Masked interferon fusion proteins and uses thereof

Abstract

Provided herein are masked interferon fusion proteins that can be cleaved to release human interferon type 1 (IFN) polypeptides. In various examples, a masking interferon fusion protein comprises a masking moiety, an IFN polypeptide, a cleavable linker linking the masking moiety and the IFN polypeptide, and an Ig Fc region linked to the IFN polypeptide. Also provided herein are methods of using the masked interferon fusion proteins for the treatment of cancer and an autoimmune disease or inflammatory condition. Also provided herein are interferon masking moieties that bind to human IFN, and their use for inhibiting IFN activity.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480049338.8 (22) Application Date 2024.07.25 (66) Domestic Priority Data PCT / CN2023 / 109070 2023.07.25 CN (85) PCT International Application Entering National Phase Date 2026.01.26 (86) PCT International Application Application Data PCT / CN2024 / 107511 2024.07.25 (87) PCT International Application Publication Data WO2025 / 021144 EN 2025.01.30 (71) Applicant: I-Mab Biotechnology (Shanghai) Co., Ltd. Address: 201210 Room 802, 8th Floor, Building 1, West Tower, No. 88 Shangke Road, Pudong New Area, Shanghai (72) Inventors: Chen Xi, Li Xueqiang, Min Zhili, Wang Zhengyi, Zheng Xintian, Bai Hui, Zhang Fei, Yu Qilin, Chen Mingchen, Liu Fan (74) Patent Agency: Beijing Yongxin Tongchuang Intellectual Property Agency Co., Ltd. 11376 Patent Attorneys: Luan Xingming, Cheng Dajun (51) Int.Cl. C07K 19 / 00 (2006.01) A61K 38 / 21 (2006.01) A61K 47 / 64 (2006.01) A61K 47 / 68 (2006.01) C07K 14 / 56 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title: Masked Interferon Fusion Protein and Its Use Thereof (57) Abstract This document provides a masked interferon fusion protein that is cleavable to release a human type 1 interferon (IFN) polypeptide. In various examples, the masked interferon fusion protein comprises a masking moiety, an IFN polypeptide, a cleavable linker connecting the masking moiety and the IFN polypeptide, and an Ig Fc region linked to the IFN polypeptide. This document also provides methods for using the said masked interferon fusion protein to treat cancer and autoimmune or inflammatory conditions. This document also provides an interferon masking moiety that binds to human IFN and its use for inhibiting IFN activity. Claims 4 pages, Description 38 pages, Sequence Listing (electronic publication), Drawings 10 pages, CN 121568967 A 2026.02.24 CN 1 21 56 89 67 A 1. A fusion protein comprising (a) interferon α and β receptor subunit 2 domain 1 (IFNAR2-D1) or a masking peptide having at least 55% sequence identity with IFNAR2-D1, (b) type I interferon (IFN), and (c) a cleavable linker between (a) and (b), wherein said peptide is capable of binding said type I IFN, and wherein said fusion protein does not contain the complete sequence of IFNAR2 domain 2 (IFNAR2-D2).2. The fusion protein of claim 1, wherein the cleavable linker is fused to the C-terminus of (a) and the N-terminus of (b), or the cleavable linker is fused to the N-terminus of (a) and the C-terminus of (b). 3. The fusion protein of claim 1 or 2, further comprising an immunoglobulin (Ig) Fc region fused to the C-terminus of the IFN or the N-terminus of IFNAR2-D1 or a masking peptide of (a). 4. The fusion protein of any of the preceding claims, wherein it does not contain at least 50% IFNAR2-D2, or does not contain at least 80% IFNAR2-D2. 5. The fusion protein of any of the preceding claims, wherein the IFNAR2-D1 comprises the amino acid sequence of SEQ ID NO: 8 or 3. 6. The fusion protein according to any one of the preceding claims, wherein the masking peptide comprises IFN-binding loop 1, IFN-binding loop 2, and IFN-binding loop 3, wherein IFN-binding loop 1 comprises the amino acid sequence YTIMSKPEDLK (SEQ ID NO: 74), wherein IFN-binding loop 2 comprises the amino acid sequence STHEAYVTVL (SEQ ID NO: 75), STQEIYVTVL (SEQ ID NO: 77), or STDEAYVTVL (SEQ ID NO: 78), and wherein IFN-binding loop 3 comprises the amino acid sequence SHNFWLAID (SEQ ID NO: 76) or SHEFWLAID (SEQ ID NO: 79). 7. The fusion protein according to claim 6, wherein the masking peptide has at least 85% sequence identity with any one of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. 8. The fusion protein according to any one of the preceding claims, wherein the masking peptide comprises an amino acid sequence selected from SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. 9. The fusion protein according to any one of claims 1-6, wherein the masking peptide has at least 85% sequence identity with SEQ ID NO: 8 or 3. 10. The fusion protein according to claim 9, wherein the masking peptide comprises at least one disulfide bond. 11. The fusion protein according to claim 10, wherein the masking peptide comprises R9C and E98C substitutions relative to SEQ ID NO: 8. 12. The fusion protein according to claim 10, wherein the masking peptide comprises an amino acid sequence of SEQ ID NO: 12 or 42. 13. The fusion protein according to claim 10, wherein the masking peptide comprises an amino acid of SEQ ID NO: 42.The sequence, and having one or more substitutions selected from the following: (1) L19: S, Q, E or R; (2) F22: G, N or D; (3) I46R; (4) M47: I or F; (5) S48: N, Y or Q; (6) K49Q; (7) H77: Y, Q or D; (8) A79: D, I, V, E or N; (9) V81: I or F; Claims 1 / 4 page 2 CN 121568967 A (10) V83: F, I or K; (11) N99: E, D or W; (12) W101: M or F; (13) L102: M or I; (14) I104: F or W; (15) M106: A, T, L or E; (16) F108: N, Y, T or E; (17) P110: S or E; (18) P111: S or E; and (19) combinations of the above substitutions. 14. The fusion protein of claim 13, wherein the masking peptide comprises an amino acid sequence selected from SEQ ID NO: 42, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, or 73. 15. The fusion protein of any of the preceding claims, wherein the fusion protein does not bind IFNAR2. 16. The fusion protein of any of the preceding claims, wherein the cleavable linker contains a protease cleavage site. 17. The fusion protein of claim 11, wherein the protease is selected from thrombin, neutrophil elastase, cysteine ​​protease, FAPa, cathepsin B, asparagine endopeptidase, serine proteases such as membrane-type serine protease 1 or urokinase (uPA), and matrix metalloproteinases (MMPs) such as MMP1, MMP2, MMP3, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, and MMP17. 18. The fusion protein of claim 16, wherein the protease cleavage site comprises an amino acid sequence selected from SEQ ID NO: 45 and 10. 19. The fusion protein of claim 11, wherein the cleavable linker comprises an amino acid sequence selected from SEQ ID NO: 9, 13, and 44. 20. The fusion protein of claim 1, wherein the type 1 IFN is selected from IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, and IFN-α21, IFN-β, IFN-κ, IFN-ε, or IFN-ω, and any amino acid sequence having at least 75% sequence identity with them. 21.20. The fusion protein of claim 20, wherein the IFN comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 4. 22. The fusion protein of any one of claims 3-21, wherein the Fc is an IgG1, IgG2, IgG3, or IgG4 isotype. 23. The fusion protein of claim 22, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 6. 24. The fusion protein of any one of the preceding claims, which forms a dimer. 25. The fusion protein of claim 1, comprising an amino acid sequence selected from SEQ ID NO: 41, 43, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39. 26. A composition comprising a fusion protein according to any one of claims 1-25 and a pharmaceutically acceptable carrier. 27. An isolated cell comprising one or more polynucleotides encoding the fusion protein according to any one of claims 1-25. 28. A polynucleotide encoding the fusion protein according to any one of claims 1-25. 29. A method of treating cancer in a patient in need, comprising administering to the patient the fusion protein according to any one of claims 1-25, the composition according to claim 26, the cell according to claim 27, or the polynucleotide according to claim 28. 30. The method of claim 29, wherein the cancer is selected from bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. 31. The method of claim 30, wherein the cleavable linker of the fusion protein is cleaved by an enzyme within the patient's tumor microenvironment. 32. A method of treating an autoimmune disease or inflammatory condition in a patient in need, comprising administering to the patient a fusion protein according to any one of claims 1-25, a composition according to claim 26, a cell according to claim 27, or a polynucleotide according to claim 28. 33. A polypeptide comprising interferon α and β receptor subunit 2 domain 1 (IFNAR2-D1) or a peptide having at least 55% sequence identity with IFNAR2-D1, wherein the polypeptide is capable of binding type 1 IFN, and wherein the polypeptide does not contain the complete sequence of IFNAR2 domain 2 (IFNAR2-D2).34. The polypeptide of claim 33, wherein it does not contain at least 50% IFNAR2-D2, or does not contain at least 80% IFNAR2-D2. 35. The polypeptide of claim 33 or 34, wherein the IFNAR2-D1 comprises the amino acid sequence of SEQ ID NO: 8 or 3. 36. The polypeptide of any one of claims 33-35, comprising IFN-binding ring 1, IFN-binding ring 2, and IFN-binding ring 3, wherein the IFN-binding ring 1 comprises the amino acid sequence of YTIMSKPEDLK (SEQ ID NO: 74), the IFN-binding ring 2 comprises the amino acid sequence of STHEAYVTVL (SEQ ID NO: 75), STQEIYVTVL (SEQ ID NO: 77), or STDEAYVTVL (SEQ ID NO: 78), and the IFN-binding ring 3 comprises the amino acid sequence of SHNFWLAID (SEQ ID NO: 76) or SHEFWLAID (SEQ ID NO: 79). 37. The polypeptide of claim 36, having at least 85% sequence identity with any one of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. 38. The polypeptide of any one of claims 33-37, comprising an amino acid sequence selected from SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. 39. The polypeptide of any one of claims 33-35, having at least 85% sequence identity with SEQ ID NO: 8 or 3. 40. The polypeptide of claim 33, comprising at least one disulfide bond. 41. The polypeptide of claim 40, comprising R9C and E98C substitutions relative to SEQ ID NO: 8. 42. The polypeptide of claim 40, comprising an amino acid sequence of SEQ ID NO: 12 or 42. 43. The polypeptide of claim 40, comprising the amino acid sequence of SEQ ID NO: 42, and having one or more substitutions selected from the following: (1) L19: S, Q, E, or R; (2) F22: G, N, or D; (3) I46R; (4) M47: I or F; (5) S48: N, Y, or Q; (6) K49Q; (7) H77: Y, Q, or D; (8) A79: D, I, V, E, or N; (9) V81: I or F; (10) V83: F, I, or K; (11) N99: E, D, or W; (12)W101: M or F; (13) L102: M or I; (14) I104: F or W; (15) M106: A, T, L or E; (16) F108: N, Y, T or E; (17) P110: S or E; (18) P111: S or E; and (19) combinations of the above substitutions. 44. The polypeptide according to claim 43, comprising an amino acid sequence selected from SEQ ID NO: 42, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 or 73. 45. A polynucleotide encoding a polypeptide according to any one of claims 33-44. Claims 4 / 4 Page 5 CN 121568967 A Cross-Reference to Related Applications Regarding Masked Interferon Fusion Proteins and Their Uses

[0001] This application claims the benefit of PCT application No. PCT / CN2023 / 109070, filed July 25, 2023, the contents of which are incorporated herein by reference in their entirety. Background Art

[0002] The type I interferon (IFN) system includes a form of IFNβ, several IFNα variants, and other IFNs that are not yet fully characterized, all of which transactivate IFN-stimulating genes via heterodimeric IFNα / β receptor 1 (IFNAR1)-IFNAR2 receptors. Type I IFNs regulate many important physiological processes, such as maintaining the hematopoietic stem cell microenvironment, maintaining synaptic plasticity and cognitive function, maintaining microbiota-driven optimal antiviral immunity, and regulating bone remodeling. Another important regulatory function of type I IFNs is inducing antiviral states, such as inhibiting viral protein synthesis, degrading viral RNA, and capturing viral components. Type I IFNs are rapidly expressed after infection, participate in the defense against pathogens, and act as a bridge between innate and adaptive immune responses.

[0003] The interferon-α (IFN-α) protein family has been shown to be useful in the treatment of a variety of diseases. For example, interferon-α2a and 2b (trade names Roferon and Intron A, respectively) have been used to treat chronic hepatitis B, C, and D (life-threatening viral liver diseases), condyloma acuminata (genital warts), AIDS-related Kaposi's sarcoma, hairy cell leukemia, malignant melanoma, basal cell carcinoma, multiple myeloma, renal cell carcinoma, herpes type I and II, varicella-zoster virus, and mycosis fungoides. The efficacy of interferon-α-containing treatment regimens in prostate cancer and chronic myeloid leukemia has also been studied. IFN-α has been reported to induce apoptosis in malignant cells via the caspase pathway. There is a need for IFN therapies with cancer-targeting potential.

[0004] This disclosure provides a masked interferon fusion protein comprising an interferon masking portion that binds to type 1 interferon (IFN).The masking peptide comprises an IFN polypeptide that retains type 1 interferon activity when not bound by the masking portion, an immunoglobulin (Ig) Fc region, and a cleavable linker connecting the masking portion and the IFN polypeptide, wherein the IFN polypeptide is linked to the Ig Fc region. These masked interferon fusion proteins can be used to treat diseases and conditions such as cancer, autoimmune diseases, or inflammatory conditions.

[0005] In one aspect, this disclosure provides a fusion protein comprising (a) an interferon α and β receptor subunit 2 domain 1 (IFNAR2-D1) or a masking peptide having at least 55% sequence identity with IFNAR2-D1, (b) type 1 interferon (IFN), and (c) a cleavable linker between (a) and (b), wherein the peptide is capable of binding the type 1 IFN, and wherein the fusion protein does not contain the complete sequence of IFNAR2 domain 2 (IFNAR2-D2).

[0006] In some embodiments, the cleavable linker is fused to the C-terminus of (a) and the N-terminus of (b), or the cleavable linker is fused to the N-terminus of (a) and the C-terminus of (b). In some embodiments, the fusion protein further comprises an immunoglobulin (Ig) Fc region fused to the C-terminus of the IFN. In some embodiments, the fusion protein further comprises an immunoglobulin (Ig) Fc region fused to the N-terminus of (a).

[0007] In some embodiments, the fusion protein does not contain at least 50% IFNAR2-D2, or does not contain at least 80% IFNAR2-D2. In some embodiments, the IFNAR2-D1 comprises the amino acid sequence of SEQ ID NO: 8 or 3. Specification page 1 / 38 6 CN 121568967 A

[0008] In some embodiments, the masking peptide comprises IFN-binding ring 1, IFN-binding ring 2, and IFN-binding ring 3, wherein IFN-binding ring 1 comprises the amino acid sequence of YTIMSKPEDLK (SEQ ID NO: 74), IFN-binding ring 2 comprises the amino acid sequence of STHEAYVTVL (SEQ ID NO: 75), STQEIYVTVL (SEQ ID NO: 77), or STDEAYVTVL (SEQ ID NO: 78), and IFN-binding ring 3 comprises the amino acid sequence of SHNFWLAID (SEQ ID NO: 76) or SHEFWLAID (SEQ ID NO: 79). In some embodiments, the masking peptide is associated with SEQ ID NO: 18, 20, 22, 24, ...Any one of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40 has at least 85% sequence identity. In some embodiments, the masking peptide comprises an amino acid sequence selected from SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

[0009] In some embodiments, the masking peptide has at least 85% sequence identity with SEQ ID NO: 8 or 3.

[0010] In some embodiments, the masking peptide comprises at least one disulfide bond. In some embodiments, the masking peptide comprises R9C and E98C substitutions relative to SEQ ID NO: 8. In some embodiments, the masking peptide comprises an amino acid sequence of SEQ ID NO: 12 or 42.

[0011] In some embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO: 42 and has one or more substitutions selected from the following: (1) L19: S, Q, E or R; (2) F22: G, N or D; (3) I46R; (4) M47: I or F; (5) S48: N, Y or Q; (6) K49Q; (7) H77: Y, Q or D; (8) A79: D, I, V, E or N; (9) V81: I or F; (10) V83: F, I or K; (11) N99: E, D or W; (12) W101: M or F; (13) L102: M or I; (14) I104: F or W; (15) M106: A, T, L or E; (16) F108: N, Y, T or E; (17) P110: S or E; (18) P111: S or E; and (19) combinations of the above substitutions.

[0012] In some embodiments, the masking peptide comprises an amino acid sequence selected from SEQ ID NO: 42, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, or 73.

[0013] In some embodiments, the fusion protein does not bind IFNAR2.

[0014] In some embodiments, the cleavable linker contains a protease cleavage site. In some embodiments, the protease is selected from thrombin, neutrophil elastase, cysteine ​​protease, FAPa, cathepsin B, legumain, serine proteases such as membrane-type serine protease 1 (matriptase) or urokinase (uPA), and matrix metalloproteinases (MMPs) such as MMP1, MMP2, MMP3, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, and MMP17.

[0015] In some embodiments, the protease cleavage site comprises an amino acid sequence selected from SEQ ID NO: 45 and 10.

[0016] In some embodiments, the cleavable adapter comprises an amino acid sequence selected from SEQ ID NO: 9, 13, and 44.

[0017] In some embodiments, the type I IFN is selected from IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, and IFN-α21, IFN-β, IFN-κ, IFN-ε, or IFN-ω, and any amino acid sequence having at least 75% sequence identity with it. In some embodiments, the IFN comprises an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 4.

[0018] In some embodiments, the Fc region is an isotype of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 6.

[0019] In some embodiments, the fusion protein forms a dimer.

[0020] In some embodiments, the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 41, 43, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39.

[0021] In one aspect, this disclosure provides a composition comprising the fusion protein and a pharmaceutically acceptable carrier.

[0022] In one aspect, this disclosure provides an isolated cell comprising one or more polynucleotides encoding the fusion protein provided herein.

[0023] In one aspect, this disclosure provides a polynucleotide encoding the fusion protein provided herein.

[0024] In one aspect, this disclosure provides a method of treating cancer in a patient in need, comprising administering to the patient the fusion protein provided herein, the composition provided herein, the cells provided herein, or the polynucleotide provided herein. In some embodiments, the cancer is selected from bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0025] In some embodiments, the cleavable linker of the fusion protein is cleaved by an enzyme within the patient's tumor microenvironment.

[0026] In one aspect, this disclosure provides a method of treating autoimmune diseases or inflammatory conditions in a patient in need.Methods comprising administering to a patient the fusion protein provided herein, the composition provided herein, the cell provided herein, or the polynucleotide provided herein.

[0027] In one aspect, this disclosure provides a polypeptide comprising interferon α and β receptor subunit 2 domain 1 (IFNAR2-D1) or a peptide having at least 55% sequence identity with IFNAR2-D1, wherein the polypeptide is capable of binding type 1 IFN, and wherein the polypeptide does not contain the complete sequence of IFNAR2 domain 2 (IFNAR2-D2).

[0028] In some embodiments, the polypeptide does not contain at least 50% IFNAR2-D2, or does not contain at least 80% IFNAR2-D2.

[0029] In some embodiments, IFNAR2-D1 comprises the amino acid sequence of SEQ ID NO: 8 or 3.

[0030] In some embodiments, the polypeptide comprises IFN-binding loop 1, IFN-binding loop 2, and IFN-binding loop 3, wherein IFN-binding loop 1 comprises the amino acid sequence of YTIMSKPEDLK (SEQ ID NO: 74), IFN-binding loop 2 comprises the amino acid sequence of STHEAYVTVL (SEQ ID NO: 75), STQEIYVTVL (SEQ ID NO: 77), or STDEAYVTVL (SEQ ID NO: 78), and IFN-binding loop 3 comprises the amino acid sequence of SHNFWLAID (SEQ ID NO: 76) or SHEFWLAID (SEQ ID NO: 79). Specification 3 / 38 pages 8 CN 121568967 A

[0031] In some embodiments, the polypeptide has at least 85% sequence identity with any one of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. In some embodiments, the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

[0032] In some embodiments, the polypeptide has at least 85% sequence identity with SEQ ID NO: 8 or 3.

[0033] In some embodiments, the polypeptide comprises at least one disulfide bond.

[0034] In some embodiments, the polypeptide comprises R9C and E98C substitutions relative to SEQ ID NO: 8. In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 12 or 42.

[0035] In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 42 and has one or more substitutions selected from: (1) L19: S, Q, E, or R; (2)F22: G, N or D; (3) I46R; (4) M47: I or F; (5) S48: N, Y or Q; (6) K49Q; (7) H77: Y, Q or D; (8) A79: D, I, V, E or N; (9) V81: I or F; (10) V83: F, I or K; (11) N99: E, D or W; (12) W101: M or F; (13) L102: M or I; (14) I104: F or W; (15) M106: A, T, L or E; (16) F108: N, Y, T or E; (17) P110: S or E; (18) P111: S or E; and (19) combinations of the above substitutions.

[0036] In some embodiments, the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 42, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, or 73. In one aspect, this disclosure provides a polynucleotide encoding the polypeptide provided herein. Brief Description of the Drawings

[0037] Figure 1 illustrates an exemplary form of the masked IFN fusion protein.

[0038] Figure 2 illustrates the human IFNAR2 binding efficiency of I-09 as detected by ELISA (A), and the inhibitory activity of I-09 and I-09 cleavage (masked IFN-Fc incubated with MMP-14 labeled "I-XX cut") on Daudi cell proliferation (B).

[0039] Figures 3A-C illustrate the binding affinity of the de novo-designed masked IFN-Fc fusion protein to human IFNAR2.

[0040] Figures 4A-C show the inhibitory activity of the de novo masked IFN-Fc fusion protein on the proliferation of Daudi cells with or without the masked portion (masked IFN-Fc incubated with MMP-14 is labeled "I-XX cut").

[0041] Figure 5 shows the sequence alignment of M-09 and M-140 (A), and the comparison of protein expression and purity of I-09 and I-40 by SDS-PAGE and size exclusion chromatography (SEC) (B).

[0042] Figure 6 shows the binding affinity (ELISA) of I-140 and I-140 cut (I-140 incubated with MMP-14 is labeled "I-140 cut") to human IFNAR2 (A), and its inhibitory activity on the proliferation of Daudi cells (B).

[0043] Figures 7A-C show the binding affinity of the I-140-derived site mutation-masked IFN-Fc fusion protein to human IFNAR2, as determined by ELISA.

[0044] Figures 8A-C illustrate the inhibitory activity of the I-140-derived site mutation-masked IFN-Fc fusion protein on Daudi cell proliferation. Detailed Description

[0045] Definitions

[0046] It should be noted that the term “an” or “a” entity refers to one or more of the entity; for example, “a polypeptide” should be understood to mean one or more polypeptides. Thus, the terms “an” (or “a”), “one or more” and “at least one” are used interchangeably herein.

[0047] As used herein, the term “CH2 region” or “CH2 domain” means the CH2 region of an immunoglobulin, including, for example, the portion of the heavy chain molecule extending from approximately residue 244 to residue 360 ​​of the antibody, using a conventional numbering scheme (residues 244-360, Kabat numbering system; residues 231-340, EU numbering system; see Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983)). For example, the CH2 region of a human IgG1 antibody corresponds to amino acids 228-340 in the EU numbering system. However, the CH2 region may also be any other subtype described herein.

[0048] As used herein, the term “CH3 region” or “CH3 domain” refers to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 in the EU numbering system. However, the CH3 region may also be any other subtype described herein.

[0049] As used herein, the term “hinge region” includes the heavy chain molecule portion that connects the CH1 domain and the CH2 domain. The hinge region contains about 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper hinge domain, the middle hinge domain, and the lower hinge domain (Roux et al., J. Immunol 161:4083 (1998)).

[0050] The term "immunoglobulin" or "Ig" refers to a class of structurally related glycoproteins composed of two pairs of polypeptide chains: a pair of low-molecular-weight light (L) chains and a pair of heavy (H) chains, all four chains potentially interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). In short, each heavy chain typically contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically contains three domains, CH1, CH2, and CH3. Between the heavy chains...The light chains are interconnected by disulfide bonds in so-called “hinge regions.” Each light chain typically contains a light chain variable region (abbreviated as VL in this paper) and a light chain constant region. The light chain constant region typically contains a domain CL. The VH and VL regions can be further subdivided into highly variable regions (or highly variable regions whose sequence and / or structurally defined ring forms are highly variable), also known as complement-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically contains three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or contradicted by the context, the amino acids in the constant region sequence specification 5 / 38 page 10 CN 121568967 A are based on the EU index number (described in Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition – US Department of Health and Human Services, NIH publication No. 91-3242, pp 662,680,689 (1991)).

[0051] As used herein, the term “inhibition” or “inhibition of…” means a reduction or complete prevention to a measurable degree.

[0052] As used herein, the term “interferon” refers to a group of signaling proteins produced and released by host cells in response to the presence of various viruses. Typically, virus-infected cells release interferons, causing nearby cells to enhance their antiviral defenses. IFNs belong to a large class of proteins called cytokines, which are used for intercellular communication to trigger protective defense mechanisms of the immune system, thereby helping to eradicate pathogens.

[0053] For the purposes of this disclosure, when referring to the masking portion of an IFN, the term "mask" or "masking" means any polypeptide or protein that blocks cytokine interaction and / or activation between IFN and IFNAR. Within the scope of this invention, "masking" can be modified by recombinant means. Amino acid modification includes the deletion, addition, and substitution of amino acids.

[0054] As used herein, the terms "masked IFN" or "binding IFN" refer to a type I interferon in which a polypeptide attaches to or binds to an IFN, thereby reducing the ability of the IFN to bind to IFNAR.

[0055] As used herein, the term "affinity" refers to the affinity of one molecule (e.g., a polypeptide) for another molecule (e.g., a polypeptide) at a single site.The binding strength of the target or ligand, such as the monovalent binding of a single target site of the masked portion to the IFN peptide.

[0056] As used herein, the term “treat” or “treatment” refers to therapeutic treatment and prophylactic or preventative measures aimed at preventing or slowing (mitigating) undesirable physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also refer to extended survival compared to expected survival without treatment. Subjects requiring treatment include those who already have a condition or disease and those who are susceptible to a condition or disease, or those who want to prevent a condition or disease.

[0057] “Subject” or “individual” or “animal” or “patient” or “mammal” means any subject requiring diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, livestock, farm animals and zoo, racing or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, etc.

[0058] As used herein, phrases such as “patients in need of treatment” or “subjects in need of treatment” include subjects who would benefit from administration of the polypeptides or compositions of this disclosure (e.g., for detection, for diagnostic procedures and / or for treatment), such as mammalian subjects.

[0059] “Pharmaceutical acceptable” means a composition that is non-toxic, inert and / or physiologically compatible with humans or other mammals.

[0060] Type I interferon and IFN-α / β receptor (IFNAR)

[0061] As used herein, the term “type I interferon” or “type I interferon” refers to a large subset of interferon proteins that help regulate the activity of the immune system. Human type I IFN belongs to a polygenic family, consisting of 13 IFN-α subtypes (IFN-α1 (Uniprot No. P01562), IFN-α2 (Uniprot No. P01563), IFN-α4 (Uniprot No. P05014), IFN-α5 (Uniprot No. P01569), IFN-α6 (Uniprot No. P05013), IFN-α7 (Uniprot No. P01567), IFN-α8 (Uniprot No. P32881), IFN-α10 (Uniprot No. P01566), IFN-α13 (Uniprot No. P01562), IFN-α14 (Uniprot No. P01566), IFN-α13 (Uniprot No. P01562), IFN-α14 (Uniprot No. P01562), IFN-α15 (Uniprot No. P01563), IFN-α14 ...The IFN-α16 (Uniprot No. P05015), IFN-α17 (Uniprot No. P05017), and IFN-α21 (Uniprot No. P01568) are all present, along with only one IFN-β (Uniprot No. P01574), IFN-ε (Uniprot No. Q86WN2), IFN-κ (Uniprot No. Q9P0W0), and IFN-ω (Uniprot No. P05000) on page 6 / 38 of the specification (CN 121568967 A). All type I IFNs are structurally similar, with highly conserved protein sequences (75-99% amino acid sequence identity). All type I IFNs bind to a specific cell surface receptor complex called the IFN-α receptor (IFNAR), which consists of IFNAR1 and IFNAR2 chains. Within the scope of this disclosure, type 1 IFN can be modified by recombinant means. Amino acid modification includes the deletion, addition, and substitution of amino acids.

[0062] IFN-α and IFN-β are secreted by a variety of cell types, including lymphocytes (NK cells, B cells, and T cells), macrophages, fibroblasts, endothelial cells, osteoblasts, etc. They stimulate macrophages and NK cells to trigger antiviral responses and also have antitumor activity. Plasma cell-like dendritic cells have been identified as the most potent producers of type 1 interferon in response to antigens and are therefore referred to as natural IFN-producing cells. Current research findings suggest that by forcing tumor-infiltrating macrophages to express IFN-α, it may be possible to induce more effective dendritic cell activation and cytotoxicity of immune effector cells.

[0063] In some embodiments, the type 1 IFN of this disclosure may be a natural IFN protein derived from humans or animals, or may be a recombinant IFN derived from transformed cells. Preferably, recombinant human IFN is prepared using transformed Escherichia coli (E. coli). Unless their biological activity deviates significantly from that of the wild type, mutants formed by amino acid substitution, deletion, or insertion are also included in the range of interferon α. ​​

[0064] In some embodiments, human type 1 IFN comprises IFN-α subtype, IFN-β, IFN-ε, IFN-κ, or IFN-ω, or any amino acid sequence having at least 75% (or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity. In some embodiments, type 1 IFN comprises IFN-α2β, or any amino acid sequence having at least 75% (or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity. In some embodiments, the type 1 IFN of this disclosure comprises SEQ ID NO:The amino acid sequence of 5 or any amino acid sequence having at least 75% (or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity.

[0065] The interferon-α / β receptor (IFNAR) is a receptor that binds type I interferon. It is a heterodimeric cell surface receptor composed of two subunits, namely IFNAR1 (Homo sapiens, Uniprot No. P17181) and IFNAR2 (Homo sapiens, Uniprot No. P48551). When type I IFN binds, IFNAR activates the JAK-STAT signaling pathway. Interferon stimulation classically leads to an antiviral immune response.

[0066] A human IFNAR2 model expressed in E. coli showed that there is a major hydrophobic patch on the receptor that interacts with a matching hydrophobic surface on IFN-α. Subsequently, adjacent motif guide proteins composed of charged side chains form a tight complex. The binding interface may determine the receptor's cross-reactivity and ligand specificity. The high-affinity IFNAR2 chain adopts a dual-domain D1 / D2 receptor structure, where D1 contributes most of the binding affinity.

[0067] Masked Interferon Fusion Proteins

[0068] As shown in the experimental examples, the binding affinity of various masked IFN fusion proteins designed and prepared herein to human IFNAR is significantly reduced, typically by hundreds or even thousands of times. However, after removing the masking portion, the released IFN-Fc protein still retains excellent binding affinity to human IFNAR and can effectively inhibit Daudi cell proliferation.

[0069] According to one embodiment of the present disclosure, a masked IFN fusion protein is provided, comprising a masking portion fused to the N-terminus of a type I IFN via a cleavable linker, the type I IFN optionally fused to an immunoglobulin Fc region.

[0070] In some embodiments, the masked IFN fusion protein of this disclosure comprises, from N-terminus to C-terminus, a masking portion, a cleavable linker, type I IFN, and an immunoglobulin Fc region. In some embodiments, the masked IFN fusion protein of this disclosure comprises, from N-terminus to C-terminus, a masking portion, a cleavable linker, type I IFN, a second linker, and an immunoglobulin Fc region.

[0071] Type I IFN Masking Portion

[0072] This disclosure provides a masking portion that has binding specificity to human type I IFN and can effectively block the binding between human type I IFN and IFNAR. In some embodiments, IFNAR includes IFNAR1 and IFNAR2.

[0073] A polypeptide comprising the masking portion disclosed herein is also provided, optionally in combination with other protein elements.

[0074] To impart a “masking” function to type 1 IFNs, the IFN masking portion needs to maintain a high binding affinity for type 1 IFNs. It has been observed that IFNAR2 domain 1 (IFNAR2-D1) interacts with type 1 interferons through its three binding loops and contributes most of the affinity for type 1 IFN binding. Therefore, in some embodiments, the masking portion of this disclosure comprises IFNAR2-D1 or a derivative (a mutant described below) and does not include part or all of the remaining sequence of IFNAR2. In one embodiment, the masking portion does not contain complete IFNAR2-D2 (SEQ ID NO: 16), or does not contain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of IFNAR2-D2 (SEQ ID NO: 16).

[0075] In some embodiments, IFNAR2-D1 is mutated to provide better masking activity, or to aid expression or stability. In some embodiments, the binding affinity of IFNAR2-D1 can be improved by mutating the binding loop, thereby enhancing the masking activity. Table 1 summarizes the amino acid sequences of the three binding loops in IFNAR2-D1 that form a binding interface with type 1 IFN.

[0076] Table 1. Sequences of IFN binding loops

[0077] In some embodiments, IFN binding loops 1-3 are selected from any group of IFN binding loops 1-3 shown in Table 1. In some embodiments, IFN binding loops 1-3 are selected from those derived from the same IFN masking portion in the examples.

[0078] In some embodiments, at least one, two, or three of the above-described IFN binding loops 1-3 are modified by the addition, deletion, substitution, or combination thereof of one, two, or three amino acids.

[0079] In some embodiments, the type 1 IFN masking portion comprises IFN-binding loop 1: YTIMSKPEDLK (SEQ ID NO: 74), IFN-binding loop 2: STHEAYVTVL (SEQ ID NO: 75), and IFN-binding loop 3: SHNFWLAID (SEQ ID NO: 76).

[0080] The tools used for redesigning peptides may be machine learning (ML) tools applied to mutagenesis-based protein engineering or other artificial intelligence (AI)-based computational tools, enabling the de novo design of variant libraries containing proteins with desired functions that have never existed in nature (Saito et al., ACS Catal., 11, 14615–14624 (2021); Eisenstein, Nature biotechnology., 41, 303–305 (2023)).

[0081] With the assistance of ML or AI-based computational tools, mutations can be made within the binding loop to improve IFN-IFNAR2-D1 binding affinity, thereby enhancing masking efficiency and / or peptide expression. Table 2 lists some redesigned binding loop sequences.

[0082] Table 2. Sequences of mutated IFN binding loops

[0083] In some embodiments, the type 1 IFN masking portion comprises IFN binding loop 1: YTIMSKPEDLK (SEQ ID NO: 74), IFN binding loop 2: STQEIYVTVL (SEQ ID NO: 77), and IFN binding loop 3: SHNFWLAID (SEQ ID NO: 76).

[0084] In some embodiments, the Type 1 IFN masking portion includes IFN binding ring 1: YTIMSKPEDLK (SEQ ID NO: 74), IFN binding ring 2: STQEIYVTVL (SEQ ID NO: 77), and IFN binding ring 3: SHEFWLAID (SEQ ID NO: 79).

[0085] In some embodiments, the Type 1 IFN masking portion includes IFN binding ring 1: YTIMSKPEDLK (SEQ ID NO: 74), IFN binding ring 2: STDEAYVTVL (SEQ ID NO: 78), and IFN binding ring 3: SHNFWLAID (SEQ ID NO: 76).

[0086] In some embodiments, the type 1 IFN masking portion comprises IFN-binding loop 1: YTIMSKPEDLK (SEQ ID NO: 74), IFN-binding loop 2: STHEAYVTVL (SEQ ID NO: 75), and IFN-binding loop 3: SHEFWLAID (SEQ ID NO: 79).

[0087] The IFNAR2-D1 backbone region can be redesigned using ML or AI-based computational tools to improve the spatial structural stability of the peptide. As used herein, “IFNAR2-D1 backbone” refers to the region of the supporting structure in IFNAR2-D1 in which the IFN-binding loop is inserted, and the IFNAR2-D1 backbone region does not participate in binding to type 1 IFNs. In one aspect of this disclosure, the type 1 IFN masking portion provided herein is an IFNAR2-D1 backbone redesigned based on the IFNAR2-D1 structure comprising the amino acid sequence of SEQ ID NO: 8.

[0088] In some embodiments, the redesigned Type 1 IFN masking portion comprises having at least 55% (or at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%) of the same as SEQ ID NO: 8.The amino acid sequence has at least 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity and is still capable of binding type 1 IFN.

[0089] In some embodiments, the type 1 IFN masking portion comprises an amino acid sequence of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In some embodiments, the masking portion comprises a peptide having at least 55% (or at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) sequence identity with any of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40. In some embodiments, such amino acid sequences retain one or more of the original or mutated IFN-binding loops 1, 2, and / or 3.

[0090] In another aspect of this disclosure, the type 1 IFN masking moiety provided herein is derived from sequence optimization based on IFNAR2-D1 containing an N-terminal extension of the amino acid sequence SEQ ID NO: 3. One or more further substitutions may be made to enhance the properties of the type 1 IFN masking moiety polypeptide, such as protein expression and masking efficiency.

[0091] In some embodiments, one or more disulfide bonds are introduced into the type 1 IFN masking moiety polypeptide to stabilize the polypeptide. As used herein, the term "disulfide bond" refers to a covalent bond with an R-S-S-R structure formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or disulfide bridge with a second thiol group from another cysteine ​​residue. Incorporating disulfide bonds into proteins is one of the ways in nature to improve protein stability; a correlation has been found between the abundance of disulfide bonds in thermophilic organisms and their maximum growth temperature, indicating the importance of disulfide bonds for protein stability under high temperature conditions (Mallick P, et al, 2002, Proc. Natl. Acad. Sci. USA, 99, 9679-9684.; Ladenstein R, et al, 2006, FEBS J., 273, 4170-4185).

[0092] The number of disulfide bonds in a protein can be readily determined by precise whole mass measurement. Disulfide bond connections can be verified (determined) by standard techniques known in the art (such as peptide mapping analysis).

[0093] In some embodiments of this disclosure, by […] at the R20C and E109C positions corresponding to SEQ ID NO: 3Amino acid substitutions are performed to introduce a pair of cysteine ​​residues to form a disulfide bond. In some embodiments, the type I IFN masking portion comprises the amino acid sequence of SEQ ID NO: 42, as described on page 9 / 38 of the specification, CN 121568967 A.

[0094] In some embodiments, one or more substitutions are introduced into the type I IFN masking portion, and the type I IFN masking portion comprises an amino acid sequence having at least 90% (or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with SEQ ID NO: 3 and is still capable of binding type I IFN.

[0095] In some embodiments, the type 1 IFN masking portion comprises the amino acid sequence of SEQ ID NO: 42 and has one or more substitutions selected from the following: (1) L19: S, Q, E, or R; (2) F22: G, N, or D; (3) I46R; (4) M47: I or F; (5) S48: N, Y, or Q; (6) K49Q; (7) H77: Y, Q, or D; (8) A79: D, I, V, E, or N; (9) V81: I or F; (10) V83: F, I, or K; (11) N99: E, D, or W; (12) W101: M or F; (13) L102: M or I; (14) I104: F or W; (15) M106: A, T, L, or E; (16) F108: N, Y, T or E; (17) P110: S or E; (18) P111: S or E; and (19) combinations of the above substitutions.

[0096] In some embodiments, the type 1 IFN masking portion comprises the amino acid sequence of SEQ ID NO: 42 and has the following amino acid substitutions: (1) R20C, M47I, H77Q, A79, and E109C; (2) R20C, H77D, A79I, N99E, and E109C; (3) R20C, H77D, and E109C; (4) R20C, N99E, and E109C; (5) L19S, R20C, F22G, M106A, F108N, E109C, P110S, and P111S; (6) L19Q, R20C, F22N, M106T, F108Y, and E109C; (7) (8) L19E, R20C, F22G, M106L, F108T, E109C, P110S and P111S; (9) L19R, R20C, F22D, F108E and E109C; (10) L19R, R20C, F22N, M106E, E109C, P110S and P111S;R20C, F22G, M106T, E109C, P110S and P111S; (11) L19S, R20C, F22N, M106L, F108E and E109C; (12) L19E, R20C, F22G and E109C; Instruction manual 10 / 38 pages 15 CN 121568967 A (13) R20C, F22N, F108T, E109C, P110S and P111S; or (14) R20C, F22G, F108N and E109C.

[0097] In some embodiments, the type 1 IFN masking portion comprises an amino acid sequence of SEQ ID NO: 42, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, or 73. In some embodiments, the masking portion comprises a peptide having at least 55% (or at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) sequence identity with any one of SEQ ID NO: 42, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, or 73). In some embodiments, such amino acid sequences retain one or more of the mutations provided above.

[0098] In some embodiments, the IFN masking portion of this disclosure is fused to the immunoglobulin Fc region. In some embodiments, the masking portion is fused to the Ig Fc region directly or via a linker. In some embodiments, the C-terminus or N-terminus of the IFN masking portion is fused to the N-terminus of the Ig Fc region. In some embodiments, the C-terminus or N-terminus of the IFN masking portion is fused to the C-terminus of the Ig Fc region.

[0099] Linker

[0100] Linkers within the scope of this disclosure can be characterized based on their amino acid composition, length, rigidity, and secondary structure. Linkers within the scope of this disclosure separate type I interferon from another functional polypeptide (such as the masking portion or the Ig Fc region) and allow each domain to fold correctly and function. In this way, linkers can be customized for a specific type I interferon and another functional polypeptide. According to one aspect, functional independence of the domain (i.e., type I interferon) from the fused (heterologous) domain is maximized by a suitable linker to limit steric interference between domains during bacterial cell export and assembly. According to one aspect, a longer and more flexible (GGGGS)n-type connector is exemplary, where n is an integer from 1 to 20 (hereinafter referred to as "GS repeat", SEQ ID NO: 1). According to another aspect, cellular stress is minimized by limiting the total length of the fusion protein. LongerThe linker sequence and high induction levels can cause stress to the cell's biosynthetic mechanisms, inhibiting cell growth and, in extreme cases, leading to cell lysis.

[0101] The linkers within the scope of this disclosure facilitate the function of the type 1 interferon domain and another functional peptide. The linkers within the scope of this disclosure allow efficient protein processing and export via the bacterial curli secretion mechanism and provide appropriate spatial and physicochemical separation for type 1 interferon and another functional peptide to preserve their respective functions.

[0102] The linkers within the scope of this disclosure comprise amino acid residues. The amino acid residues can be any naturally occurring amino acid residues. The amino acid residues can also be synthetic amino acids known to those skilled in the art. Representative amino acids that can be used for linkers include glycine, alanine, valine, leucine, isoleucine, serine, cysteine, selenocysteine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, asparagine, and glutamine.

[0103] The linkers within the scope of this disclosure contain cleavage sites. Cleavage sites and cleavage enzymes are known to those skilled in the art. According to this embodiment, the cleavable linker is cleaved by enzymes within the patient's tumor microenvironment. As used herein, the "tumor microenvironment" refers to a "complex network" composed of different cell types, soluble factors, signaling molecules, and extracellular matrix components that synergistically regulate the fate of tumor progression. Proteases expressed by cells in the tumor microenvironment (e.g., tumor cells and tumor-associated macrophages) (and thus tumor-enriched) cleave the linker at the cleavage site, releasing functional polypeptides from the linker into the surrounding environment, for example, for therapeutic or diagnostic purposes.

[0104] Proteases are a class of enzymes involved in the cleavage or hydrolysis of various proteins. The proteases are tumor-enriched proteases, such as matrix tissue proteases, caspases, and metalloproteinases. Exemplary enzymes include enzymes from the matrix metalloproteinase (MMP) family (which have their own recognition sequences), thrombin, neutrophil elastase, cysteine ​​protease, cathepsin B, asparagine endopeptidase, serine proteases (such as membrane-type serine protease 1 or urokinase (uPA)), or matrix metalloproteinases (MMPs) such as MMP1, MMP2, MMP3, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, or MMP17. Other enzymes are proteases secreted by pathogens, such as CD2830 from Clostridium difficile.

[0105] The cleavage site sequences of tumor-enriched MMPs include, but are not limited to, the following sequences: SGVFSIPLTA (SEQ ID NO: 143), ...144)、VPQSLTMG (SEQ ID NO: 145)、VPRSLTMG (SEQ ID NO: 146)、VPLGLTMG (SEQ ID NO: 80)、VPLSITMG (SEQ ID NO: 81)、VPLSLAMG (SEQ ID NO: 82)、VPLSLDMG (SEQ ID NO: 83)、VPLSLTGG (SEQ ID NO: 84)、GPQGIAGQ (SEQ ID NO: 85)、GPQGIAGQ (SEQ ID NO: 86)、VPMSMRGG (SEQ ID NO: 87)、IPVSLRSG (SEQ ID NO: 88)、RPFSMIMG (SEQ ID NO: 89)、VPLSLTMG (SEQ ID NO: 90)、VPLSLYSG (SEQ ID NO: 91)、IPESLRAG (SEQ ID NO: 92)、IPENFFGV (SEQ ID NO: 93)、VPYGLGSP (SEQ ID NO: 94)、HPSAFSEA (SEQ ID NO: 95)、GPQGIAGQ (SEQ ID NO: 96)、GPQGLLGA (SEQ ID NO: 97)、GPAGLSVL (SEQ ID NO: 98)、GPAGIVTK (SEQ ID NO: 99)、DAASLLGL (SEQ ID NO: 100)、RPAVMTSP (SEQ ID NO: 101)、PPGAYHGA (SEQ ID NO: 102)、LRAYLLPA (SEQ ID NO: 103)、GPYELKAL (SEQ ID NO: 104)、TAAALTSC (SEQ ID NO: 105)、GPEGLRVG (SEQ ID NO: 106)、GHARLVHV (SEQ ID NO: 107)、QPVGINTS (SEQ ID NO: 108)、ELGTYNVI (SEQ ID NO: 109)、DVAQFVLY (SEQ ID NO: 110)、DVANYNFF (SEQ ID NO: 111)、HPVGLLAR (SEQ ID NO: 112)、KPQQFFGL (SEQ ID NO: 113)、IPVSLRSG (SEQ ID NO: 114)、HPVGLLAR (SEQ ID NO: 115)、QPVGINTS (SEQ ID NO:116), RPAVMTSP (SEQ ID NO: 117), GPQGIAGQ (SEQ ID NO: 118), HVLNLRST (SEQ ID NO: 119), DPESIRSE (SEQ ID NO: 120), DPLEFKSH (SEQ ID NO: 121), RPIPITAS (SEQ ID NO: 122), RVLGLKAH (SEQ ID NO: 123), KVLNLTDN (SEQ ID NO: 124), PPEALRGI (SEQ ID NO: 125), IVAMLRAP (SEQ ID NO: 126), TAAAITGA (SEQ ID NO: 127), SGRIGFLRTA (SEQ ID NO: 128), SGAIGFLRTA (SEQ ID NO: 129), SGRAMHMYTA (SEQ ID NO: 130), SGAAMHMYTA (SEQ ID NO: 131), SGRSENIRTA (SEQ ID NO: 10), SGASENIRTA (SEQ ID NO: 132), SGRPENIRTA (SEQ ID NO: 133), SGAPENIRTA (SEQ ID NO: 134), SGARYRWLTA (SEQ ID NO: 135), SGLISHSITA (SEQ ID NO: 136), SGNLRSKLT (SEQ ID NO: 137), VPLSLTMG (SEQ ID NO: 138), GPLSLTMG (SEQ ID NO: 139), GVLSLTMG (SEQ ID NO: 140), GGLSLTMG (SEQ ID NO: 141), and VHLSLTMG (SEQ ID NO: 142).

[0106] According to one aspect, when linking type 1 interferon protein and functional peptide, the adapter length can be any length that a cell (e.g., bacterial cell) can express. According to one aspect, when linking to type 1 interferon protein via adapter, the length of functional peptide can be any length that a cell (e.g., bacterial cell) can express.

[0107] According to one aspect, the adapter sequence is a polypeptide sequence consisting of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48 or more amino acids. In some embodiments, the adapter sequence comprises about 3 amino acids to about 50 amino acids. In some embodiments, the adapter sequence comprises about 3 amino acids to about 40 amino acids. In some embodiments, the adapter sequence comprises...The linker sequence comprises about 5 amino acids to about 30 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 25 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 24 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 23 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 22 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 21 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 20 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 19 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 18 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 17 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 16 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 15 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 14 amino acids. In some embodiments, the adapter sequence comprises about 5 amino acids to about 13 amino acids. In some embodiments, the adapter sequence comprises about 5 amino acids to about 12 amino acids. In some embodiments, the adapter sequence comprises about 5 amino acids to about 11 amino acids. In some embodiments, the adapter sequence comprises about 5 amino acids to about 10 amino acids.

[0108] In some embodiments, the adapter sequence comprises a flexible polypeptide, such as a polypeptide that does not have a rigid secondary and / or tertiary structure. In some embodiments, the adapter sequence comprises glycine and serine residues. In some embodiments, at least 50% of the amino acids contained in the adapter sequence are glycine or serine residues, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more are glycine or serine residues. In some embodiments, the adapter sequence consists of glycine and serine residues.

[0109] In some embodiments, the adapter connecting the type 1 IFN to the masking portion is a cleavable adapter that comprises one or more protease cleavage sites intercalated within the adapter peptide sequence. The more than one protease cleavage site may be the same or different. In some embodiments, the cleavable adapter includes a protease cleavage site. In some embodiments of this disclosure, the adapter includes a single GS repeat, and the protease cleavage site is located within that single GS repeat. In some embodiments of this disclosure, the adapter includes more than one GS repeat, and the protease cleavage site is located after at least one GS repeat. After one or more GS repeats, the single protease cleavage site may be located within a GS repeat sequence, or by replacing the GS repeat.The GS repeats at the initiation and / or termination of the linker may be shortened GS repeats containing one, two, three, or four amino acid residues.

[0110] In some embodiments of this disclosure, the protease is MMP-14, and the cleavage site contains the amino acid sequence of SGQLLGFLTA (SEQ ID NO: 45) and / or SGRSENIRTA (SEQ ID NO: 10). In some embodiments, the sequences of the cleavable linkers provided in this disclosure are provided in Table 3.

[0111] Table 3. Cleavable linkers containing protease cleavage sites:

[0112] In some embodiments, the second linker connecting the type I IFN and the Ig Fc region contains 3-50 amino acids. In some embodiments, the second linker contains 3-30 amino acids. In some embodiments, the second linker contains 3-20 amino acids. In some embodiments, the second linker contains 3-10 amino acids. In some embodiments, the second linker contains 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the second linker comprises three amino acids. In some embodiments, the second linker comprises three glycine residues.

[0113] Immunoglobulin Fc Region

[0114] The masked IFN fusion protein of this disclosure comprises an immunoglobulin Fc region. As used herein, the term "immunoglobulin Fc region" refers to an immunoglobulin fragment lacking the variable regions of the light and heavy chains, constant region 1 (CH1) of the heavy chain, and constant region 1 (CL1) of the light chain, i.e., a fragment consisting of constant regions 2 and 3 (CH2 and CH3) of the heavy chain. Optionally, the immunoglobulin Fc region may also comprise a hinge region. Furthermore, the immunoglobulin Fc region of this disclosure can be an extended Fc region, which, in addition to the constant regions 2 and 3 (CH2 and CH3) of the heavy chain, includes a portion of the constant region 1 (CH1) of the heavy chain and / or the constant region 1 (CL1) of the light chain, or the entirety of the region described on page 13 / 38 of the specification, CN 121568967 A, provided that it exhibits substantially the same or better effects as the classic Fc region. Furthermore, the immunoglobulin Fc region of this disclosure can be composed of CH2 and / or CH3 lacking most of their amino acid sequences.

[0115] Therefore, the immunoglobulin Fc region of this disclosure may include: 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; 5) a combination of one or more constant domains with an immunoglobulin hinge region (or a portion of the hinge region); or 6) a dimer of each constant domain of the heavy chain with a constant region of the light chain. In some embodiments, the immunoglobulin Fc region includes a CH2 structure.The Fc region includes the CH2 domain, CH3 domain, and immunoglobulin hinge region (or partial hinge region). In some embodiments, the immunoglobulin Fc region comprises a dimer of the CH2 and CH3 domains and an immunoglobulin hinge region (or partial hinge region).

[0116] Furthermore, amino acid sequence mutants of wild-type Fc may be included within the scope of the immunoglobulin Fc region of this disclosure. As used herein, the term "amino acid sequence mutant" refers to an amino acid sequence that differs from the wild-type due to the deletion, insertion, conserved or non-conserved substitution, or combination thereof of one or more amino acid residues. For example, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 of IgG Fc, which are known to be important for linkage, may be used as suitable modification sites.

[0117] Various derivatives may be used in this disclosure, such as derivatives prepared by removing disulfide bond sites, removing certain N-terminal amino acids of the natural Fc, or adding methionine to the N-terminus of the natural Fc. In addition, complement binding sites (e.g., C1q binding sites) or ADCC sites can be eliminated to remove the effector function of the native Fc region. Techniques for preparing amino acid sequence mutants of the Fc region of immunoglobulins are disclosed in International Patent Publications WO 97 / 34631 and WO 96 / 32478.

[0118] The above-mentioned Fc derivatives exhibit the same biological activity as the wild type, but with improved structural stability under heat and pH conditions.

[0119] The type I interferon of this disclosure is fused to the Fc region to improve its biological properties, such as increased solubility, prolonged serum half-life, and increased binding affinity (when not bound to the masking portion) to target cells (e.g., cells of target organs) and receptors (e.g., IFNAR1 and IFNAR2) compared to the unfused protein. In some embodiments, the type I IFN is fused to the Ig Fc region directly or through a linker as described above. In some embodiments of this disclosure, the C-terminus of the type I IFN is fused to the N-terminus of the Fc region.

[0120] Compared to masked IFNs with unfused Fc regions, the masked IFN fusion proteins of this disclosure exhibit improved solubility and prolonged serum half-life. Upon cleavage of the masking portion, the fused interferon and Fc region can exhibit enhanced binding affinity to target cells (e.g., cells of target organs) and receptors (e.g., IFNAR1 and IFNAR2) compared to the unfused interferon.

[0121] In some embodiments of this disclosure, the IFN masking portion is fused to the Fc region. The fused IFN masking portion exhibits improved biological properties, such as improved solubility, prolonged serum half-life, and enhanced binding affinity to its ligands (e.g., interferon). In some embodiments, the interferon comprises human type 1 IFN.

[0122] Specific modification of amino acids in the Fc region of human IgG is an active area of ​​research, resulting in structure-function...The relational information relates to the development of therapeutic proteins (particularly monoclonal antibodies) (e.g., see U.S. Patent No. 6,165,745 and PCT Publication No. WO2004 / 035752, which relate to changes in the serum half-life of peptides operatively linked to the Fc region; and U.S. Patent No. 6,737,056 and PCT Publication No. WO2004 / 029207, which relate to changes in the effector function of monoclonal antibodies comprising modified Fc regions).

[0123] Immunoglobulin Fc regions that can be used in this disclosure can be glycosylated to the same, higher, or lower degree as in the native form, or can be deglycosylated or glycosylated. Increased or decreased glycosylation or deglycosylation of immunoglobulin regions can be achieved by typical methods, such as chemical methods, enzymatic methods, or genetic engineering methods. In this document, when deglycosylated, the complement (C1q) binding affinity of the immunoglobulin Fc region is significantly reduced, and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is also reduced or eliminated, thus it does not induce an unwanted immune response in vivo.

[0124] As used herein, the term “deglycosylation” refers to the enzymatic removal of glycans from the Fc region. When used in conjunction with the Fc region, the term “aglycosylation” refers to an Fc region without glycans, expressed by prokaryotes (preferably Escherichia coli).

[0125] The immunoglobulin Fc region used in this disclosure has the amino acid sequence of the human immunoglobulin Fc region or closely related analogues thereof. The Fc region can be obtained from natural forms isolated from animals including cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs. Furthermore, the immunoglobulin Fc region can be derived from the Fc regions of IgG, IgA, IgD, IgE, and IgM, or constituted by combinations or hybrid forms of them. Preferably, it is derived from IgG or IgM, the most abundant proteins in human blood, and most preferably from IgG, which is known to prolong the serum half-life of ligand-binding proteins. In this document, immunoglobulin Fc can be obtained from natural immunoglobulins by isolating intact immunoglobulins from human or animal organisms and treating them with proteolytic enzymes, or it can be a recombinant or its derivative obtained from transformed animal cells or microorganisms. Preferably, it is recombinant human immunoglobulin Fc produced by Escherichia coli transformants.

[0126] Among the four IgG subclasses, IgG1 and IgG3 induce the strongest Fc effector function. However, because IgG1 has the longest half-life and is more stable than IgG3, most therapeutic antibodies with Fc-mediated function are IgG1 isotypes. IgG2 and IgG4 isotypes have significantly lower binding affinity for FcγR. Recent evidence suggests that the IgG2 isotype is not entirely devoid of effector function, while the IgG4 isotype can undergo Fab arm exchange in vivo, leading to bispecific antibodies and off-target effects.

[0127] In some embodiments, the Fc region is the IgG1 Fc region. In some embodiments, the IgG1 Fc region contains an amino acid sequence with a mutant C5A, which prevents CH1 and CL interactions, compared to the wild-type human IgG1 Fc region.

[0128] In some embodiments, the IgG1 Fc region contains the amino acid sequence of SEQ ID NO: 6 or any amino acid sequence having at least 75% (or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with it.

[0129] In some embodiments, the immunoglobulin Fc region forms a dimer containing two identical Fc polypeptides. In some embodiments, the immunoglobulin Fc region forms a dimer containing two Fc polypeptides with different modifications. As used herein, the term "dimer" refers to an associated structure formed by two molecules (such as polypeptides or proteins) through covalent or non-covalent interactions. Homodimers or homodimerizations are formed by two identical molecules, while heterodimers or heterodimerizations are formed by two different molecules.

[0130] Masked IFN Fusion Protein

[0131] The masked IFN fusion protein of this disclosure comprises, from the N-terminus to the C-terminus: a masking portion – a cleavable linker – type I IFN – an immunoglobulin Fc region, as shown in FIG1. ​​Alternatively, the masked IFN fusion protein of this disclosure comprises, from the N-terminus to the C-terminus: a masking portion – a cleavable linker – type I IFN – a second linker – an immunoglobulin Fc region.

[0132] In another embodiment, the masked IFN fusion protein of this disclosure comprises, from the N-terminus to the C-terminus: an immunoglobulin Fc region – a masking portion – a cleavable linker – type I IFN. In another embodiment, the masked IFN fusion protein of this disclosure comprises, from the N-terminus to the C-terminus: an immunoglobulin Fc region – a second linker – a masking portion – a cleavable linker – type I IFN.

[0133] The cleavable linker and the second linker may be the same or different. In some embodiments, the second linker includes a cleavable site.

[0134] In some embodiments, the second linker is non-cleavable. In some embodiments, the second linker comprises three glycine amino acids.

[0135] In some embodiments, no more than 100 amino acid residues are present between the C-terminus of the masking portion and the N-terminus of the type I IFN. In some embodiments, no more than 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acid residues are present between the C-terminus of the masking portion and the N-terminus of the type I IFN. Specification 15 / 38 pages 20 CN 121568967 A

[0136] In one embodiment, two fusion proteins may be associated covalently (e.g., via disulfide bonds, polypeptide bonds, or cross-linking agents) or non-covalently to produce a dimer protein. In a preferred embodiment, the two fusion proteins are covalently associated via at least one, more preferably two, interchain disulfide bonds through cysteine ​​residues, which are preferably located in the immunoglobulin hinge region within the immunoglobulin Fc region of each chain.

[0137] In one aspect, the masked IFN fusion protein of this disclosure may be prepared using genetic engineering techniques generally known in the art, and the resulting fusion protein is preferably synthesized in cells capable of glycosylation of the Fc region at normal glycosylation sites (i.e., typically present in template antibodies). In another aspect, this disclosure provides a method for producing a masked IFN fusion protein comprising a masking portion, a cleavable linker, a type I IFN, an immunoglobulin Fc region, and a second linker optionally inserted between the type I IFN and the immunoglobulin Fc region. The method includes the following steps: (a) providing a mammalian cell containing a DNA molecule encoding the fusion protein (with or without a signal sequence), and (b) culturing the mammalian cell to produce the fusion protein. The resulting fusion protein can then be harvested, refolded if necessary, and purified using conventional purification techniques well-known and used in the art.

[0138] The amino acid sequence of the masked IFN fusion protein may be substituted at one or more residues. In some embodiments, such substitutions are conserved substitutions. A “conserved amino acid substitution” is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin polypeptides are preferably replaced with another amino acid residue from the same side chain family. In another embodiment, an amino acid sequence may be replaced with a structurally similar sequence that differs in the order and / or composition of the side chain family members.

[0139] The table below provides non-limiting examples of conserved amino acid substitutions, where a similarity score of 0 or higher indicates that two amino acids are conservedly substituted.

[0140] Amino acid similarity matrix specification, page 16 / 38, 21 CN 121568967 A Conserved amino acid substitution

[0141] Those skilled in the art will also understand that the masked IFN fusion protein disclosed herein can be modified to differ in amino acid sequence from the naturally occurring binding polypeptide from which it originates. For example, the amino acid sequence of a polypeptide or amino acid derived from a specified protein may be similar to the starting sequence, for example, it may have a certain percentage of similarity to the starting sequence, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

[0142] In some embodiments, the masked IFN fusion protein comprises an amino acid sequence or one or more portions thereof. For example, the masked IFN fusion protein of this disclosure may include additional flexible linker sequences or may be modified to add additional functional portions (e.g., PEG, drugs, toxins, or markers). In some embodiments, the masked IFN fusion protein or the IFN masking portion may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0143] The masked IFN fusion protein or IFN masking moiety, its variants or derivatives, of this disclosure include modified derivatives, i.e., by covalently linking any type of molecule to a polypeptide, and such covalent linking does not impede the function of the polypeptide. For example, but not as a limitation, the masked IFN fusion protein or IFN masking moiety can be modified, for example by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Any of the many chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the masked IFN fusion protein or IFN masking moiety may contain one or more non-classical amino acids.

[0144] The masked IFN fusion protein or IFN masking motif of this disclosure can be conjugated or fused to a therapeutic agent, which may include detectable markers (such as radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutics or diagnostics, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radiolabels, combinations thereof, and other such substances known in the art.

[0145] Polynucleotides encoding polypeptides and methods for preparing polypeptides

[0146] This disclosure also provides isolated polynucleotide or nucleic acid molecules encoding polypeptides of this disclosure (such as the masked IFN fusion protein or IFN masking motif of this disclosure), variants thereof, or derivatives thereof.

[0147] The DNA encoding the polypeptide can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding the polypeptide). The encoding DNA can also be obtained by synthetic methods.

[0148] This disclosure provides vectors (e.g., expression vectors) containing isolated polynucleotides provided herein. Vectors can be used to transform, transduce, or transfect host cells, thereby expressing genetic elements carried therein within the host cells. Vectors may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication. Vectors may also contain materials that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coatings. Vectors may be expression vectors or cloning vectors.

[0149] In some embodiments, the expression vectors provided herein contain a polynucleotide encoding a polypeptide provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operatively linked to the polynucleotide sequence, and at least one selection marker.

[0150] Vectors containing a polynucleotide sequence encoding a masked IFN fusion protein or an IFN masking portion can be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotic cells, yeast cells, or higher eukaryotic cells as described above. Examples of available mammalian host cell lines are CHO, BHK, NSO, 293, and their derivatives.

[0151] Host cells are transformed with the above-described expression or cloning vectors to produce polypeptides and cultured in conventional nutrient media appropriately modified for inducing promoters, screening transformants, or amplifying genes encoding desired sequences. In another embodiment, the polypeptides can be produced by homologous recombination methods known in the art.

[0152] Host cells used to produce the polypeptides provided herein can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), RPMI-1640 (Sigma), and Duchenne Modified Eagle Medium (DMEM, Sigma), are suitable for culturing host cells after the addition of necessary hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements (defined as inorganic compounds typically present in micromolar final concentrations), and glucose or equivalent energy sources. Any other necessary supplements may also be added at appropriate concentrations known to those skilled in the art. Culture conditions (such as temperature, pH, etc.) are consistent with those previously used for the selected host cells used for expression, and will be apparent to those skilled in the art.

[0153] When using recombinant technology, the peptide can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the peptide is produced intracellularly, the first step is to remove particulate debris (whether from host cells or lysed fragments), for example by centrifugation or ultrafiltration. The peptide prepared from the cell can then be purified using any suitable purification technique known in the art.

[0154] Treatment and Diagnostic Methods

[0155] As described herein, the masked IFN fusion protein or IFN masking portion, variant, or derivative of this disclosure can be used in certain therapeutic and diagnostic methods.

[0156] This disclosure also relates to peptide-based therapies involving administration of the masked IFN fusion protein of this disclosure to a patient (such as an animal, mammal, or human) to treat one or more of the diseases or conditions described herein. Therapeutic compounds of this disclosure include, but are not limited to, the masked IFN fusion protein of this disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the masked IFN fusion protein of this disclosure (including variants and derivatives thereof as described herein).

[0157] This disclosure also relates to peptide-based therapies involving administration of the IFN masking portion of this disclosure to a patient (such as an animal, mammal, or human) to treat one or more of the diseases or conditions described herein. Therapeutic compounds of this disclosure include, but are not limited to, the IFN masking portion of this disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the IFN masking portion of this disclosure (including variants and derivatives thereof as described herein).

[0158] In some embodiments, a method of treating cancer in a patient in need is provided. In one embodiment, the method includes administering an effective amount of the masked IFN fusion protein of this disclosure to the patient.

[0159] In some embodiments, the use of the masked IFN fusion protein of this disclosure in the preparation of a medicament for treating cancer in a patient in need is provided.

[0160] In some embodiments, the masked IFN fusion protein of this disclosure is provided for treating cancer in a patient in need.

[0161] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0162] Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed, and / or prognosed by the polypeptides, variants, or derivatives thereof disclosed herein, including but not limited to the progression and / or metastasis of malignant tumors and related conditions such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including medulloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, etc. Endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcomaTumors, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymal carcinoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. (Instruction manual 19 / 38 pages 24 CN 121568967 A)

[0163] The specific dosage and treatment regimen for any particular patient depends on a variety of factors, including the specific peptide used, its variants or derivatives, the patient's age, weight, general health condition, sex and diet, as well as the timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of these factors by healthcare professionals is a matter of routine skill in the art. Dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by principles of pharmacology and pharmacokinetics well known in the art.

[0164] Methods of administration of the masked IFN fusion protein or IFN masking portion of this disclosure include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding peptide or composition can be administered via any convenient route, such as by infusion or bolus injection, absorption through an epithelial or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be co-administered with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides of this disclosure can be administered orally, rectally, parenterally, intracisionally, vaginally, intraperitoneally, topically (e.g., via powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0165] As used herein, the term “parenterical” refers to administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0166] Administration can be systemic or local. Furthermore, it may be necessary to introduce the peptides or compositions of this disclosure into the central nervous system via any suitable route, including intraventricular and intrathecal injections; intraventricular injections may be assisted by intraventricular catheters (e.g., connected to a reservoir, such as the Ommaya reservoir). Lung administration may also be used, for example, by using an inhaler or nebulizer and formulation with an aerosol.

[0167] It may be necessary to locally apply the masked IFN fusion protein, IFN masking portion, or composition of this disclosure to areas requiring treatment.The site of treatment; this can be achieved (e.g., but not limited to) local infusion during surgery, topical application (e.g., postoperative use in conjunction with wound dressings), injection, via catheter, via suppository, or via implantation, said implantation being a porous, non-porous, or gel-like material, including membranes (e.g., silicone rubber membranes) or fibers. Preferably, when administering the proteins of this disclosure (including masked IFN fusion proteins or IFN masking portions), care must be taken to use materials that do not adsorb to the protein.

[0168] In some embodiments, a method is also provided for in vivo / ex vivo detection of the expression of human type 1 IFN target cells (e.g., cells of target organs) and receptors (e.g., IFNAR1 and IFNAR2) in a subject, said method comprising administering the IFN masking portion of this disclosure to a subject, or contacting an ex vivo sample with the IFN masking portion of this disclosure, and detecting the binding of the expression of type 1 IFN target cells or their receptors in the sample.

[0169] In some embodiments, a method for detecting the expression of human type 1 IFN protein in a sample is also provided, the method comprising contacting the sample with the IFN masking portion of the present disclosure and detecting binding indicating type 1 IFN expression in the sample.

[0170] In some embodiments, the use of the IFN masking portion of the present disclosure in the preparation of a kit for detecting the expression of human type 1 IFN protein in a sample is provided.

[0171] Compositions

[0172] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of a masked IFN fusion protein or an IFN masking portion and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0173] As used herein, the term “pharmaceuticalally effective amount” means an amount of a pharmaceutical composition that is sufficient to treat a disease given a reasonable benefit / risk ratio applicable to any medical treatment. Effective dosage can vary depending on a variety of factors, including the severity and type of the disease being treated, the patient's age and sex, drug activity, drug sensitivity, timing of administration, route of administration, excretion rate, duration of treatment, co-administration with other drugs, and other parameters well known in the pharmaceutical and medical fields.

[0174] In one specific embodiment, the term "pharmaceuticalally acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for animal (especially human) use. Furthermore, a "pharmaceuticalally acceptable carrier" is typically a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of pharmaceutical excipient.

[0175] The term "carrier" refers to a diluent, adjuvant, excipient, or medium administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils (including oils of petroleum, animal, plant, or synthetic origin, such as flower oils).Oils such as vegetable oil, soybean oil, mineral oil, and sesame oil are suitable carriers. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous solutions of dextran and glycerol, can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers such as acetates, citrates, or phosphates. It is also envisioned to include antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and osmolarity regulators such as sodium chloride or dextran. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into a suppository using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in EW. Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide (preferably in a purified form) and a suitable amount of carrier to provide a form suitable for administration to a patient. The formulation should be suitable for the manner of administration. Parenteral formulations may be packaged in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0176] In one embodiment, the composition is formulated according to conventional methods to be a pharmaceutical composition suitable for intravenous administration to a human. Typically, compositions for intravenous administration are solutions in sterile isotonic buffer solutions. If necessary, the composition may also include a solubilizer and a local anesthetic (such as lidocaine) to reduce pain at the injection site. Typically, the components are supplied separately or mixed together in unit dosage forms, for example, as dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet), with the amount of active substance indicated. In the case of administration of the composition by infusion, the composition may be provided in an infusion bottle containing sterile pharmaceutical-grade water or saline. In the case of administration of the composition by injection, ampoules of sterile water or saline for injection may be provided so that the components can be mixed prior to administration.

[0177] The compounds of this disclosure may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions (such as anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.) and salts formed with cations (such as cations derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0178] Examples

[0179] Example 1.AI-Guided Design of Masking Parts Based on IFNAR2 Domain 1

[0180] This embodiment illustrates the design, preparation, and testing of masking parts based on IFNAR2 domain 1.

[0181] Three masking parts were tested, including (1) a wild-type IFNAR2-D1 core sequence (“M-09”); (2) an IFNAR2-D1 core sequence corresponding to the IFNAR2-D1 peptide (SEQ ID NO: 3) with disulfide bonds introduced at R20C and E109C (“M-10”); and (3) a wild-type IFNAR2-D1 core sequence with an extended domain 2 loop at the C-terminus (“M-11”), each of which was fused to human type 1 IFNα2β-Fc via a cleavable linker (SEQ ID NO: 9). The Fc fragment used included a C5A mutation in the hinge region.

[0182] The corresponding fusion proteins are named I-09 (M-09 + adapter + IFNα2β-Fc), I-10 (M-10 + adapter + IFNα2β-Fc), I-11 (M-11 + adapter + IFNα2β-Fc) and I-31 (M-10 + extended adapter + IFNα2β-Fc). The sequences of these fusion proteins and the various parts of the fusion proteins are shown in Table 4-1 below.

[0183] Table 4-1. Sequences of the various parts of the masked IFN fusion proteins Specification 22 / 38 pages 27 CN 121568967 A Specification 23 / 38 pages 28 CN 121568967 A

[0184] For IFNAR2 D1 as the masking part, the IFN-Fc fusion protein I-09 with the D1 core sequence masked was evaluated. In the IFNAR2 binding assay, I-09 showed a significantly lower affinity for human IFNAR2 compared to the unmasked IFNα2β-Fc protein (or simply "IFN-Fc", I-01) (EC50 was approximately 4.4 times that of IFN-Fc, see Figure 2A). Treatment of Daudi cells with human interferon for 3 days gradually inhibited cell proliferation and induced apoptosis in Daudi cells. In the Daudi proliferation inhibition assay, the in vitro activity of I-09 was approximately 400 times lower than that of IFN-Fc, but its activity was significantly restored after the masking portion was removed (see Figure 2B).

[0185] The masking efficiency of the other three fusion proteins, I-10, I-11, and I-31, was also tested. As shown in Figures 3A and 4A, similar to I-09, I-10, I-11, and I-31, in their fusion forms, also exhibited significantly reduced binding affinity to IFNAR2, and their activity was greatly weakened in the Daudi anti-proliferation experiment. However, their activity was restored once the masked portion was removed.

[0186] De novo design

[0187] The novel masking sequence was designed using an AI sequence design tool based on the IFNAR2-D1 backbone region, while retaining the residues of the IFN binding interface. Wild-type human IFNAR2 domain 1 (D1) expression levels are very low because domain 1 and domain 2 are originally co-expressed and interact closely through a hydrophobic interface. Removing domain 2 exposes the hydrophobic surface of domain 1, leading to instability issues.

[0188] To stabilize domain 1 and maintain its masking function, the IFN binding loop of IFNAR2 domain 1 was retained, and the domain 1 sequence, excluding the IFN binding loop, was redesigned using an AI sequence design method. In this method, a neural network is used to process a large amount of available protein structure data and identify sequence-structure patterns. A novel domain 1 sequence with desired properties was generated using the trained model. The sequence identity between the AI-designed sequences and wild-type domain 1 was approximately 55-60% (including the fixed IFN binding loop). Of the 50 designed sequences, 12 sequences M-40, M-41, M-55, M-60, M-62, M-65, M-66, M-67, M-82, M-83, M-87, and M-89 were expressed at high levels.

[0189] All obtained masked portions were fused to human type 1 IFNα2β-Fc via extended cleavable adapters (SEQ ID NO: 13). The sequences of the various parts of the fusion protein are shown in Table 4-2 below.

[0190] Table 4-2. Sequences of various parts of the masked IFN fusion protein (Pages 25 / 38, CN 121568967 A, Pages 26 / 38, CN 121568967 A, Pages 27 / 38, CN 121568967 A, Pages 28 / 38, CN 121568967 A)

[0191] All de novo redesigned masked IFN-Fc fusion proteins, such as I-09, I-10, I-11, and I-31, showed reduced binding to human IFNAR2 (see Figures 3A-C). Except for I-87, the other 11 de novo redesigned sequences showed extremely low binding signals at concentrations up to 1000 nM. In the Daudi proliferation inhibition assay, the activity of all the above-mentioned masked IFN-Fc fusion proteins was significantly reduced. After incubation with MMP-14 to cleave the linker and remove the masking portion, the activity of most molecules was fully or nearly fully restored (see Figures 4A-C).

[0192] Site Mutation Design

[0193] In this experiment, the N-terminal extended (11 amino acids) IFNAR2-D1 sequence was used as the starting sequence (SEQ ID NO: 3). To improve expression levels and purity, a disulfide bond was introduced into this sequence (SEQ ID NO: 42, see Figure 5A).

[0194] After the N-terminal 11 amino acid extension and the introduction of the disulfide bond, the protein expression levels and purity of I-140 were much higher than those of I-09 (see Figure 5B). I-140 showed no binding signal to hIFNAR2 at concentrations up to 1000 nM (see Figure 6A), and its in vitro activity in inhibiting Daudi proliferation was about 7500 times lower than that of IFN-Fc. However, the activity was greatly restored after the masking portion was removed (see Figures 6A and 6B).

[0195] To improve the protein expression profile and enhance the masking efficiency, the masking portion sequence was further mutated. The DDG values ​​of 20 amino acids were calculated using the AI-trained DDG prediction tool neoFormer / neoDDG / neoPPI, and the surface region sequence of the masked portion and the IFN / IFNAR2-D1 interaction interface were optimized. Fourteen sequences were obtained: M-148, M-150, M-151, M-152, M-153, M-155, M-156, M-157, M-158, M-159, M-160, M-161, M-162, and M-163.

[0196] All obtained masked portions were fused with human type 1 IFNα2β-Fc via cleavable linkers. The sequences of each part of the fusion protein are shown in Table 4-3 below.

[0197] Table 4-3. Sequences of various parts of the masked IFN fusion protein (Pages 30 / 38, CN 121568967 A, Pages 31 / 38, CN 121568967 A, Pages 32 / 38, CN 121568967 A, Pages 33 / 38, CN 121568967 A, Pages 34 / 38, CN 121568967 A, Pages 35 / 38, CN 121568967 A, Pages 40 / 36 / 38, CN 121568967 A, Pages 41 / 37 / 38, CN 121568967 A)

[0198] All fourteen I-140-derived site mutations were used to design masked IFN-Fc fusion proteins at concentrations up to 1000... Both at nM showed extremely low binding affinity to human IFNAR2 (see Figures 7A-C), and their activity in inhibiting Daudi proliferation was significantly reduced compared to IFN-Fc. However, after removing the masking portion (by incubating with MMP-14 to cleave the masking portion),The activity of most molecules was fully restored (see Figures 8A-C).

[0199]

[0200] The scope of this disclosure is not limited to the specific embodiments described, which are merely individual examples of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations of this disclosure, provided they fall within the scope of the appended claims and their equivalents.

[0201] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated as incorporated by reference. Instruction manual page 38 / 38, page 43, CN 121568967 A, Figure 1; Instruction manual figure 1 / 10 page, page 44, CN 121568967 A, Figure 2; Instruction manual figure 2 / 10 page, page 45, CN 121568967 A, Figure 3; Instruction manual figure 3 / 10 page, page 46, CN 121568967 A, Figure 4; Instruction manual figure 4 / 10 page, page 47, CN 121568967 A, Figure 4 (continued); Instruction manual figure 5 / 10 page, page 48, CN 121568967 A, Figure 5; Instruction manual figure 6 / 10 page, page 49, CN 121568967 A, Figure 6; Instruction manual figure 7 / 10 page, page 50, CN 121568967 A, Figure 7; Instruction manual figure 8 / 10 page, page 51, CN 121568967 A, Figure 8; Instruction manual figure 9 / 10 page, page 52, CN 121568967 A Figure 8 (Continued from the instruction manual, page 10 / 10, 53 CN 121568967 A)

Claims

1. A fusion protein comprising (a) interferon α and β receptor subunit 2 domain 1 (IFNAR2-D1) or a masking peptide having at least 55% sequence identity with IFNAR2-D1, (b) type I interferon (IFN), and (c) a cleavable linker between (a) and (b), wherein the peptide is capable of binding the type I IFN, and wherein the fusion protein does not contain the complete sequence of IFNAR2 domain 2 (IFNAR2-D2).

2. The fusion protein of claim 1, wherein the cleavable connector is fused to the C-terminus of (a) and the N-terminus of (b), or the cleavable connector is fused to the N-terminus of (a) and the C-terminus of (b).

3. The fusion protein according to claim 1 or 2, further comprising an immunoglobulin (Ig) Fc region, said immunoglobulin (Ig) Fc region being fused to the C-terminus of said IFN or the N-terminus of (a) IFNAR2-D1 or masking peptide.

4. The fusion protein according to any one of the preceding claims, wherein it does not contain at least 50% or at least 80% IFNAR2-D2.

5. The fusion protein according to any one of the preceding claims, wherein the IFNAR2-D1 comprises the amino acid sequence of SEQ ID NO:8 or 3.

6. The fusion protein according to any one of the preceding claims, wherein the masking peptide comprises IFN-binding loop 1, IFN-binding loop 2 and IFN-binding loop 3, wherein IFN-binding loop 1 comprises the amino acid sequence YTIMSKPEDLK (SEQ ID NO: 74), wherein IFN-binding loop 2 comprises the amino acid sequence STHEAYVTVL (SEQ ID NO: 75), STQEIYVTVL (SEQ ID NO: 77) or STDEAYVTVL (SEQ ID NO: 78), and wherein IFN-binding loop 3 comprises the amino acid sequence SHNFWLAID (SEQ ID NO: 76) or SHEFWLAID (SEQ ID NO: 79).

7. The fusion protein of claim 6, wherein the masking peptide has at least 85% sequence identity with any one of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

8. The fusion protein according to any one of the preceding claims, wherein the masking peptide comprises an amino acid sequence selected from SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

9. The fusion protein according to any one of claims 1-6, wherein the masking peptide has at least 85% sequence identity with SEQ ID NO: 8 or 3.

10. The fusion protein of claim 9, wherein the masking peptide comprises at least one disulfide bond.

11. The fusion protein of claim 10, wherein the masking peptide comprises R9C and E98C substitutions relative to SEQ ID NO:

8.

12. The fusion protein of claim 10, wherein the masking peptide comprises the amino acid sequence of SEQ ID NO: 12 or 42.

13. The fusion protein of claim 10, wherein the masking peptide comprises the amino acid sequence of SEQ ID NO: 42 and has one or more substitutions selected from: (1) L19: S, Q, E or R; (2) F22: G, N, or D; (3) I46R; (4) M47: I or F; (5) S48: N, Y, or Q; (6) K49Q; (7) H77: Y, Q, or D; (8) A79: D, I, V, E or N; (9) V81: I or F; (10) V83: F, I, or K; (11) N99: E, D or W; (12) W101: M or F; (13) L102: M or I; (14) I104: F or W; (15) M106: A, T, L or E; (16) F108: N, Y, T or E; (17) P110: S or E; (18) P111: S or E; and (19) The above-mentioned substitution combinations.

14. The fusion protein of claim 13, wherein the masking peptide comprises an amino acid sequence selected from SEQ ID NO: 42, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 or 73.

15. The fusion protein according to any one of the preceding claims, wherein the fusion protein does not bind IFNAR2.

16. The fusion protein according to any one of the preceding claims, wherein the cleavable adapter contains a protease cleavage site.

17. The fusion protein of claim 11, wherein the protease is selected from thrombin, neutrophil elastase, cysteine ​​protease, FAPa, cathepsin B, asparagine endopeptidase, serine proteases such as membrane-type serine protease 1 or urokinase (uPA), and matrix metalloproteinases (MMPs) such as MMP1, MMP2, MMP3, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14 and MMP17.

18. The fusion protein of claim 16, wherein the protease cleavage site comprises an amino acid sequence selected from SEQ ID NO:45 and 10.

19. The fusion protein of claim 11, wherein the cleavable linker comprises an amino acid sequence selected from SEQ ID NO: 9, 13 and 44.

20. The fusion protein of claim 1, wherein the type 1 IFN is selected from IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17 and IFN-α21, IFN-β, IFN-κ, IFN-ε or IFN-ω and any amino acid sequence having at least 75% sequence identity with it.

21. The fusion protein of claim 20, wherein the IFN comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 75% sequence identity with SEQ ID NO:

4.

22. The fusion protein according to any one of claims 3-21, wherein the Fc is an IgG1, IgG2, IgG3 or IgG4 isotype.

23. The fusion protein of claim 22, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:

6.

24. The fusion protein according to any one of the preceding claims, which forms a dimer.

25. The fusion protein according to claim 1, comprising an amino acid sequence selected from SEQ ID NO: 41, 43, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39.

26. A composition comprising the fusion protein according to any one of claims 1-25 and a pharmaceutically acceptable carrier.

27. An isolated cell comprising one or more polynucleotides, said polynucleotides encoding a fusion protein according to any one of claims 1-25.

28. A polynucleotide encoding a fusion protein according to any one of claims 1-25.

29. A method of treating cancer in a patient in need, comprising administering to the patient a fusion protein according to any one of claims 1-25, a composition according to claim 26, a cell according to claim 27, or a polynucleotide according to claim 28.

30. The method of claim 29, wherein the cancer is selected from bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

31. The method of claim 30, wherein the cleavable linker of the fusion protein is cleaved by an enzyme within the patient's tumor microenvironment.

32. A method of treating an autoimmune disease or inflammatory condition in a patient in need, comprising administering to the patient a fusion protein according to any one of claims 1-25, a composition according to claim 26, a cell according to claim 27, or a polynucleotide according to claim 28.

33. A polypeptide comprising interferon α and β receptor subunit 2 domain 1 (IFNAR2-D1) or a peptide having at least 55% sequence identity with IFNAR2-D1, wherein the polypeptide is capable of binding type 1 IFN, and wherein the polypeptide does not contain the complete sequence of IFNAR2 domain 2 (IFNAR2-D2).

34. The polypeptide of claim 33, wherein it does not contain at least 50% IFNAR2-D2, or does not contain at least 80% IFNAR2-D2.

35. The polypeptide according to claim 33 or 34, wherein the IFNAR2-D1 comprises the amino acid sequence of SEQ ID NO: 8 or 3.

36. The polypeptide according to any one of claims 33-35, comprising an IFN-binding ring 1, an IFN-binding ring 2, and an IFN-binding ring 3, wherein the IFN-binding ring 1 comprises the amino acid sequence YTIMSKPEDLK (SEQ ID NO: 74), the IFN-binding ring 2 comprises the amino acid sequence STHEAYVTVL (SEQ ID NO: 75), STQEIYVTVL (SEQ ID NO: 77), or STDEAYVTVL (SEQ ID NO: 78), and the IFN-binding ring 3 comprises the amino acid sequence SHNFWLAID (SEQ ID NO: 76) or SHEFWLAID (SEQ ID NO: 79).

37. The polypeptide of claim 36, having at least 85% sequence identity with any one of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

38. The polypeptide according to any one of claims 33-37, comprising an amino acid sequence selected from SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

39. The polypeptide according to any one of claims 33-35, having at least 85% sequence identity with SEQ ID NO: 8 or 3.

40. The polypeptide of claim 33, wherein it comprises at least one disulfide bond.

41. The polypeptide of claim 40, wherein it comprises R9C and E98C substitutions relative to SEQ ID NO:

8.

42. The polypeptide of claim 40, comprising the amino acid sequence of SEQ ID NO: 12 or 42.

43. The polypeptide of claim 40, comprising the amino acid sequence of SEQ ID NO: 42, and having one or more substitutions selected from: (1) L19: S, Q, E or R; (2) F22: G, N, or D; (3) I46R; (4) M47: I or F; (5) S48: N, Y, or Q; (6) K49Q; (7) H77: Y, Q, or D; (8) A79: D, I, V, E or N; (9) V81: I or F; (10) V83: F, I, or K; (11) N99: E, D or W; (12) W101: M or F; (13) L102: M or I; (14) I104: F or W; (15) M106: A, T, L or E; (16) F108: N, Y, T or E; (17) P110: S or E; (18) P111: S or E; and (19) The above-mentioned substitution combinations.

44. The polypeptide of claim 43, comprising an amino acid sequence selected from SEQ ID NO: 42, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 or 73.

45. A polynucleotide encoding a polypeptide according to any one of claims 33-44.

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