marc Roy Stall Michelle Marlow Lissowski Eric Fift

<120> Modified interleukin 21 receptor protein

<130> PC71892A

<140> PCT/IB2014/to be assigned

<141> 2014-12-22

<150> US 61/931438

<151> 2014-01-24

<160> 29

<170> PatentIn version 3.5

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Claims (18)

A modified interleukin 21 receptor (IL21R) protein extracellular domain (ECD) comprising a modification selected from the group consisting of: a. at position 148 of the amino acid sequence set forth as SEQ ID NO: Substituting one or more amino acids at or within the four amino acid residues of tryptophan acid (W148); b. at position 148 of the amino acid of the amino acid sequence SEQ ID NO: Inserting one or more amino acids; and c. deleting the tryptophan residue at position 148 of the amino acid sequence SEQ ID NO: 1; and further wherein the substituted or inserted amino acid is a naturally occurring amino acid selected from the group consisting of glycine, alanine, leucine, methionine, phenylalanine, lysine, glutamic acid, glutamic acid, serine , valine, valine, isoleucine, cysteine, tyrosine, histidine, arginine, aspartic acid, aspartic acid and threonine. The modified IL21R ECD of claim 1, wherein a. the substituted amino acid at position 148 is selected from the group consisting of serine or aspartate; b. the ECD further comprises the SEQ ID NO Substituting at least one amino acid of the amino acid positions 147, 149 and 150 of the amino acid sequence; c. the amino acid substitution comprises glycine at position 147, a leucine at position 148 and Glycine at position 149 (SEQ ID NO: 2); d. the amino acid substitution comprises aspartic acid at position 148 and serine at position 150 (SEQ ID NO: 3); e. The amino acid substitution comprises aspartic acid at position 148, glycine at position 149 and serine at position 150 (SEQ ID NO: 4); f. the amino acid substitution comprises a serine at position 148 and a glycine at position 149 (SEQ ID NO: 7); g. the modified IL21R ECD comprises a serine at position 148 (SEQ ID NO: 6); h. The modified IL21R ECD comprises an amino acid substitution at the amino acid position 147, an amino acid substitution at the amino acid position 149, an aspartic acid at position 148, in position 150 of serine, and further comprising aspartic acid at position 49 (SEQ ID NO: 5); i. the modified IL21R ECD comprises glycine at position 147, a serine at position 148 a glycine acid at position 149, an amino acid substitution at position 150 of the amino acid, and an amino acid substitution at position 122, both relative to the amino acid sequence SEQ ID NO: 1; j. The IL21R ECD comprises glycine at position 147, a serine at position 148, a glycine at position 149, an amino acid at position 150 of the amino acid, and an alanine at position 122, both of which are relative The amino acid sequence of SEQ ID NO: 1; k. the modified IL21R ECD comprises glycine at position 147, at Position 148 of a serine, a glycine at position 149, an amino acid substitution at position 150 of the amino acid, and an isokinetic acid at position 122, both relative to the amino acid sequence SEQ ID NO: 1 The modified IL21R ECD comprises glycine at position 147, a serine at position 148, a glycine at position 149, an amino acid at position 150 of the amino acid, and at position 122. Tryptophan, all relative to the amino acid sequence SEQ ID NO: 1; m. The modified IL21R ECD comprises glycine at position 147, a serine at position 148, and a glycine at position 149 Acid, at the position of the amino acid 150 Amino acid substituted and phenylalanine at position 122, both relative to the amino acid sequence SEQ ID NO: 1; n. The modified IL21R ECD comprises a glycine at position 147, a silk amine at position 148 The acid, the glycine at position 149, the amino acid at position 150 of the amino acid, and the tyrosine at position 122 are all relative to the amino acid sequence SEQ ID NO: 1. A fusion protein comprising the modified IL21R ECD of claim 2, which is fused to a heterologous amino acid sequence via a peptidyl linker, wherein the linker comprises a sequence selected from the group consisting of GSGEGEGSEGSG (SEQ ID NO: 13 ), GGSEGEGSEGGS (SEQ ID NO: 14) and GGGGS (SEQ ID NO: 15). The fusion protein of claim 3, wherein the heterologous amino acid sequence: a. comprises a human IgG1 Fc domain comprising an amino acid sequence set forth as SEQ ID NO: 16; b. comprising a human IgG1 Fc domain, comprising Described as the amino acid sequence of SEQ ID NO: 19; c. comprising a human IgG1 Fc domain comprising the amino acid sequence set forth as SEQ ID NO: 17; d. comprising a human IgG1 Fc domain comprising the set forth as SEQ ID NO: amino acid sequence of 18. The fusion protein of claim 4, which comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. A fusion protein consisting of the amino acid sequence SEQ ID NO:20. A pharmaceutical composition comprising the fusion protein of claim 5 and a pharmaceutically acceptable agent. The pharmaceutical composition according to claim 7, wherein the composition comprises 0.5-200 mg/mL of the fusion protein of claim 5, 10-50 mM tromethamine, 0.01-0.10 mg/mL EDTA, 20-120 mg/mL sucrose, and 0.01-0.4 mg/mL polysorbate 80. An isolated nucleic acid encoding the modified IL21R ECD of claim 2. An isolated nucleic acid encoding the fusion protein of claim 5. An isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO:31. A vector comprising the nucleic acid of claim 10. A host cell comprising the nucleic acid of claim 10 or the vector of claim 12. A method of making a fusion protein comprising a modified IL21R ECD comprising growing a host cell of claim 13 under conditions which express the fusion protein. A modified IL21R-Fc fusion protein encoded by a nucleic acid insert contained in a vector of ATCC Accession No. PTA-120480. A modified IL21R-Fc fusion protein comprising an amino acid sequence encoded by a nucleic acid insert contained in a vector deposited under ATCC Accession No. PTA-120480. A use of the fusion protein of claim 5 for the manufacture of a medicament for the treatment of a disease or condition mediated by the interaction of IL21 with IL21R. The use of claim 17, wherein the disease or condition is an inflammatory or autoimmune disease or condition selected from the group consisting of: transplant rejection, graft versus host disease (GVHD), multiple sclerosis, allergy, and different Allergies, diabetes, arthritic conditions, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, spondyloarthropathy, multiple sclerosis, encephalomyelitis, severe Muscle weakness, systemic lupus erythematosus, cutaneous lupus erythematosus, autoimmune thyroiditis, dermatitis, atopic dermatitis, eczema dermatitis, psoriasis, Sjogren's syndrome, IBD, Crohn's disease, ulcerative colitis, asthma, endogenous snarl Asthma, allergic asthma, scleroderma, vasculitis, and Behcet's Disease.