阅读说明：本技术 白介素12融合蛋白及其组合物和治疗方法 (Interleukin 12 fusion proteins and compositions and methods of treatment thereof ) 是由 傅阳心 彭华 薛娣媛 于 2019-04-24 设计创作，主要内容包括：本发明提供了可用于治疗各种不同疾病和障碍(例如增生、实体肿瘤或造血系统恶性肿瘤)的新的白介素12的融合蛋白及其前体药物、组合物和制备方法。(The present invention provides novel fusion proteins of interleukin 12 and prodrugs, compositions and methods of preparation thereof, which are useful for treating a variety of different diseases and disorders, such as hyperplasia, solid tumors or hematopoietic malignancies.)

1. A fusion protein comprising:

a first structural unit: one or two subunits of interleukin 12(IL12) selected from the group consisting of P35 and P40 subunits, wherein the first building block is located at the N-terminus of the fusion protein;

a second structural unit: an antibody Fc fragment, wherein the second building block is located at the C-terminus of the fusion protein; and

a first linker segment covalently linking the first and second building units or covalently linking the two subunits of the first building unit.

2. The fusion protein of claim 1, wherein the P35 and P40 subunits are derived from a mammal selected from the group consisting of a human, monkey, mouse, dog, rat, cow, pig, and sheep.

3. The fusion protein of claim 2, wherein the mammal is a mouse.

4. The fusion protein of claim 2, wherein the mammal is a human.

5. The fusion protein of any one of claims 1-4, wherein the antibody Fc fragment comprises a human Fc fragment.

6. The fusion protein of any one of claims 1-5, wherein the antibody Fc fragment comprises human IgG 1.

7. The fusion protein of any one of claims 1-6, wherein the human IgG1 is a human Fc-knob or a human Fc-hole.

8. The fusion protein of any one of claims 1-3 and 5-7, wherein the P35 subunit has the amino acid sequence set forth in SEQ ID No. 3.

9. The fusion protein of any one of claims 1, 2, and 4-7, wherein the P35 subunit has the amino acid sequence set forth in SEQ ID No. 4.

10. The fusion protein of any one of claims 1-3 and 5-8, wherein the P40 subunit has the amino acid sequence set forth in SEQ ID No. 1.

11. The fusion protein of any one of claims 1, 2, and 4-9, wherein the P40 subunit has the amino acid sequence set forth in SEQ ID No. 2.

12. The fusion protein of any one of claims 1-11, wherein the first linker segment (L1) has the amino acid sequence set forth in SEQ ID No. 12.

13. The fusion protein of any one of claims 1-12, further comprising a signal peptide modified at the N-terminus of the first building block.

14. The fusion protein of claim 13, wherein the signal peptide modified at the N-terminus of the P35 subunit is the first signal peptide comprising the amino acid sequence set forth in SEQ ID No.27 (SP 1).

15. The fusion protein of claim 13, wherein the signal peptide modified at the N-terminus of the P35 subunit is the first signal peptide comprising the amino acid sequence set forth in SEQ ID No.28 (SP 1).

16. The fusion protein of claim 13, wherein the signal peptide modified at the N-terminus of the P40 subunit is a second signal peptide comprising the amino acid sequence set forth in SEQ ID No.29 (SP 2).

17. The fusion protein of claim 13, wherein the signal peptide modified at the N-terminus of the P40 subunit is a second signal peptide comprising the amino acid sequence set forth in SEQ ID No.30 (SP 2).

18. The fusion protein of any one of claims 1-17, further comprising:

a subsequence of interleukin 12 receptor (IL12R) linked to the N-terminus of the first building block; and

covalently linking IL12R to the second linker segment of the first building block (L2).

19. The fusion protein of claim 18, wherein the IL12R is selected from IL12R β 1(R β 1) and IL12R β 2(R β 2).

20. The fusion protein of claim 18 or 19, wherein the second linker segment L2 is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

21. The fusion protein of claim 19 or 20, wherein R β 1 comprises the amino acid sequence set forth in SEQ ID No.8 and R β 2 comprises the amino acid sequence set forth in SEQ ID No. 10.

22. The fusion protein of claim 19 or 20, wherein R β 1 comprises the amino acid sequence set forth in SEQ ID No.9 and R β 2 comprises the amino acid sequence set forth in SEQ ID No. 11.

23. The fusion protein of any one of claims 20-22, wherein the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase.

24. The fusion protein of claim 23, wherein the matrix metalloproteinase is matrix metalloproteinase 14(MMP 14).

25. The fusion protein of any one of claims 18-24, wherein the linker segment L2 comprises the amino acid sequence set forth in SEQ ID nos. 13-26.

26. The fusion protein of any one of claims 18-25, wherein

The C-terminus of the IL12R is linked to the N-terminus of the first building block by the linker segment L2; and is

The C-terminus of the first building block and the N-terminus of the second building block are connected by the linker segment L1;

wherein the first building block comprises two subunits, the C-terminus of the first subunit and the N-terminus of the second subunit are connected by linker segment L1.

27. A homodimeric or heterodimeric protein comprising the fusion protein of any one of claims 1-26.

28. The homodimeric or heterodimeric protein of claim 27 which is a homodimer of monomers that are fusion proteins comprising the P40 subunit, linker segment L1, P35 subunit, linker segment L1, human IgG1 and have the amino acid sequence set forth in SEQ ID No. 31.

29. The homodimeric or heterodimeric protein of claim 27 which is a homodimer of monomers that are fusion proteins comprising the P40 subunit, linker segment L1, P35 subunit, linker segment L1, human IgG1 and have the amino acid sequence set forth in SEQ ID No. 32.

30. The homodimeric or heterodimeric protein of claim 27 which is a heterodimer of the following monomers:

a first monomer: a fusion protein comprising a P40 subunit, a linker segment L1, and a human Fc-knob and having the amino acid sequence set forth in SEQ ID No. 18; and

a second monomer: a fusion protein comprising the P35 subunit containing linker segment L1 and the human Fc-socket and having the amino acid sequence structure set forth in SEQ ID No. 35.

31. The homodimeric or heterodimeric protein of claim 27 which is a heterodimer of the following monomers:

a first monomer: a fusion protein comprising a P40 subunit, a linker segment L1, and a human Fc-knob and having the amino acid sequence set forth in SEQ ID No. 18; and

a second monomer: a fusion protein comprising the P35 subunit containing linker segment L1 and the human Fc-socket and having the amino acid sequence structure set forth in SEQ ID No. 36.

32. The homodimeric or heterodimeric protein of claim 27 which is a homodimer of the following monomers: IL12R β 1, linker segment L2, P40 subunit, linker segment L1, P35 subunit, linker segment L1 and human IgG1, and having the amino acid sequence set forth in SEQ ID No. 37.

33. The homodimeric or heterodimeric protein of claim 27 which is a homodimer of the following monomers: IL12R β 1, linker segment L2, P40 subunit, linker segment L1, P35 subunit, linker segment L1 and human IgG1, and having the amino acid sequence set forth in SEQ ID No. 38.

34. The homodimeric or heterodimeric protein of claim 27 which is a homodimer of the following monomers: IL12R β 2, linker segment L2, P40 subunit, linker segment L1, P35 subunit, linker segment L1 and human IgG1, and having the amino acid sequence set forth in SEQ ID No. 39.

35. The homodimeric or heterodimeric protein of claim 27 which is a homodimer of the following monomers: IL12R β 2, linker segment L2, P40 subunit, linker segment L1, P35 subunit, linker segment L1 and human IgG1, and having the amino acid sequence set forth in SEQ ID No. 40.

36. The homodimeric or heterodimeric protein of claim 27, which is a heterodimer consisting of the following monomers:

a first monomer: a fusion protein of R β 1, linker segment L2, P40 subunit, linker segment L1, human Fc-knob, and having the amino acid sequence set forth in SEQ ID No. 41; and

a second monomer: a fusion protein of R β 2, linker segment L2, P35 subunit, linker segment L1 and the Fc-mortar of human IL12 and having the amino acid sequence set forth in SEQ ID No. 43.

37. The homodimeric or heterodimeric protein of claim 27 which is a heterodimer of the following monomers:

a first monomer: a fusion protein of R β 1, linker segment L2, P40 subunit, linker segment L1, human Fc-knob, and having the amino acid sequence set forth in SEQ ID No. 42; and

a second monomer: a fusion protein of R β 2, linker segment L2, P35 subunit, linker segment L1 and the Fc-mortar of human IL12 and having the amino acid sequence set forth in SEQ ID No. 44.

38. The homodimeric or heterodimeric protein of claim 27 which is a heterodimer of the following monomers:

a first monomer: a fusion protein of R β 1, linker segment L2, P40 subunit, linker segment L1, human Fc-knob, and having the amino acid sequence set forth in SEQ ID No. 45; and

a second monomer: comprising a fusion protein comprising the P35 subunit of signal peptide SP1, linker segment L1 and the human Fc-mortar, and having the amino acid sequence set forth in SEQ ID No. 47.

39. The homodimeric or heterodimeric protein of claim 27 which is a heterodimer of the following monomers:

a first monomer: a fusion protein of R β 1, linker segment L2, P40 subunit, linker segment L1, human Fc-knob, and having the amino acid sequence set forth in SEQ ID No. 46; and

a second monomer: comprising a fusion protein comprising the P35 subunit of signal peptide SP1, linker segment L1 and the human Fc-mortar, and having the amino acid sequence set forth in SEQ ID No. 48.

40. The homodimeric or heterodimeric protein of claim 27 which is a heterodimer of the following monomers:

a first monomer: a fusion protein comprising a P40 subunit, a linker segment L1, and a human Fc-knob, and having the amino acid sequence set forth in SEQ ID No. 49; and

a second monomer: a fusion protein comprising R β 2, linker segment L2, P35 subunit, linker segment L1 and the human Fc-socket, and having the amino acid sequence set forth in SEQ ID No. 51.

41. The homodimeric or heterodimeric protein of claim 27 which is a heterodimer of the following monomers:

a first monomer: a fusion protein comprising a P40 subunit, a linker segment L1, and a human Fc-knob, and having the amino acid sequence set forth in SEQ ID No. 50; and

a second monomer: a fusion protein comprising R β 2, linker segment L2, P35 subunit, linker segment L1 and the human Fc-socket, and having the amino acid sequence set forth in SEQ ID No. 52.

42. The homodimeric or heterodimeric protein of any of claims 27-41 that is hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

43. A substantially purified protein of any one of claims 1-42.

44. A polynucleotide encoding the protein of any one of claims 1-43.

45. An expression vector comprising the polynucleotide of claim 44.

46. A pharmaceutical composition comprising a protein according to any one of claims 1-43 and a pharmaceutically acceptable excipient, carrier or diluent.

47. A method of treating a disease or disorder, the method comprising:

administering to a patient in need thereof a therapeutically effective amount of a protein of any one of claims 1-43 or a pharmaceutical composition of claim 46,

wherein the disease or disorder is selected from the group consisting of hyperplasia, solid tumors, and hematopoietic malignancies.

48. The method of claim 47, further comprising administering to the subject one or more of chemotherapy, radiation therapy, targeted therapy, or immunotherapy.

49. The method of claim 48, comprising administering a chemotherapeutic agent.

50. The method of claim 48, comprising administering radiation therapy.

51. Use of a protein according to any one of claims 1 to 43 for the treatment or alleviation of a disease or a disorder.

52. Use of a protein according to any one of claims 1 to 43 and a pharmaceutically acceptable excipient, carrier or diluent in the manufacture of a medicament for treating or ameliorating a disease or disorder.

53. The use of claim 51 or 52, wherein the disease or disorder is selected from head and neck cancer, endometrial cancer, colorectal cancer, ovarian cancer, breast cancer, melanoma, lung cancer, renal cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain tumor, gastric cancer, testicular cancer, pancreatic cancer, thyroid cancer.

54. Use of a protein according to any one of claims 1 to 43 for the preparation of a medicament.

55. The use of claim 54, wherein the medicament is an anti-neoplastic agent.

56. The use of claim 55, wherein the anti-neoplastic agent is effective for treating B-cell lymphoma or anti-colorectal cancer.

57. A cell line comprising a polynucleotide encoding the protein of any one of claims 1-43.

58. A method of making a protein, the method comprising culturing the cell line of claim 57.

59. The method of claim 58, further comprising purifying or isolating the produced protein.

60. A method of making a protein, the method comprising:

providing an expression vector encoding the protein of any one of claims 1-43;

introducing the expression vector into a host cell;

culturing said host cell in a culture medium under conditions sufficient for expression of said protein; and

purifying the protein from the host cell or culture medium.

61. The method of claim 60, wherein the host cell is 293F.

62. The method of claim 60, wherein the host cell is CHO.

63. The method of any one of claims 60-62, wherein the introduction of the expression vector is by transient transfection or a stable cell line.

64. The method of any one of claims 60-63, wherein the purification of the protein is by affinity chromatography or pore size exclusion of protein A/G.

65. An isolated protein produced by the method of any one of claims 60-64.

66. The isolated protein of claim 65, which is substantially pure.

Technical Field

The present invention relates generally to novel fusion proteins and their therapeutic uses. More particularly, the present invention provides novel fusion proteins of interleukin 12 and prodrugs, compositions and methods of preparation thereof that are useful for treating a variety of different diseases and disorders, such as hyperplasia, solid tumors or hematopoietic malignancies.

Background

Interleukin-12 (IL12), also known as Cytotoxic Lymphocyte Maturation Factor (CLMF), was first identified in 1989 as a Natural Killer (NK) cell stimulating factor with multiple biological activities on peripheral blood lymphocytes. IL12 is produced by a variety of different cells of the immune system, including phagocytes, B cells, and activated dendritic cells, in response to infection (Colombo et al, 2002Cytokine & Growth factors Reviews 13: 155-168). IL12 plays an essential role in mediating the interactions of the innate and adaptive arms of the immune system, acting on T cells and Natural Killer (NK) cells, enhancing the proliferation and activity of cytotoxic lymphocytes and the production of other inflammatory cytokines, particularly interferon-gamma.

IL12 is a heterodimeric molecule consisting of an alpha chain (p35 subunit) and a beta chain (p40 subunit) covalently linked by disulfide bridges, forming a biologically active 74kDa heterodimer. In humans and mice, IL12 has been shown to be a potent activator of Natural Killer (NK) cell activity (Kobayashi et al, 1989J.exp.Med.170: 827-. IFN-. gamma.is also an essential mediator of the anti-angiogenic effect due to IL12 (Voest et al, 1995J.Natl Cancer Inst.87: 581-; Majewski et al, 1996J.invest.Dermatol.106: 1114-). 1118).

Studies have shown that IL12 enhances tumor cell killing (antibody-dependent cellular cytotoxicity, ADCC) mediated by immune cells directed specifically against tumor targets by anti-tumor antibodies (Lieberman et al, 1991J. Surg. Res.50: 410-415). IL12 stimulates nitric oxide production in vivo and causes a delay in tumor progression in mice (Wigginton et al, 1996Cancer Res.56: 1131-1136). It has also been shown that the production of endogenous IL12 gradually decreases with increasing tumor burden (Handel-Fernandez et al 1997J.Immunol.158:280-286), providing a rationale for providing IL12 to cancer patients to reconstitute cell-mediated anti-tumor responses.

IL12 has also been reported to be a potent inhibitor of tumor-driven angiogenesis (Voest et al, 1995 supra; Majewski et al, 1996 supra) demonstrating significant in vivo inhibition of tumor angiogenesis mediated by IFN- γ inducible protein-10 (IP-10) in mice (Sgadari et al, 1996Blood 87:3877-3882), said IP-10 being a chemokine with a potent antiangiogenic effect on the vasculature of growing tumors (Angiolello et al, 1996Ann NY Acad.Sci.795: 158-167; Arenberg et al, 1996 J.exp.Med.981-992). In vitro, it inhibits endothelial cell formation into tubular structures (Angiolello et al, 1995J.exp. Med.182: 155-162). In vivo, induction of IP-10 by IL-2 leads to central tumor necrosis and peripheral vessels show partial to complete occlusion of the vessel lumen by thrombosis ((angioillo et al, 1996 supra; Dias et al, 1998int. J. cancer 75: 151-.

Although IL12 has shown potent anti-tumor effects in preclinical models, systemic administration of recombinant IL12 has resulted in severe side effects such as fever, gastrointestinal reactions, lymphopenia and severe liver dysfunction in clinical trials, and death in existing patients has been attributed to IL12 administration due to its severe toxicity (Lasek et al, 2014Cancer immunological Immunol Immunother.63(5): 419-435).

The current treatments and methods available for hyperplasias, solid tumors, or hematopoietic malignancies are inadequate. There remains an urgent and ongoing need for new and improved therapeutic agents that are effective in treating such diseases and conditions.

Disclosure of Invention

The present invention is based, in part, on the surprising discovery of novel fusion proteins and their therapeutic uses. Disclosed herein are novel fusion proteins of IL12 and prodrugs thereof, compositions and methods of preparation thereof that are useful for the treatment of various diseases and disorders, such as hyperplasia, solid tumors, or hematopoietic malignancies, with reduced side effects and off-target toxicity.

In one aspect, the invention relates generally to a fusion protein. The fusion protein comprises: a first structural unit: one or two subunits of IL12 selected from the group consisting of P35 and P40 subunits, wherein said first building block is located at the N-terminus of said fusion protein; a second structural unit: an antibody Fc fragment, wherein the second building block is located at the C-terminus of the fusion protein; and a first linker segment covalently linking the first and second building units or covalently linking the two subunits of the first building unit.

In another aspect, the invention relates generally to a homodimeric or heterodimeric protein comprising a fusion protein disclosed herein.

In another aspect, the invention relates generally to a substantially purified protein, such as a fusion protein or fragment disclosed herein.

In another aspect, the invention relates generally to a polynucleotide encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof.

In another aspect, the invention relates generally to an expression vector comprising a polynucleotide encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof.

In another aspect, the present invention relates generally to a pharmaceutical composition comprising a protein, such as a fusion protein disclosed herein, or a fragment thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

In another aspect, the present invention relates generally to a pharmaceutical composition comprising a polynucleotide encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

In another aspect, the invention relates generally to a method of treating a disease or condition. The method comprises administering to a patient in need thereof a therapeutically effective amount of a polynucleotide encoding a protein, e.g., a fusion protein disclosed herein, or a fragment thereof, wherein the disease or disorder is selected from the group consisting of a hyperplasia, a solid tumor, or a hematopoietic malignancy.

In another aspect, the invention relates generally to the use of a protein, such as a fusion protein disclosed herein, or a fragment thereof, for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

In another aspect, the invention relates generally to the use of a polynucleotide encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof, for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

In another aspect, the invention relates generally to the use of a protein, such as a fusion protein disclosed herein or a fragment thereof, and a pharmaceutically acceptable excipient, carrier or diluent in the manufacture of a medicament for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

In another aspect, the invention relates generally to the use of a polynucleotide encoding a protein, such as a fusion protein disclosed herein or a fragment thereof, and a pharmaceutically acceptable excipient, carrier or diluent in the manufacture of a medicament for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

In another aspect, the invention relates generally to a cell line comprising a polynucleotide encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof.

In another aspect, the invention relates generally to a method of producing a protein, the method comprising culturing the cell line. In certain embodiments, the methods further comprise purifying or isolating the produced protein, such as a fusion protein disclosed herein, or a fragment thereof.

In another aspect, the invention relates generally to a method of making a protein. The method comprises the following steps: providing an expression vector encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof; introducing the expression vector into a host cell; culturing said host cell in a culture medium under conditions sufficient for expression of said protein; and purifying the protein from the host cell or culture medium.

In another aspect, the present invention relates generally to an isolated protein produced by the methods disclosed herein.

Drawings

FIG. 1 shows a schematic diagram of the structure of a prodrug of IL12-Fc dimer: homodimer-IL 12-Fc (Homo IL12) in tandem.

Figure 2 shows a schematic of the structure of a prodrug of IL12-Fc dimer: heterodimer-IL 12-Fc in parallel form (Het IL12), Fc-k is an abbreviation for Fc-pestle, and Fc-h is an abbreviation for Fc-mortar.

FIG. 3 shows a schematic representation of the structure of the homodimer-IL 12-R β 1 dimer prodrug (Homo-R1).

FIG. 4 shows a schematic representation of the structure of the homodimer-IL 12-R β 2 dimer prodrug (Homo-R2).

FIG. 5 shows a schematic of the structure of the heterodimer-IL 12-R β 1/R β 2 dimer prodrug (Het-R1/R2), Fc-k being an abbreviation for Fc-knob, and Fc-h being an abbreviation for Fc-hole.

FIG. 6 shows a schematic of the structure of the heterodimer-IL 12-R β 1 dimer prodrug (Het-R1), Fc-k being an abbreviation for Fc-knob, and Fc-h being an abbreviation for Fc-hole.

FIG. 7 shows a schematic of the structure of the heterodimer-IL 12-R β 2 dimer prodrug (Het-R2), Fc-k being an abbreviation for Fc-knob, and Fc-h being an abbreviation for Fc-hole.

FIG. 8 shows exemplary data of SDS-PAGE electrophoresis results of the expression of the 7 fusion proteins in FIGS. 1-7.

Figure 9 shows exemplary data that injection of IL12-Fc not linked to the IL12 receptor completely abolished MC38 tumors, and Het IL12 had a stronger clearance than Homo IL 12.

Fig. 10 shows exemplary data for Het IL12 having higher cytotoxicity than Homo IL 12.

FIG. 11 shows exemplary data for Het-R1, Het-R2, and Het-R1/R2 all effectively eliminating MC38 tumors.

Figure 12 shows exemplary data that Het IL12 prodrugs linked to IL12 receptors have fewer side effects when administered systemically.

FIG. 13 shows exemplary data for SDS-PAGE electrophoresis results of expression of human Het-R1 and Het-R1/R2 digested with or without MMP 14.

Figure 14 shows (a) human Het-R1 exhibiting similar activity to Het IL12 and recombinant IL12 in vitro after MMP14 digestion, (B) exemplary data for human Het-R1/R2 exhibiting similar activity to Het IL12 and recombinant IL12 after MMP14 digestion using HEK Blue-IL12 reporter cell line.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following terms, unless otherwise indicated by the context in which they are present, are intended to have the following meanings.

When tradenames are used herein, they include product dosage forms, common name drugs, and active pharmaceutical ingredients of the tradename product, unless the context indicates otherwise.

Ranges provided herein are to be understood as being shorthand for all values falling within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, "at least" a particular value is understood to mean that value and all values greater than that value.

As used herein, "more than 1" is understood to mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 100, etc., or any value in between.

In this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

Unless otherwise indicated or apparent from the context, the term "about" when used herein is to be understood as a normal range of tolerance in the art, e.g., within 2 standard deviations of the mean. About may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. All numerical values provided herein may be modified by the term about, unless otherwise clear from the context.

The term "or" as used herein is to be understood as being inclusive unless specified otherwise or clear from the context.

The term "comprising" when used in defining compositions and methods means that the compositions and methods include the recited elements, but do not exclude other elements. The term "consisting essentially of … …" when used to define compositions and methods means that the compositions and methods include the recited elements and exclude other elements having any significance to the compositions and methods. For example, "consisting essentially of … …" means that a pharmacologically active agent that is not specifically recited is administered and excludes pharmacologically active agents that are not specifically recited. The term consisting essentially of … … does not exclude pharmacologically inactive or pharmacologically inert agents such as pharmaceutically acceptable excipients, carriers or diluents. The term "consisting of … …" when used to define compositions and methods is meant to exclude trace amounts of other ingredients and elements of substantial method steps. Embodiments defined by each of these transitional terms are within the scope of the present invention.

As used herein, the term "agonist" refers to a compound that, in combination with a receptor, can produce a cellular response. An agonist may be a ligand that binds directly to the receptor. Alternatively, an agonist may be indirectly combined with a receptor, for example, by: (a) form a complex with another molecule that directly binds to the receptor, or (b) otherwise result in modification of another compound such that the other compound directly binds to the receptor.

As used herein, the term "antagonist" refers to a compound that competes with an agonist or inverse agonist for binding to a receptor, thereby blocking the effect of the agonist or inverse agonist on the receptor. However, antagonists have no effect on constitutive receptor activity.

As used herein, the term "antibody" refers to a molecule capable of binding an epitope or antigenic determinant. The term is intended to include whole antibodies and antigen-binding fragments thereof. The term encompasses polyclonal, monoclonal, chimeric, Fab, Fv, single chain antibody and single or multiple immunoglobulin variable chain or CDR domain designs, as well as bispecific and multispecific antibodies. The antibody may be from any animal source. Preferably, the antibody is mammalian, e.g., human, murine, rabbit, goat, guinea pig, camel, horse, etc., or other suitable animal. Antibodies can recognize polypeptide or polynucleotide antigens. The term includes active fragments, including, for example, antigen-binding fragments of immunoglobulins, variable and/or constant regions of heavy chains, variable and/or constant regions of light chains, complementarity determining regions (cdr), and framework regions. The term includes polyclonal and monoclonal antibody preparations, as well as hybrid antibodies, altered antibodies, chimeric antibodies, hybrid antibody molecules, F (ab)₂And f (ab) fragments, Fv molecules (e.g., non-covalent heterodimers), dimeric and trimeric antibody fragment constructs, minibodies, humanized antibody molecules, and any functional fragments obtained from these molecules, wherein the fragments retain specific binding.

As used herein, the term "antigen" is used herein to refer to any substance that causes the immune system to produce antibodies or a specific cell-mediated immune response directed against it. A disease-associated antigen is any substance associated with any disease that causes the immune system to produce antibodies or specific cell-mediated immune responses directed against it. The antigen is capable of being recognized by the immune system and/or is capable of inducing a humoral immune response and/or a cellular immune response leading to the activation of B and/or T lymphocytes. The antigen may have one or more epitopes (B and/or T cell epitopes). The antigen preferably reacts, typically in a highly selective manner, with its corresponding antibody or TCR, and does not react with numerous other antibodies or TCRs that may be evoked by other antigens. An antigen as used herein may also be a mixture of several individual antigens.

As used herein, the term "biologically active" entity or entity having "biological activity" is an entity that has the structural, regulatory, or biochemical functions of a naturally occurring molecule or any function associated with or concomitant with a metabolic or physiological process. Biologically active polypeptides or fragments thereof include polypeptides or fragments thereof that may participate in a biological process or reaction and/or may produce a desired effect. The biological activity may include an increase in a desired activity or a decrease in an undesired activity. For example, an entity exhibits biological activity when it participates in a molecular interaction with another molecule, when it has therapeutic value in alleviating a disease condition, when it has prophylactic value in inducing an immune response, or when it has diagnostic and/or prognostic value in determining the presence of a molecule. The biologically active protein or polypeptide may be naturally occurring, or it may be synthesized from known components, for example by recombinant or chemical synthesis, and may include heterologous components.

As used herein, the terms "cancer" and "carcinoma" refer to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of cancer include, but are not limited to, epithelial carcinoma, lymphoma, sarcoma, blastoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, hepatoma, breast cancer, colon cancer and head and neck cancer.

As used herein, the term "cell" refers to any prokaryotic, eukaryotic primary cell or immortalized cell line, any population of cells in, for example, a tissue or organ. Preferably, the cells are of mammalian (e.g., human) origin and can be infected by one or more pathogens.

As used herein, the term "co-administration" refers to the simultaneous presence of two agents in the blood. The two agents may be administered simultaneously or sequentially.

The term "co-expressed" as used herein means that two different polypeptides are simultaneously expressed in a host cell such that the two polypeptides can interact or bind and form a complex in the host cell or in the culture medium of the host cell.

As used herein, the term "disease" or "disorder" refers to a pathological condition, such as one that may be identified as deviating from a healthy or normal state by symptoms or other identifying factors. The term "disease" includes disorders, syndromes, conditions and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immunological, metabolic, infectious, and ischemic diseases.

As used herein, the term "effective amount" of an active agent refers to an amount sufficient to elicit a desired biological response. As will be appreciated by one of ordinary skill in the art, the effective amount of a compound of the present invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease to be treated, the mode of administration, and the patient.

As used herein, the term "expression of a nucleic acid molecule" refers to the conversion of the information contained in the nucleic acid molecule into a gene product. The gene product can be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a peptide or polypeptide produced by translation of mRNA. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

As used herein, the term "host cell" refers to an individual cell or cell culture that may be or has been the recipient of any recombinant vector or isolated polynucleotide. The host cell may be a transfected, transformed, transduced or infected cell of any origin, including prokaryotic, eukaryotic, mammalian, avian, insect, plant or bacterial cells, or it may be a cell of any origin useful for propagation of the nucleic acids described herein. Host cells include progeny of a single host cell, and such progeny may not necessarily be identical (in morphology or in DNA repertoire) to the original parent cell due to natural, accidental, or deliberate mutation and/or alteration. Host cells include cells transfected or infected in vivo or in vitro with a recombinant vector or polynucleotide of the invention. A host cell comprising a recombinant vector of the invention may be referred to as a "recombinant host cell".

Host cells include, but are not limited to, mammalian, plant, insect, fungal and bacterial cells. Bacterial cells include, but are not limited to, cells of gram-positive bacteria such as species of bacillus, streptomyces, and staphylococcus, and cells of gram-negative bacteria such as cells of escherichia and pseudomonas. Fungal cells preferably include yeast cells such as Saccharomyces (Saccharomyces), Pichia pastoris (Pichia pastoris) and Hansenula polymorpha (Hansenula polymorpha). Insect cells include, but are not limited to, Drosophila cells and Sf9 cells. Plant cells include cells from crop plants such as cereals, medicinal and ornamental plants or bulbs, and the like. Mammalian cells suitable for use in the present invention include epithelial cell lines (pig etc.), osteosarcoma cell lines (human etc.), neuroblastoma cell lines (human etc.), epithelial cancers (human etc.), glial cells (murine etc.), liver cell lines (monkey etc.), CHO cells (chinese hamster ovary), COS cells, BHK cells, HeLa cells, 911, AT1080, a549, 293 or per.c6, human ECC NTERA-2 cells, D3 cells of mESC cell line, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3 cells, 293T, REH and MCF-7 and hMSC cells.

As used herein, the term "Fc" refers to a molecule or sequence comprising the sequence of a non-antigen binding fragment of an intact antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any immunoglobulin (e.g., IgG1, IgG 2). Native Fc consists of monomeric polypeptides that can be joined into dimeric or multimeric forms by covalent (i.e., disulfide bond) and non-covalent bonding. The number of intermolecular disulfide bonds between the monomer subunits of the native Fc molecule depends on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2), and ranges from 1 to 4.

As used herein, the term "Fc domain" or "Fc region" means the "fragment crystallizable" region of an immunoglobulin heavy chain. Typically, an Fc domain is capable of interacting with a second Fc domain to form a dimeric complex. The Fc domain may be capable of binding to cell surface receptors known as Fc receptors and/or proteins of the complement system, or may be modified to reduce or enhance these binding activities. The Fc domain may be derived from IgG, IgA, IgD, IgM, or IgE antibody isotypes and perform immune activities including opsonization, cell lysis, degranulation of mast cells, basophils, and eosinophils, and other Fc receptor-dependent processes, activation of complement pathways, and in vivo protein stability.

"Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined herein. As with Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from intact antibodies or produced by recombinant gene expression or other means.

Fc fusion proteins have been reported to combine the Fc region of IgG with domains of another protein, such as various cytokines and soluble receptors (e.g., Capon et al, 1989Nature 337: 525-531; Chamow et al, 1996Trends Biotechnol.14: 52-60; U.S. Pat. Nos. 5,116,964 and 5,541,087).

The use of Fc fusions is known in the art (e.g., U.S. patent nos. 7,754,855, 5,480,981, 5,808,029, WO7/23614, WO98/28427, and references cited therein). Fc fusion proteins can include variant Fc molecules (e.g., as described in U.S. patent No.7,732,570). Fc fusion proteins can be solubilized in the cytoplasm or can bind to the cell surface of cells with specific Fc receptors.

As used herein, the term "Fc variant" refers to a molecule or sequence that is modified from an original Fc but still comprises a binding site that salvages the receptor FcRn. International applications WO 97/34631 (published 1997, 9/25) and WO 96/32478 describe exemplary Fc variants and interactions with salvage receptors and are incorporated herein by reference. Thus, the term "Fc variant" encompasses molecules or sequences that are humanized from Fc that is not human in origin. In addition, the native Fc comprises sites that can be removed due to structural features or biological activity that are not required to provide the fusion molecules of the invention. Thus, in certain embodiments, the term "Fc variant" comprises a molecule or sequence that lacks one or more of the native Fc sites or residues that affect or participate in the following processes: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity upon expression in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in more detail below.

As used herein, the term "fusion protein" refers to a polypeptide comprising two or more regions from different or heterologous proteins covalently linked (i.e., "fused") by recombinant, chemical, or other suitable means. If desired, the fusion molecule may be fused at one or several sites by peptide or other linker segments or sequences. For example, one or more peptide linkers can be used to facilitate the construction of a fusion protein.

As used herein, the term "GC content" refers to the percentage of deoxyguanosine (G) and/or deoxycytidine (C) deoxyribonucleoside or guanosine (G) and/or cytidine (C) ribonucleoside residues comprised by a nucleic acid sequence.

As used herein, the term "high dose" means at least 5% (e.g., at least 10%, 20%, 50%, 100%, 200%, or even 300%) higher than the highest standard recommended dose of a particular compound for treating any human disease or condition.

As used herein, the term "immune response" refers to the process of stimulating and/or recruiting immune cells from the blood to lymphoid or non-lymphoid tissues by a multifactorial process involving different adhesion and/or activation steps. The activation profile results in the release of cytokines, growth factors, chemokines and other factors, up-regulates the expression of adhesion and other activating molecules on immune cells, promotes adhesion, morphological changes and/or extravasation with concomitant chemotaxis through tissues, increases cell proliferation and cytotoxic activity, stimulates antigen presentation, and provides other phenotypic changes, including the production of memory cell types. An immune response also means an activity of an immune cell to suppress or modulate the inflammatory or cytotoxic activity of other immune cells. An immune response refers to the activity of an immune cell in vivo or in vitro.

The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (e.g., the region of IL12 or IL12R sequences) when compared and aligned for maximum correspondence over a comparison window or specified region), when measured using the BLAST or BLAST 2.0 sequence comparison algorithms using default parameters described below, or by manual alignment and visual inspection. These sequences are therefore said to be "substantially identical". This definition also refers to or may be applied to the complement of the test sequence. The definition also includes sequences with deletions and/or additions as well as sequences with substitutions. As described below, the preferred algorithm may take into account gaps, etc. Preferably, identity exists over a region of at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, typically over a region of 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length or over the full length of an amino acid or nucleic acid sequence.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters may be used or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

Preferred examples of algorithms suitable for determining sequence identity and percent sequence similarity are the BLAST algorithms described in Altschul et al, 1977Nuc. acids Res.25: 3389-. BLAST software is publicly available at the world wide web site ncbi.nlm.nih.gov/through the National Center for Biotechnology Information. Default parameters or other non-default parameters may be used. As default parameters, the BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M-5, N-4 and a comparison of the two strands. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10 and a BLOSUM62 scoring matrix of 50 (see Henikoff & Henikoff, proc. natl. acad. sci. usa 89:10915(1989)) alignment (B), an expectation (E) of 10, M5, N-4 and two-strand comparison as default parameters.

As used herein, the term "inhibit" refers to any measurable decrease in biological activity. Thus, "inhibition" as used herein may be referred to as a percentage of normal activity level.

As used herein, the term "interleukin 12" or "IL 12" refers to a biologically active polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the native mammalian IL12 amino acid sequence, meaning that the mutated protein ("mutein") has a similar (75% or greater) functionality to the native IL12 protein in at least one functional assay.

Exemplary functional assays for IL12 polypeptides include inducing interferon- γ (IFN-. gamma.) production by, for example, T cells or Natural Killer (NK) cells and promoting differentiation of helper T1(Th1) cells. Helper T cells that differentiate into Th1 cells can be identified by secretion of IFN- γ. IFN- γ secreted by IL-12-stimulated T cells or NK cells can be conveniently detected, for example, in serum or cell culture using ELISA. ELISA methods and techniques are well known in the art, and kits for detecting IFN- γ are commercially available (e.g., R & D Systems, Minneapolis, Minn.; Peprotech, Rocky Hill, N.J.; and Biosource Intl., Camarillo, Calif.). See also Coligan et al, "Current Methods in Immunology", 1991-; harlow and Lane, handbook of Laboratory with Antibodies (Using Antibodies: A Laboratory Manual), 1998, Cold Spring Harbor Laboratory Press; and "ELISA Manual" (The ELISA Guideboost), edited by Crowther, 2000, Humana Press.

As used herein, the term "isolated" molecule (e.g., a polypeptide or polynucleotide) is a molecule that has been manipulated to exist at a higher concentration than in nature or that has been removed from its original environment. For example, a subject antibody is isolated, purified, substantially isolated, or substantially purified when at least 10% or 20% or 40% or 50% or 70% or 90% of the non-subject antibody material that accompanies the subject antibody in nature has been removed. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated. Furthermore, for the purposes of the present invention, the recombinant DNA molecules contained in the vector are considered to be isolated. Isolated RNA molecules include in vivo or in vitro RNA replication products of DNA and RNA molecules. Isolated nucleic acid molecules also include synthetically produced molecules. In addition, the vector molecule contained in the recombinant host cell is also isolated. Thus, not all "isolated" molecules must be "purified".

As used herein, the term "linker" or "linking segment" refers to a molecule or group that links two other molecules or groups. Peptide linkers may allow the linked molecules or groups to attain a functional configuration. The linker peptide preferably comprises at least 2 amino acids, at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or about 100 amino acids.

The components of the fusion protein, such as cytokines or other biologically active molecules and any peptide linkers, can be organized in almost any manner so long as the fusion protein has its intended function. In particular, each component of the fusion protein can be separated from another component by at least one suitable peptide linker segment or sequence, if desired. In addition, the fusion protein may include a tag, for example, to facilitate modification, identification, and/or purification of the fusion protein. More specific fusion proteins are described in the examples below.

As used herein, the term "low dose" refers to at least 5% (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) lower than the minimum standard recommended dose of a particular compound formulated for a given route of administration for the treatment of any human disease or disorder. For example, a low dose of a pharmaceutical agent formulated for administration by inhalation is different from a low dose of the same pharmaceutical agent formulated for oral administration.

As used herein, the term "culture medium" includes any medium, solution, solid, semi-solid, or rigid support that can support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, escherichia coli or pseudomonas host cells, and cellular contents. Thus, the term may encompass media in which the host cell has been grown, e.g., media in which the polypeptide has been secreted, including media before or after the proliferation step. The term also encompasses buffers or reagents containing host cell lysates, for example, where a polypeptide is produced intracellularly and the host cell is lysed or disrupted to release the polypeptide.

As used herein, the term "modulate" refers to the direct or indirect production of an increase or decrease, stimulation, inhibition, interference, or blocking of a measured activity when compared to a suitable control. A "modulator" of a polypeptide or polynucleotide refers to an agent that affects, e.g., increases, decreases, stimulates, inhibits, interferes with, or blocks the measured activity of the polypeptide or polynucleotide when compared to a suitable control. For example, a "modulator" may bind and/or activate or inhibit a target with a measurable affinity, or directly or indirectly affect normal regulation of receptor activity.

The term "operably linked" refers to a functional linkage between a first nucleic acid sequence and a second nucleic acid sequence such that the first and second nucleic acid sequences are transcribed as a single nucleic acid sequence. Operably linked nucleic acid sequences need not be physically adjacent to each other. The term "operably linked" also refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter or array of transcription factor binding sites) and a transcribable nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the transcribable sequence.

As used herein, the term "pharmaceutically acceptable" excipient, carrier or diluent refers to a pharmaceutically acceptable material, composition or medium, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the dosage form and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a phosphate buffer solution; and other non-toxic compatible materials used in pharmaceutical dosage forms. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate, magnesium stearate and polyoxyethylene-polyoxypropylene copolymers, as well as coloring agents, mold release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

As used herein, the terms "polynucleotide," "nucleic acid molecule," "nucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, including ribonucleotides, and deoxyribonucleotides. They may include double-stranded, single-stranded or triple-helical sequences, and may include, but are not limited to, cDNA, mRNA from viral, prokaryotic and eukaryotic sources, genomic DNA sequences from viral (e.g., DNA viruses and retroviruses) or prokaryotic sources, RNAi, cRNA, antisense molecules, recombinant polynucleotides, ribozymes and synthetic DNA sequences. The term also encompasses sequences that include any known base analogs of DNA and RNA. Nucleotides may be referred to by their commonly accepted single letter codes.

Polynucleotides are not limited to those occurring in nature, but also include polynucleotides in which non-natural nucleotide analogs and internucleotide linkages occur. The nucleic acid molecule may comprise a modified nucleic acid molecule (e.g., a modified base, sugar, and/or internucleotide linkage). Non-limiting examples of this type of non-natural structure include polynucleotides in which the sugar is not ribose, polynucleotides in which 3 '-5' and 2 '-5' phosphodiester linkages occur, polynucleotides in which reverse linkages (3 '-3' and 5 '-5') and branched structures occur. In addition, polynucleotides of the invention include non-natural internucleotide linkages such as Peptide Nucleic Acids (PNA), Locked Nucleic Acids (LNA), C1-C4 alkylphosphonate linkages of methylphosphonate, C1-C6 alkylphosphonate triesters, phosphorothioates and phosphorodithioate types. In any event, the polynucleotides of the invention maintain the ability to hybridize to a target nucleic acid in a manner similar to a native polynucleotide.

Unless otherwise indicated or otherwise evident from the context, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as such the explicitly indicated sequences. Degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more (or all) of the selected codons is replaced by mixed base and/or deoxyinosine residues (Batzer et al, 1991Nucleic Acid Res.19: 5081; Ohtsuka et al, 1985J.biol.chem.260: 2605-2608; Rossolini et al, 1994mol.cell.Probes 8: 91-98).

As used herein, the term "prevention" refers to a method for preventing, delaying, diverting or halting the onset, occurrence, severity or recurrence of a disease or disorder. For example, a method is considered prophylactic if there is a reduction or delay in the onset, occurrence, severity, or recurrence of the disease or disorder, or one or more symptoms thereof, in a subject susceptible to the disease or disorder as compared to a subject not receiving the method. The disclosed methods are also considered prophylactic if in a subject susceptible to a disease or disorder there is a reduction or delay in the onset, occurrence, severity, or recurrence of one or more symptoms of the disease or disorder after receiving the method as compared to the development of the subject prior to receiving treatment. The reduction or delay in the onset, occurrence, severity, or recurrence of osteoporosis may be a reduction of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount therebetween.

Prevention and the like do not mean that the subject is permanently prevented from suffering from the particular disease or disorder. Prophylaxis may require multiple administrations of the agent. Prevention may include preventing recurrence of the disease in a subject in which all disease symptoms have been eliminated or preventing recurrence in a relapsing-remitting disease.

As used herein, the term "promoter" refers to a DNA regulatory region capable of binding RNA polymerase in mammalian cells and initiating transcription of a downstream (3' direction) coding sequence operably linked thereto. The promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of the gene of interest at a detectable level above background. Within the promoter sequence there may be a transcription initiation site as well as a protein binding domain (consensus sequence) responsible for the binding of RNA polymerase. Eukaryotic promoters typically, but not always, contain "TATA" and "CAT" boxes. Promoters include promoters that are naturally adjacent to nucleic acid molecules and promoters that are not naturally adjacent to nucleic acid molecules. In addition, the term "promoter" includes inducible promoters, conditionally active promoters such as cre-lox promoter, constitutive promoters, and tissue-specific promoters.

As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, etc. are included within the definition. The definition covers both full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. In addition, a polypeptide may refer to a protein that contains modifications, such as deletions, additions, and substitutions (typically conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate or may be accidental. Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission.

The term "purified" as used herein means that a protein may be substantially or essentially free of components normally associated with or interacting with the protein found in its naturally occurring environment, i.e., in the case of a recombinantly produced protein, in the native cell or host cell. Proteins that may be substantially free of cellular material include preparations of proteins having less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein, which may be present in an amount of about 30%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cell when the protein or variant thereof is recombinantly produced by host cells. When the protein or variant thereof is recombinantly produced by a host cell, the protein may be present in the culture medium at a dry weight concentration of about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about 1mg/L or less. Thus, a "substantially purified" protein may have a purity level of at least about 80%, particularly a purity level of at least about 85%, more particularly a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or more, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

The proteins and prodrugs of the invention are preferably isolated and/or purified after their preparation to obtain a composition containing an amount equal to or higher than 80% by weight ("substantially pure"), which is then used or formulated as described herein. In certain embodiments, the compounds of the invention are greater than 95% pure.

As used herein, the term "receptor" refers to a protein, including a glycoprotein or fragment thereof, that is capable of interacting with another molecule, called a ligand. The ligand may belong to any class of biochemical or chemical compounds. The ligand is typically an extracellular molecule that upon binding to a receptor typically triggers a cellular response, e.g., triggers a signal transduction pathway. The receptor need not be a membrane bound protein.

As used herein, the term "recombinant" with respect to a nucleic acid molecule means that a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, due to its origin or manipulation, is not accompanied by all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. The term "recombinant" when used in reference to a host cell means a host cell into which a recombinant polynucleotide has been introduced.

As used herein, the term "recombinant virus" refers to a virus that has been genetically modified by manual manipulation. The term covers any virus known in the art.

As used herein, the term "sample" refers to a sample or research sample from a human, animal, such as a cell, tissue, organ, fluid, gas, aerosol, serum, gel, or coagulate. The "sample" may be tested in vivo, e.g., without removal from the human or animal, or it may be tested in vitro. The sample may be tested after processing, for example by histological methods. "sample" also refers to, for example, cells that make up or are isolated from a fluid or tissue sample. "sample" may also refer to cells, tissues, organs or fluids freshly obtained from a human or animal, or processed or stored.

As used herein, the term "soluble" means that the fusion molecule, and in particular the fusion protein, does not readily settle from an aqueous buffer, such as cell culture medium, under low G-force centrifugation (e.g., less than about 30,000 revolutions per minute in a standard centrifuge). The fusion molecule is soluble if it remains in aqueous solution at a temperature above about 5-37 ℃, at or near neutral pH, in the presence of low concentrations or in the absence of anionic or nonionic detergents. Under these conditions, soluble proteins typically have a sedimentation value, for example, less than about 10 to 50 svedberg units.

The aqueous solutions referred to herein typically have a buffering compound to establish a pH typically in the pH range of about 5-9 and an ionic strength range between about 2mM to 500 mM. Sometimes a protease inhibitor or a mild non-ionic detergent is added. In addition, if desired, a carrier protein (e.g., bovine serum albumin) may be added. Exemplary aqueous buffers include standard phosphate buffered saline, tris buffered saline, or other well known buffer and cell culture media formulations.

As used herein, the term "stimulating" refers to enhancing, amplifying, increasing, enhancing a physiological activity, such as an immune response. The stimulus may be a positive change. For example, the increase may be 5%, 10%, 25%, 50%, 75% or even 90-100%. Other exemplary increases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

As used herein, the terms "subject" and "patient" are used interchangeably herein to refer to a living animal (human or non-human). The subject may be a mammal. The term "mammal" refers to any animal in the taxonomic classification mammalian class. The mammal may be a human or non-human mammal such as dogs, cats, pigs, cows, sheep, goats, horses, rats and mice. The term "subject" does not exclude an individual who is completely normal or normal in all respects to the disease or disorder.

As used herein, the term "inhibit" refers to a decrease, attenuation, decrease, cessation, or stabilization of a physiological activity, such as an immune response. Inhibition may be a negative change. For example, the reduction may be 5%, 10%, 25%, 50%, 75% or even 90-100%. Exemplary reductions include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

As used herein, the term "therapeutically effective amount" refers to a dose of one or more therapeutic agents sufficient to achieve the targeted therapeutic effect with little or no unwanted side effects. A therapeutically effective amount can be readily determined by a skilled practitioner, for example, by first administering a low dose of the agent and then escalating the dose until the desired therapeutic effect is achieved with little or no unwanted side effects.

As used herein, the term "transfected" means having DNA or RNA introduced, with or without any concomitant facilitator such as a lipofectin. Methods known in the art for transfection include, for example, calcium phosphate transfection, DEAE dextran transfection, protoplast fusion, electroporation, and lipofection.

As used herein, the term "treating" a disease or disorder refers to alleviating, delaying or ameliorating such a condition or one or more symptoms of such a disease or condition, either before or after onset. Treatment may be directed to one or more effects or symptoms of the disease and/or underlying disease. Treatment may be any alleviation, and may be, but is not limited to, complete elimination of the disease or disease symptoms. The degree of such reduction or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as compared to an equivalent untreated control, as measured by any standard technique.

As used herein, the term "tumor" refers to any malignant or neoplastic cell.

As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering genetic material to a host cell or organism. The vector may be composed of DNA or RNA. The vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases (which can be used to insert foreign DNA), and usually carries a selectable marker such as a gene encoding antibiotic resistance, and usually contains a recognition sequence (e.g., a promoter) for expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors.

Any of the compositions or methods disclosed herein can be combined with one or more of any of the other compositions and methods provided herein.

Detailed Description

The present invention provides novel fusion proteins and therapeutic uses thereof. More specifically, the present invention provides novel IL12 fusion proteins and prodrugs thereof, compositions and methods of preparation thereof that are useful in the treatment of a variety of different diseases and disorders, such as hyperplasia, solid tumors, or hematopoietic malignancies, with reduced off-target toxicity and side effects during treatment.

In one aspect, the invention relates generally to a fusion protein. The fusion protein comprises: a first structural unit: one or two subunits of IL12 selected from the group consisting of P35 and P40 subunits, wherein said first building block is located at the N-terminus of said fusion protein; a second structural unit: an antibody Fc fragment, wherein the second building block is located at the C-terminus of the fusion protein; and a first linker segment covalently linking the first and second building units or covalently linking the two subunits of the first building unit.

In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from a mammal selected from the group consisting of human, monkey, mouse, dog, rat, cow, pig, and sheep.

In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from a human. In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from monkeys. In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from a mouse. In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from dog. In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from a mammal selected from a rat. In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from a cow. In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from porcine. In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from ovine.

Any suitable antibody Fc fragment may be used.

In certain embodiments of the fusion protein, the antibody Fc fragment comprises a human Fc fragment. In certain embodiments, the antibody Fc fragment comprises the amino acid sequence set forth in SEQ ID No. 5.

In certain embodiments, the antibody Fc fragment comprises human IgG 1. In certain embodiments, the antibody Fc fragment comprises the amino acid sequence set forth in SEQ ID No. 6.

In certain embodiments, the human IgG1 is a human Fc-knob or a human Fc-hole.

In certain embodiments, the human IgG1 comprises the amino acid sequence set forth in SEQ ID No. 7.

In certain embodiments of the fusion protein, the mouse P35 subunit has the amino acid sequence set forth in SEQ ID No. 3.

In certain embodiments of the fusion protein, the human P35 subunit has the amino acid sequence set forth in SEQ ID No. 4.

In certain embodiments of the fusion protein, the mouse P40 subunit has the amino acid sequence set forth in SEQ ID No. 1.

In certain embodiments of the fusion protein, the human P40 subunit has the amino acid sequence set forth in SEQ ID No. 2.

In certain embodiments of the fusion protein, the first linker segment L1 has the amino acid sequence set forth in SEQ ID No. 12.

In certain embodiments, the fusion protein further comprises a signal peptide modified at the N-terminus of the first building block.

In certain embodiments, the signal peptide SP1 modified at the N-terminus of the mouse P35 subunit comprises the amino acid sequence set forth in SEQ ID No. 27.

In certain embodiments, the signal peptide SP1 modified at the N-terminus of the human P35 subunit comprises the amino acid sequence set forth in SEQ ID No. 28.

In certain embodiments, the signal peptide SP2 modified at the N-terminus of the mouse P40 subunit comprises the amino acid sequence set forth in SEQ ID No. 29.

In certain embodiments, the signal peptide SP2 modified at the N-terminus of the human P40 subunit comprises the amino acid sequence set forth in SEQ ID No. 30.

In certain embodiments, the fusion protein further comprises a portion of interleukin 12 receptor (IL12R) covalently linked to the N-terminus of the first building block by a second linker segment (L2). In certain embodiments, the IL12R is selected from R β 1 and R β 2.

In certain embodiments, the mouse R β 1 comprises the amino acid sequence set forth in SEQ ID No.8 and the mouse R β 2 comprises the amino acid sequence set forth in SEQ ID No. 10.

In certain embodiments, the human R β 1 comprises the amino acid sequence set forth in SEQ ID No.9 and the human R β 2 comprises the amino acid sequence set forth in SEQ ID No. 11.

In certain embodiments of the fusion protein, the second linker segment L2 is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

In certain embodiments, the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase such as matrix metalloproteinase 14(MMP 14).

In certain embodiments of the fusion protein, the second linker segment L2 comprises the amino acid sequence set forth in SEQ ID nos. 13-26.

In certain embodiments of the fusion protein, the C-terminus of IL12R is linked to the N-terminus of the first building block through the L2; and the C-terminus of the first building block and the N-terminus of the second building block are connected via the L1. When the first building block comprises two subunits, the C-terminus of the first subunit and the N-terminus of the second subunit are connected by linker segment L1.

In another aspect, the invention relates generally to a homodimeric or heterodimeric protein comprising a fusion protein disclosed herein.

In certain embodiments, the homodimeric or heterodimeric protein is a homodimer of monomers comprising a fusion protein of mouse P40 subunit, L1 linker, mouse P35 subunit, L1 linker, and human IgG1 and having an amino acid sequence set forth, for example, in SEQ ID No. 31.

In certain embodiments, the homodimeric or heterodimeric protein is a homodimer of monomers comprising a fusion protein of human P40 subunit, L1 linker, human P35 subunit, L1 linker, and human IgG1 and having an amino acid sequence set forth, for example, in SEQ ID No. 32.

In certain embodiments, the homodimeric or heterodimeric protein is a heterodimer consisting of the following monomers: a first monomer: a fusion protein comprising a mouse P40 subunit, an L1 linker, and a human Fc-pestle and having the amino acid sequence set forth in SEQ ID No. 33; and a second monomer: a fusion protein comprising a mouse P35 subunit, an L1 linker, and a human Fc-socket and having an amino acid sequence structure as set forth, for example, in SEQ ID No. 35.

In certain embodiments, the homodimeric or heterodimeric protein is a heterodimer consisting of the following monomers: a first monomer: a fusion protein comprising a human P40 subunit, an L1 linker, and a human Fc-knob and having the amino acid sequence set forth in SEQ ID No. 34; and a second monomer: a fusion protein comprising a human P35 subunit, an L1 linker, and a human Fc-socket and having an amino acid sequence structure as set forth, for example, in SEQ ID No. 36.

In certain embodiments, the homodimeric or heterodimeric protein is a homodimer consisting of the following monomers: mouse IL12R β 1, L2 linker, mouse P40 subunit, L1 linker, mouse P35 subunit, L1 linker and human IgG1, and having an amino acid sequence set forth, for example, in SEQ ID No. 37.

In certain embodiments, the homodimeric or heterodimeric protein is a homodimer consisting of the following monomers: human IL12R β 1, L2 linker, human P40 subunit, L1 linker, human P35 subunit, L1 linker and human IgG1, and having an amino acid sequence set forth, for example, in SEQ ID No. 38.

In certain embodiments, the homodimeric or heterodimeric protein is a homodimer consisting of the following monomers: mouse IL12R β 2, L2 linker, mouse P40 subunit, L1 linker, mouse P35 subunit, L1 linker and human IgG1, and having the amino acid sequence set forth, for example, in SEQ ID No. 39.

In certain embodiments, the homodimeric or heterodimeric protein is a homodimer consisting of the following monomers: human IL12R β 2, L2 linker, human P40 subunit, L1 linker, human P35 subunit, L1 linker and human IgG1, and having an amino acid sequence set forth, for example, in SEQ ID No. 40.

In certain embodiments, the homodimeric or heterodimeric protein is a heterodimer consisting of the following monomers: a first monomer: a fusion protein of mouse IL12R β 1, L2 linker, mouse P40 subunit, L1 linker, human Fc-pestle and having an amino acid sequence as set forth, for example, in SEQ ID No. 41; and a second monomer: a fusion protein of mouse IL12R β 2, L2 linker, mouse P35 subunit, L1 linker and Fc-socket of human IL12 and having the amino acid sequence as set forth, for example, in SEQ ID No. 43.

In certain embodiments, the homodimeric or heterodimeric protein is a heterodimer consisting of the following monomers: a first monomer: a fusion protein of human IL12R β 1, L2 linker, human P40 subunit, L1 linker, human Fc-pestle and having an amino acid sequence set forth, for example, in SEQ ID No. 42; and a second monomer: human IL12R β 2, L2 linker, human P35 subunit, L1 linker and Fc-socket fusion protein of human IL12 and having the amino acid sequence set forth, for example, in SEQ ID No. 44.

In certain embodiments, the homodimeric or heterodimeric protein is a heterodimer consisting of the following monomers: a first monomer: a fusion protein of mouse IL12R β 1, L2 linker, mouse P40 subunit, L1 linker, human Fc-pestle and having an amino acid sequence as set forth, for example, in SEQ ID No. 45; and a second monomer: a fusion protein comprising a mouse P35 subunit containing the SP1 signal peptide, an L1 linker, and a human Fc-socket, and having an amino acid sequence set forth, for example, in SEQ ID No. 47.

In certain embodiments, the homodimeric or heterodimeric protein is a heterodimer consisting of the following monomers: a first monomer: a fusion protein of human IL12R β 1, L2 linker, human P40 subunit, L1 linker, human Fc-pestle and having an amino acid sequence set forth, for example, in SEQ ID No. 46; and a second monomer: a fusion protein comprising a human P35 subunit containing an SP1 signal peptide, an L1 linker, and a human Fc-socket, and having an amino acid sequence as set forth, for example, in SEQ ID No. 48.

In certain embodiments, the homodimeric or heterodimeric protein is a heterodimer consisting of the following monomers: a first monomer: a fusion protein comprising a mouse P40 subunit, an L1 linker, and a human Fc-knob, and having an amino acid sequence as set forth, for example, in SEQ ID No. 49; and a second monomer: a fusion protein comprising mouse IL12R β 2, L2 linker, mouse P35 subunit, L1 linker and human Fc-socket and having an amino acid sequence as set forth, for example, in SEQ ID No. 51.

In certain embodiments, the homodimeric or heterodimeric protein is a heterodimer consisting of the following monomers: a first monomer: a fusion protein comprising a human P40 subunit, an L1 linker, and a human Fc-knob, and having an amino acid sequence set forth, for example, in SEQ ID No. 50; and a second monomer: a fusion protein comprising human IL12R β 2, L2 linker, human P35 subunit, L1 linker and human Fc-socket, and having an amino acid sequence as set forth, for example, in SEQ ID No. 52.

In certain embodiments, the homodimeric or heterodimeric protein is hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

In another aspect, the invention relates generally to a substantially purified protein, such as a fusion protein or fragment disclosed herein.

In another aspect, the invention relates generally to a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein.

In another aspect, the present invention relates generally to an expression vector comprising a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein.

In another aspect, the present invention relates generally to a pharmaceutical composition comprising a protein, such as a fusion protein or fragment disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

In another aspect, the present invention relates generally to a pharmaceutical composition comprising a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

In another aspect, the invention relates generally to a method of treating a disease or condition. The method comprises administering to a patient in need thereof a therapeutically effective amount of a polynucleotide encoding a protein, e.g., a fusion protein or fragment disclosed herein, wherein the disease or disorder is selected from the group consisting of a hyperplasia, a solid tumor, or a hematopoietic malignancy.

In certain embodiments, the disease or disorder to be treated is hyperplasia.

In certain embodiments, the disease or disorder to be treated is a solid tumor.

In certain embodiments, the disease or disorder to be treated is a hematopoietic malignancy.

In certain embodiments, the subject to be treated is further administered one or more of chemotherapy, radiation therapy, targeted therapy, immunotherapy, or hormonal therapy.

In certain embodiments, the method further comprises administering to the subject a chemotherapeutic agent

In certain embodiments, the method further comprises administering radiation therapy to the subject.

In certain embodiments, the method further comprises administering targeted therapy to the subject.

In certain embodiments, the method further comprises administering immunotherapy to the subject.

In certain embodiments, the method further comprises administering hormone therapy to the subject.

As used herein, the term "chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents include erlotinib (b: (a))Genentech/OSI Pharm.), bortezomib (Millennium Pharm, fulvestrant (AstraZeneca), sotriptan (SU11248, Pfizer), letrozole (I), (II)Novartis), imatinib mesylate (Novartis), PTK787/ZK 222584(Novartis), oxaliplatin (A) ((B)Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus,wyeth), lapatinib (GSK572016, Glaxo Smith Kline), lonafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs), and gefitinib (R: (R) ((R))AstraZeneca), AG1478, AG1571(SU 5271; sugen), alkylating agents such as thiotepa andcyclophosphamide, alkyl sulfonates such as busulfan, improsulfan and piposulfan, aziridines such as benzodidopa, carboquone, miltdopa (metedopa) and metopa, ethyleneimine and methylmelamine such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine, annonaceous acetogenins (especially bullatacin and bullatacin), camptothecins (including the synthetic analogues topotecan), bryostatin, kelitin (calastatin), CC-1065 (including its adolesin, kalesin and bizelesin synthetic analogues), cryptophycins (especially cryptophycin 1 and cryptophytin 8), uroleporin, duocarmycin (including the synthetic analogues KW-9 and CB 1-1), picropodophyllin, chlorambucil, latrunculin, nitrogen mustards such as chlorambucil, chlorophosphamide, estramustine, ifosfamide, dichloromethyldiethylamineDichloromethyldiethanolamine oxide hydrochloride, melphalan, neoenbisine, benzene mustard cholesterol, prednisetum, trilobacil, uracil mustard, nitrosoureas such as carmustine, chlorouramicin, fotemustine, lomustine, nimustine and ranimustine, antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 11 and calicheamicin omega 11(Angew chem. Intl. Ed. Engl. (1994)33: minus 186), daptomycin (dynemicin) including daptomycin A (dynemicin A), bisphosphonates such as clodronate, esperamicin (esperamicin), and neocarcinomacin and related chromophoric proteins enediyne chromophores), aclacinomycin (acamlysin), actinomycin, amtricin, leptomycin, lepimericin, actinomycin C, carmustine, carbapenem antibiotics (carbapenem), carbapenem antibiotics, and carbapenem antibiotics chromophores (carbapenem antibiotics), aclacinin, Tryptophin, actinomycin D, daunomycin, ditetracycline, 6-diazo-5-oxo-L-norleucine,(doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin (idarubicin), idarubicin, marijumycin, mitomycins such as mitomycin C, mycophenolic acid, nogomycin, olivomycin, pelomycin, porfiomycin, puromycin, doxorubicin (quelamycin), rodobicin, streptonigrin, streptozotocin, tubercidin, ubenimex, netretastatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogs such as difenomic acid, methotrexate, pteropterin, trimetrexate, purine analogs such as fludarabine, 6-mercaptopurine, thioprimine, thioguanine, pyrimidine analogs such as ancitabine, doxorubicin, idarubicin, doxorubicin, doxorubi, Azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine, fluorouridine, androgens such as dimethyltestosterone, drotaandrosterone propionate, epithioandrostanol, meindrotane, testolactone, antiadrench agents such as aminoglutethimide, mitotane, trostane, folic acid supplements such as folinic acid, acetyl glucalEsters, aldphosphoramide glycosides, aminoacetylpropionic acid, eniluracil, amsacrine, betanidine (benzarbucil), bisantrene, edatrexate, deflazamine (defofamine), dimecortin, diazequinone, efonide (elfomitine), etiloamine, epothilone, etoglutu, gallium nitrate, hydroxyurea, lentinan, lonidamine (lonidamine), maytansinoids such as maytansine and ansamitocins, mitoguazone, mitoxantrone, mupidamol (mopidanmol), nitrazine (nitrarine), pentastatin, methionine mustard (phenomet), pirarubicin, losoxantrone, podophyllic acid, 2-ethyl hydrazide, procarbazine,polysaccharide complexes (JHS Natural Products, Eugene, Oreg.), Razoxan, rhizomycin, sizofuran, germanium spire (spirogemanium), alternospironic acid, triimine, 2,2',2 "-trichlorotriethylamine, trichothecenes (especially T-2 toxin, Virasurin A (verrucarin A), Myrothecin A and serpentinine), uratan, vindesine, dacarbazine, mannomustine, dibromomannitol, dibromodulcitol, pipobroman, ganciclovir, arabinoside (" Ara-C "), cyclophosphamide, thiotepa, taxanes such as taxanes(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.),(unhydrogenated castor oil), albumin-engineered paclitaxel nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, Illinois) and(docetaxel; Rhone-Poulenc Rorer, Antony, France), chlorambucil,(gemcitabine), 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin, vinblastine, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine,(vinorelbine), noscapine (novantrone), teniposide, idatrofloxacin, daunomycin, aminopterin, capecitabineIbandronate, CPT-11, topoisomerase inhibitor RFS 2000, Difluoromethylornithine (DMFO), retinoids such as retinoic acid, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

Examples of the second (or additional) agent or therapy may include, but are not limited to, immunotherapy (e.g., PD-1 inhibitors (pembrolizumab, nivolumab, cimepriazumab), PD-L1 inhibitors (atelizumab, ovuzumab, delbruzumab), CTLA4 antagonists (ipilimumab), cell signaling inhibitors (e.g., imatinib, gefitinib, bortezomib, erlotinib, sorafenib, sunitinib, dasatinib, vorinostat, lapatinib, sirolimus, nilotinib, everolimus, pazopanib, trastuzumab, bevacizumab, cetuximab, ranibizumab, pegaptanib, panitumumab, etc.), mitotic inhibitors (e.g., paclitaxel, vincristine, vinblastine, etc.), alkylating agents (e.g., cisplatin, cyclophosphamide, chlorambucil, vincristine, vinblastine, etc.), alkylating agents (e.g., cisplatin, cyclophosphamide, melphalan, bemustine, bemusa, carmustine, etc.), antimetabolites (e.g., methotrexate, 5-FU, etc.), intercalating anticancer agents (e.g., actinomycin, anthracycline, bleomycin, mitomycin-C, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, teniposide, etc.), immunotherapy agents (e.g., interleukins, interferons, etc.), and anti-hormonal agents (e.g., tamoxifen, raloxifene, etc.).

In another aspect, the invention relates generally to the use of a protein, such as a fusion protein or fragment disclosed herein, for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

In another aspect, the invention relates generally to the use of polynucleotides encoding proteins, such as fusion proteins or fragments disclosed herein, for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

In another aspect, the invention relates generally to the use of a protein, such as a fusion protein or fragment disclosed herein, and a pharmaceutically acceptable excipient, carrier or diluent in the manufacture of a medicament for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

In another aspect, the invention relates generally to the use of a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein, and a pharmaceutically acceptable excipient, carrier or diluent in the manufacture of a medicament for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

In certain embodiments, the drug is an anti-cancer drug.

In certain embodiments, the disease or disorder is one or more selected from head and neck cancer, endometrial cancer, colorectal cancer, ovarian cancer, breast cancer, melanoma, lung cancer, renal cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain tumor, gastric cancer, testicular cancer, pancreatic cancer, and thyroid cancer.

In certain embodiments, the anti-cancer drug is effective to treat B-cell lymphoma or anti-colorectal cancer.

In another aspect, the invention relates generally to a cell line comprising a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein.

In another aspect, the invention relates generally to a method of producing a protein, the method comprising culturing the cell line. In certain embodiments, the methods further comprise purifying or isolating the produced protein, e.g., a fusion protein or fragment disclosed herein.

In another aspect, the invention relates generally to a method of making a protein. The method comprises the following steps: providing an expression vector encoding a protein, such as a fusion protein or fragment disclosed herein; introducing the expression vector into a host cell; culturing said host cell in a culture medium under conditions sufficient for expression of said protein; and purifying the protein from the host cell or culture medium.

Any suitable expression vector may be used. An exemplary expression vector is the pee12.4 expression vector.

Any suitable host cell may be used, for example 293F and CHO cells.

Introduction of the expression vector may be achieved by any suitable transfection method, and may be by transient transfection or a stable cell line.

Any suitable purification method may be used. Exemplary purification methods are affinity chromatography or pore size exclusion methods by protein A/G.

In another aspect, the present invention relates generally to an isolated protein produced by the methods disclosed herein.

In certain embodiments, the isolated protein is substantially pure.

As disclosed herein, linker sequences can be used to link two or more of the biologically active polypeptides to produce a single chain molecule having a desired functional activity.

Any suitable linker may be used. Exemplary peptide linker sequences include peptide linker sequences having about 7 to 20 amino acids, e.g., about 8 to 16 amino acids. The linker sequence is preferably flexible so as not to hold the biologically active polypeptide or effector molecule in a single, unwanted conformation. The linker sequence may be used, for example, to separate a restriction site from the fused molecule. In particular, the peptide linker sequence may be positioned to provide molecular flexibility. The linker preferably comprises mainly amino acids with small side chains such as glycine, alanine and serine to provide flexibility.

In general, preparation of the fusion protein complexes of the invention can be accomplished by the procedures disclosed herein and by recognized recombinant DNA techniques involving, for example, polymerase chain amplification reaction (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of DNA into a suitable cell, transformation or transfection of a host, culture of a host. In addition, the fusion molecules can be isolated and purified using chaotropic agents and well-known methods of electrophoresis, centrifugation and chromatography. (for disclosures related to these methods, see Sambrook et al, A Laboratory Manual of Molecular Cloning (2 nd edition, 1989), and Ausubel et al, modern methods of Molecular Biology (Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989)).

The invention also provides nucleic acid sequences and DNA sequences encoding the fusion proteins of the invention. The DNA sequence may be carried by a vector suitable for extrachromosomal replication, such as a phage, virus, plasmid, phagemid, cosmid, YAC or episome. For example, DNA vectors encoding the desired fusion proteins can be used to facilitate the preparation methods described herein, and to obtain significant amounts of the fusion proteins or components thereof. The DNA sequence may be inserted into a suitable expression vector, i.e., a vector containing the elements necessary for transcription and translation of the inserted protein coding sequence. A variety of different host-vector systems may be utilized to express the protein coding sequence. These systems may include mammalian cell systems infected with viruses (e.g., vaccinia, adenovirus, etc.), insect cell systems infected with viruses (e.g., baculovirus), microorganisms containing yeast vectors such as yeast or bacteria transformed with phage DNA, plasmid DNA, or cosmid DNA. Depending on the host-vector system utilized, any of a number of suitable transcription and translation elements may be used. (for disclosures related to these methods, see Sambrook et al, A Laboratory Manual of Molecular Cloning (2 nd edition, 1989), and Ausubel et al, modern methods of Molecular Biology (Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989)).

The fusion protein component encoded by the DNA vector may be provided in a cassette format. The term "cassette" means that each component can be readily replaced with another component by standard recombinant methods. In particular, DNA vectors configured in a cassette format are particularly desirable when the encoded fusion complex is intended for use against pathogens that may have or have the ability to develop a serous type.

To make a vector encoding the fusion protein complex, the sequence encoding the biologically active polypeptide is linked to the sequence encoding the effector peptide using a suitable ligase. The DNA encoding the proposed peptide may be obtained by isolating the DNA from natural sources, e.g.from suitable cell lines, or by known synthetic methods, e.g.the phosphotriester method (Oligonucleotide Synthesis, IRL Press, M.J.Gait eds., 1984). Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers. Once isolated, the gene encoding the biologically active polypeptide can be amplified by PCR or other means known in the art. Suitable PCR primers for amplifying the biologically active polypeptide gene may add restriction sites to the PCR product. The PCR product preferably includes a splice site for the effector peptide and leader sequences necessary for proper expression and secretion of the biologically active polypeptide-effector fusion complex. The PCR product also preferably includes sequences encoding the linker sequences or restriction enzyme sites used to ligate such sequences.

The fusion proteins described herein can be produced by standard recombinant DNA techniques. For example, once a DNA molecule encoding the biologically active polypeptide is isolated, the sequence may be ligated to another DNA molecule encoding the effector polypeptide. The nucleotide sequence encoding the biologically active polypeptide may be directly linked to the DNA sequence encoding the effector peptide or, more generally, the DNA sequence encoding the linker sequence discussed herein may be inserted between and linked to the sequence encoding the biologically active polypeptide and the sequence encoding the effector peptide using a suitable ligase. The resulting hybrid DNA molecule can be expressed in a suitable host cell to produce the fusion protein complex. The DNA molecules are linked to each other in a5 'to 3' orientation such that, upon linkage, the translation frames encoding the polypeptides are not altered (i.e., the DNA molecules are linked in frame to each other). The obtained DNA molecule encodes the in-frame fusion protein.

Other nucleotide sequences may also be included in the genetic construct. For example, a promoter sequence that controls the expression of the sequence encoded for the biologically active polypeptide fused to the effector peptide or a leader sequence that directs the fusion protein to the cell surface or culture medium may be included in the construct or present in an expression vector into which the construct is inserted.

In obtaining variant biologically active polypeptides IL12, IL12R, or Fc domain coding sequences, one of ordinary skill in the art will recognize that the polypeptides may be modified by certain amino acid substitutions, additions, deletions, and post-translational modifications without loss or reduction of biological activity. In particular, it is well known that conservative amino acid substitutions, i.e., the substitution of one amino acid with another amino acid of similar size, charge, polarity, and conformation, are unlikely to significantly alter protein function. The 20 standard amino acids that are constituent components of proteins can be broadly classified into four groups of conserved amino acids as follows: the nonpolar (hydrophobic) group includes alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine; the polar (uncharged, neutral) group includes asparagine, cysteine, glutamine, glycine, serine, threonine, and tyrosine; the positively charged (basic) group includes arginine, histidine and lysine; and the negatively charged (acidic) group contains aspartic acid and glutamic acid. The substitution of one amino acid in a protein by another in the same group is unlikely to have an adverse effect on the biological activity of the protein. In other cases, modifications to amino acid positions can be made to reduce or increase the biological activity of the protein. Such alterations may be introduced by site-specific mutagenesis, either randomly or on the basis of known or presumed structural or functional properties of the target residue. Following expression of the variant protein, changes in biological activity resulting from the modification can be readily assessed using binding or functional assays.

Homology between nucleotide sequences can be determined by DNA hybridization analysis, where the stability of a double-stranded DNA hybrid depends on the degree of base pairing that occurs. Conditions of high temperature and/or low salt content reduce the stability of the hybrid and can be altered to prevent annealing of sequences having less than a selected degree of homology. For example, for sequences having about 55% G-C content, hybridization and washing conditions of 40-50 ℃, 6 XSSC (sodium chloride/sodium citrate buffer) and 0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology, hybridization and washing conditions of 50-65 ℃,1 XSSC and 0.1% SDS indicate about 82-97% homology, and hybridization and washing conditions of 52 ℃, 0.1 XSSC and 0.1% SDS indicate about 99-100% homology. A wide variety of computer programs are also available for comparing nucleotide and amino acid sequences (and measuring the degree of homology). Readily available sequence comparison and multiple sequence Alignment algorithms are the Basic Local Alignment Search Tool (BLAST) and ClustalW programs, respectively.

A number of strategies are available for expressing the protein fusion complexes of the invention. For example, the above-described fusion protein constructs can be incorporated into a suitable vector by known means, such as making a cut in the vector for insertion of the construct using a restriction enzyme, and then ligated. The vector containing the genetic construct is then introduced into a suitable host for expression of the fusion protein (for disclosure relating to these methods see Sambrook et al, A Laboratory Manual of Molecular Cloning (2 nd edition, 1989)).

Selection of a suitable vector can be made empirically based on a variety of factors related to the cloning protocol. For example, the vector should be compatible with the host used and have a suitable replicon for use in the host. Furthermore, the vector must be able to accommodate the DNA sequence encoding the fusion protein complex to be expressed. Suitable host cells include eukaryotic and prokaryotic cells, preferably those that can be readily transformed and exhibit rapid growth in culture. In particular, preferred host cells include prokaryotes such as Escherichia coli, Bacillus subtilis, and the like, and eukaryotes such as animal cells and yeast strains such as Saccharomyces cerevisiae. Mammalian cells are generally preferred, in particular J558, NSO, SP2-O or CHO. Other suitable hosts include, for example, insect cells such as Sf 9. Conventional culture conditions were used. See Sambrook, supra. Stable transformed or transfected cell lines can then be selected. Cells expressing the fusion protein complexes of the invention can be determined by known procedures. For example, expression of a fusion protein complex linked to an immunoglobulin can be determined by ELISA specific for the linked immunoglobulin and/or by immunoblotting. Other methods for detecting the expression of fusion proteins comprising a biologically active polypeptide linked to IL12 or IL12R domain are disclosed in the examples.

The host cell may be used for production purposes to propagate nucleic acid encoding the desired fusion protein or a component thereof. Host cells may include prokaryotic or eukaryotic cells in which the fusion protein is specifically intended to be produced. Thus, host cells specifically include yeast, fly, worm, plant, frog, mammalian cells and organs capable of propagating nucleic acids encoding the fusions. Non-limiting examples of mammalian cell lines that can be used include CHO dhfr-cells (Urlaub and Chasm, 1980Proc. Natl. Acad. Sci. USA,77:4216), 293 cells (Graham et al, 1977J. Gen. Virol.,36:59) or myeloma cells such as SP2 or NSO (Galfre and Milstein, 1981meth. enzymol.,73(B): 3).

Host cells capable of propagating nucleic acids encoding the desired fusion protein complexes also encompass non-mammalian eukaryotic cells including insect (e.g., spodoptera frugiperda (sp.)), yeast (e.g., saccharomyces cerevisiae (s. cerevisiae), schizosaccharomyces (s. pombe), pichia pastoris (p. pastoris), kluyveromyces lactis (k. lactis), hansenula polymorpha (h. polymorpha); as generally reviewed by Fleer, r.,1992Current Opinion in Biotechnology,3(5): 486496), fungal, and plant cells. Certain prokaryotes such as Escherichia coli and Bacillus are also contemplated.

The nucleic acid encoding the desired fusion protein can be introduced into the host cell by standard techniques for transfecting cells. The term "transfection" is intended to encompass all conventional techniques for introducing nucleic acids into host cells, including calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, viral transduction, and/or integration.

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

Promoter selection also depends on the desired efficiency and level of peptide or protein production. In order to drastically increase the protein expression level in E.coli, an inducible promoter such as tac is generally used. Protein overexpression may be detrimental to the host cell. Thus, the host cell growth may be the first. The use of an inducible promoter system allows the host cell to be cultured to an acceptable density prior to induction of gene expression, thereby facilitating higher product yields.

Various different signal sequences may be used in accordance with the present invention. Signal sequences homologous to the biologically active polypeptide coding sequence may be used. Alternatively, signal sequences selected or designed for efficient secretion and processing in the expression host may also be used. The signal sequence may be linked directly to the protein coding sequence by a sequence encoding a signal peptidase cleavage site, or via a short nucleotide bridge.

The expression constructs may be assembled using known recombinant DNA techniques. Restriction enzyme digestion and ligation are basic steps used to join two DNA fragments. Polylinkers and adapters can be used to facilitate the joining of selected fragments. The expression constructs can generally be assembled in stages using multiple rounds of restriction, ligation and transformation of E.coli. A number of cloning vectors suitable for the construction of expression constructs are known in the art (lambda ZAP and pBLUESCRIPT SK-1, Stratagene, La Jolla, Calif.; pET, Novagen Inc., Madison, Wis.).

The expression construct may be transformed into a host with the linear or circular cloning vector construct, or may be removed from the cloning vector and used as such or introduced into a delivery vector. The delivery vector facilitates introduction and maintenance of the expression construct in the selected host cell type. The expression construct is introduced into the host cell by any known gene transfer system, such as natural competence, chemical-mediated transformation, protoplast transformation, electroporation, biolistic transformation, transfection, or conjugation. The gene transfer system chosen depends on the host cell and vector system used.

The invention also provides a production method for isolating the fusion protein of interest. In the method, a host cell (e.g., yeast, fungal, insect, bacterial, or animal cell) into which a nucleic acid encoding the protein of interest operably linked to a regulatory sequence is introduced is grown in culture on a production scale to stimulate transcription of the nucleotide sequence encoding the fusion protein of interest. Subsequently, the fusion protein of interest is isolated from the harvested host cells or from the culture medium. The protein of interest can be isolated from the culture medium or the harvested cells using standard protein purification techniques. In particular, the purification techniques can be used to express and purify a desired fusion protein on a large scale (i.e., in an amount of at least milligrams) from a variety of different equipment including roller bottles, rotary shake flasks, tissue culture plates, bioreactors, or fermentors.

The expressed protein fusion complex can be isolated and purified by known methods. Typically, the culture medium is centrifuged or filtered, and the supernatant is then purified by affinity or immunoaffinity chromatography, such as protein a or protein G affinity chromatography or an immunoaffinity protocol comprising the use of monoclonal antibodies that bind to the expressed fusion complex, e.g., the linked TCR or immunoglobulin region thereof. The fusion protein of the present invention can be isolated and purified by an appropriate combination of known techniques. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing molecular weight difference such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods utilizing charge difference such as ion exchange column chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing hydrophobicity difference such as reversed-phase high performance liquid chromatography, and methods utilizing isoelectric point difference such as isoelectric focusing electrophoresis, metal affinity columns such as Ni-NTA. (for disclosures related to these methods, see Sambrook et al, A Laboratory Manual of Molecular Cloning (2 nd edition, 1989), and Ausubel et al, modern methods of Molecular Biology (Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989)).

Preferably, the fusion protein of the invention is substantially pure. That is, the fusion protein has been separated from the cellular components with which it is naturally associated such that the fusion protein is preferably present at least 80% or 90% to 95% homogeneity (w/w). For many pharmaceutical, clinical and research applications, fusion proteins having at least 98 to 99% homogeneity (w/w) are most preferred. Once substantially purified, the fusion protein should be substantially free of contaminants for therapeutic use. Once partially purified or purified to significant purity, the soluble fusion proteins can be used therapeutically or to perform in vitro or in vivo assays disclosed herein. Significant purity can be determined by a variety of different standard techniques such as chromatography and gel electrophoresis.

The invention also provides pharmaceutical formulations comprising a therapeutically effective amount of a composition, fusion protein, polynucleotide, genetic construct, vector or host cell according to the invention and a pharmaceutically acceptable excipient or vehicle.

Preferred excipients for use in the present invention include sugars, starches, celluloses, gums and proteins. In a preferred embodiment, the pharmaceutical compositions of the present invention are formulated in pharmaceutical forms for administration as solids (e.g., tablets, capsules, troches, granules, suppositories, crystalline or amorphous sterile solids that can be reconstituted to provide liquid forms, etc.), liquids (e.g., solutions, suspensions, emulsions, elixirs, lotions, oils, etc.), or semisolids (gels, ointments, creams, etc.). The pharmaceutical compositions of the present invention may be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intracapsular, intracerebroventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal routes. Revisions to the different forms of administration of pharmaceutically active ingredients, excipients to be used and their manufacturing procedures can be found in Remington's Pharmaceutical Sciences (a.r. gennaro, Ed.), 20 th edition, Williams & Wilkins PA, USA (2000). Examples of pharmaceutically acceptable media are known in the state of the art and include saline solutions buffered with phosphate salts, water, emulsions such as oil/water emulsions, different types of wetting agents, sterile solutions and the like. The composition comprising the medium may be formulated by conventional procedures known in the state of the art.

Where the pharmaceutical composition of the invention comprises a nucleic acid (a polynucleotide, vector or genetic construct of the invention), the invention contemplates a specially prepared pharmaceutical composition for administration of the nucleic acid. The pharmaceutical composition may comprise the nucleic acid in naked form, in other words, in the absence of a compound that protects the nucleic acid from degradation by the nuclease of the organism, which has the advantage of eliminating the toxicity associated with the reagents used for transfection. For such naked compounds, suitable routes of administration include intravascular, intratumoral, intracranial, intraperitoneal, intraperitoneally, intrasplenic, intramuscular, subretinal, subcutaneous, mucosal, topical and oral routes (Templeton,2002DNA Cell biol.,21: 857-867). Alternatively, the nucleic acid may form part of a liposome, be conjugated to cholesterol or be conjugated to a compound capable of promoting translocation across cell membranes, such as the TAT peptide derived from the TAT protein of HIV-1, the third helix of the homeodomain of the antennapedia protein of Drosophila melanogaster (D.melanogaster), the VP22 protein of herpes simplex virus, oligomers of arginine and peptides such as those described in WO07069090 (Lindgren et al, 2000Trends Pharmacol. Sci21: 99-103; Schwarze et al, 2000Trends Pharmacol. Sci.21: 45-48; Lundberg et al, 2003mol. Therapy 8: 143-150; and Snyder et al, 2004Pharm. Res 393. 21: 389-). Alternatively, the polynucleotide may form part of a plasmid vector or a viral vector, preferably an adenovirus-based vector, in an adeno-associated virus or in a retrovirus, such as a Murine Leukemia Virus (MLV) -based virus, or administered on a lentivirus (HIV, FIV, EIAV).

The composition of the invention may be administered at less than 10 mg/kg body weight, preferably less than 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001mg/kg body weight, and less than 200nmol of agent, in other words about 4.4X 10¹⁶The individual copies are administered per Kg body weight or a dose of less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15 or 0.075nmol/Kg body weight. The unit dose may be administered by injection, by inhalation or by topical administration. The bifunctional polynucleotides and compositions of the invention may be administered directly to the organ in which the target mRNA is expressed, in which case between 0.00001mg and 3mg per organ, or preferably between 0.0001 and 0.001mg per organ, about 0.03 and 3.0mg per organ, about 0.1 and 3.0mg per organ, or 0.3 and 3.0mg per organ will be administered.

The administration will depend on the severity of the condition to be treated and the response to the condition, and may vary from days to months or until remission of the condition is observed. Optimal administration can be determined by periodically measuring the concentration of the agent in the patient's organism. The optimal administration can be determined from the EC50 value obtained previously by in vitro or in vivo testing in animal models. The unit dose may be administered once daily or less than once daily, preferably less than once every 2, 4,8 or 30 days. Alternatively, an initial dose may be administered followed by one or more maintenance doses, typically in lower amounts than the initial dose. The maintenance regimen may comprise treating the patient with a dose ranging between 0.01 μ g to 1.4mg/kg body weight/day, for example 1, 0.1, 0.01, 0.001 or 0.00001mg/kg body weight/day. Maintenance doses are preferably administered up to once every 5, 10 or 30 days. The treatment must last for a period of time that varies depending on the type of change experienced by the patient, its severity and the condition of the patient. After treatment, the patient's evolution must be monitored to determine whether the dose should be increased if the disease is not responsive to the treatment, or whether the dose should be decreased if amelioration of the disease or unwanted secondary effects are observed.

The daily dose may be administered in a single administration or in two or more administrations, as the case may be. If repeated or frequent administration is desired, implantation of a drug delivery device such as a pump, semi-permanent catheter (intravenous, intraperitoneal, intracisternal, or intravesical) or reservoir may be recommended.

The compositions of the present invention are administered according to methods known to those skilled in the art, including but not limited to intravenous, oral, nasal, parenteral, topical, transdermal, rectal administration and the like.

The following examples are intended to illustrate the practice of the invention and are not intended to limit it in any way.

Examples

The following examples describe certain exemplary embodiments of compounds made in accordance with the disclosed invention. It will be appreciated that the following general procedures, and other procedures known to those of ordinary skill in the art, may be applied to the compounds disclosed herein and to the subclasses and species thereof.

Example 1 design of seven IL12-Fc prodrugs

IL12 has two subunits, p35 and p 40. The Fc segment of human IgG1 human Fc-knob and human Fc-hole were used to construct the corresponding prodrugs. The prodrug design links two subunits of IL12 in series or in parallel. The specific form is as follows:

fig. 1 is a schematic diagram showing the structure of a homodimer-IL 12-Fc (Homo IL12), in which two subunits are connected in series and the dimer has a molecular weight of 175KD (MW 175 KD).

FIG. 2 is a schematic diagram showing the structure of heterodimer-IL 12-Fc (Het IL12), where two subunits are connected in parallel. The dimer has a molecular weight of 115KD (MW 115KD), and a P35 signal peptide and a P40 signal peptide are added in the N-terminus of the P35 and P40 subunits, respectively.

The prodrug form blocks the binding of IL12 to either of its receptors or to both receptors IL12R β 1 and IL12R β 2. A portion of the extracellular domain of IL12R β 1, consisting of the two fibronectin type III domains (I + II) at the N-terminus of IL12R β 1 and referred to as R β 1, was fused to the N-terminus of P40. R β 1 acts as a decoy that competitively prevents endogenous IL12R β 1 from interacting with P40. P35 was blocked using two fibronectin type III domains (I + II) at the N-terminus of IL12R β 2, called R β 2, thereby preventing P35 from interacting with endogenous IL12R β 2.

The prodrug construct of homodimer-IL 12 is shown in figure 3.

Fig. 3 is a schematic representation of the prodrug structure of the homodimer-IL 12-R β 1 (known as Homo-R1), dimer MW 232 KD.

Figure 4 is a schematic representation of the prodrug structure of the homodimer-IL 12-R β 2 (known as Homo-R2), dimer MW 250 KD.

Figure 5 is a schematic representation of the prodrug structure of heterodimer-IL 12-R β 1/R β 2 (known as Het-R1/R2), dimer MW 182 KD.

Figure 6 is a schematic representation of the prodrug structure of heterodimer-IL 12-R β 1 (designated Het-R1), dimer MW 144KD, with the addition of the P35 signal peptide to the C-terminus of the fusion segment P35 of the P35-Fc-mortar monomer in the dimer.

Figure 7 is a schematic representation of the prodrug structure of heterodimer-IL 12-R β 2 (designated Het-R2), dimer MW 153KD, with the addition of P40 signal peptide to the C-terminus of the fusion segment P40 of P40-Fc-pestle monomer in this dimer.

Example 2 construction, purification and production of prodrug of IL12

Seven proteins described in example 1 were expressed and produced. First, the recombinant DNA for each fusion protein constructed on the expression vector pee12.4 was transfected into 293F or CHO cells. Culturing the host cells and collecting the cell supernatant. The protein is then purified from the supernatant by affinity column chromatography of protein a/G.

For expression of homodimeric protein, the constructed expression vector plasmid was transformed into 293 or CHO cell hosts. The plasmid expression product in the cell may spontaneously form homodimeric protein. For expression of heterodimeric proteins, both expression vector plasmids must be transfected at the same molar ratio, and monomers expressed in the cells can also spontaneously form heterodimers.

The results of SDS-PAGE are shown in FIG. 8.

Procedures for vector construction, host cell transfection and induction of expression are discussed below.

Expression plasmids for the various fusion proteins were constructed on the PEE12.4 vector and transfected into 293F or CHO cells. The protein expressed in the culture supernatant was purified by a protein a affinity chromatography column.

The expression vector was constructed as follows:

(1) PEE12.4-HindIII-p40 (signal) -NruI-p35 (no signal) -BsiWI-hIgG1-EcoRI

(2) PEE12.4-HindIII-P35 (Signal) -BsiWI-Fch-EcoRI

(3) PEE12.4-HindIII-P40 (signal) -NruI-Fck-EcoRI

(4) PEE12.4-HindIII-IL12Rb1-BsiWI-p40 (No Signal) -NruI-p35 (No Signal) -BsiWI-hIgG1-EcoRI

(5) PEE12.4-HindIII-IL12Rb2-BsiWI-p40 (No Signal) -NruI-p35 (No Signal) -BsiWI-hIgG1-EcoRI

(6) PEE12.4-HindIII-IL12Rb1-BsiWI-p40 (No Signal) -NruI-Fck-EcoRI

(7) PEE12.4-HindIII-IL12Rb2-BstBI-p35 (No Signal) -BsiWI-Fch-EcoRI

HindIII, NruI, BsiWI and EcoRI are enzyme cleavage sites.

The linking sequence between each fusion protein segment is:

(1) homo IL 12: l1 linker between P40 and P35, L1 linker between P35 and Fc.

(2) Het IL 12: linker L1 between P40 and Fc, linker L1 between P35 and Fc.

(3) L2 as a linker between R β 1 and P40; the corresponding target sequence for the protease is SGRSENIRTA.

(4) L2 as a linker between R β 2 and P40; the target sequence for the corresponding protease is SGRSENIRTA.

(5) L2 as a linker between R β 2 and P35; the corresponding target sequence for the protease is SGRSENIRTA.

Transfection may be performed stably or transiently, and the transient transfection protocol is:

(1) cell recovery: freephyle 293F cells at 3X 10⁷Cryopreservation of individual cells/mL in CD OptiCHO^TMMedium (containing 10% DMSO). After removal from liquid nitrogen, it was rapidly dissolved in a 37 ℃ water bath and added to a solution containing 10mL of OptiCHO^TMThe medium was centrifuged at1,000 rpm for 5min in a 15mL centrifuge tube. The supernatant was discarded and the cell pellet was suspended in 30ml OptiCHO^TMIn culture medium, and at 37 deg.C and 8% CO₂And cultured at 135 rpm. After 4 days, the cells are expanded and cultured at a concentration not exceeding 3X 10⁶Individual cells/mL.

(2) 293F cells prepared from suspension culture were used at 0.6-0.8X 10 day before transfection⁶Seeding density of individual cells/mL transient transfection (200 mL).

(3) After 2 days, the transfected cell suspensions were counted (expected cell density was 2.5-3.5X 10)⁶Individual cells/mL), and the cell suspension was centrifuged at1,000 rpm for 5min, and the supernatant was discarded.

(4) The cells were resuspended in 50mL fresh Freestyle 293 media, centrifuged again at1,000 rpm for 5min, and the supernatant discarded.

(5) 293F cells were resuspended in 200mL Freestyle 293 media.

(6) Mu.g of plasmid was diluted with 5mL of Freestyle 293 medium and filter sterilized using a 0.22. mu.M filter.

(7) 1.8mg PEI was diluted with 5mL Freestyle 293 medium and filter sterilized using a 0.22. mu.M filter. Immediately 5mL of plasmid and 5mL of PEI were mixed and allowed to stand at room temperature for 5 min.

(8) The plasmid/PEI mixture was added to the cell suspension and left at 37 ℃ with 8% CO₂85rpm incubator supplemented with 50. mu.g/L of growth factor LONG^TM R3 IGF-1。

(9) After 4hrs, 200ml of E was addedX-CELL^TM293 medium and 2mM glutamine, and the speed was adjusted to 135rpm to continue the culture.

(10) After 24hrs, the cell proliferation inhibitor 3.8mM VPA was added. Supernatants were collected between 4-8 days post-transfection (cell viability at collection was confirmed to be greater than 70% for optimal protein quality).

Fusion protein collection, purification and electrophoretic verification:

(1) sample preparation: the suspension cell culture solution was transferred to a 500mL centrifuge tube and centrifuged at 8,000rpm for 20 minutes. The supernatant was discarded and impurities were removed using a 0.45 μ M filter. 0.05% NaN3 was added to prevent bacterial growth during the purification process.

(2) Assembling the columns: an appropriate amount of protein a agarose was added to the column (20 mg human Fc fusion protein per 1mL protein a) and incubated with 20% ethanol solution for about 10 minutes at room temperature. The column outlet was opened to slowly drain the ethanol solution by gravity.

(3) The column was washed and equilibrated with 10 column volumes of distilled water and binding buffer (20mM sodium phosphate +0.15M NaCl, pH 7.0), respectively.

(4) The sample was constantly pumped through the column at a flow rate of 10 column volumes per hr.

(5) The column was washed with 10 column volumes of binding buffer and washed until no protein was detected in the effluent.

(6) Elution was performed using elution buffer (0.1M glycine, pH 2.7) and the eluate was collected in 1mL increments. An appropriate amount of 1M Tris, pH 9.0 was added for neutralization (pH was adjusted to 6-8, the isoelectric point of the purified protein should be 0.5 or higher).

(7) The target protein solution was replaced with the required buffer (buffer pH was adjusted to avoid the isoelectric point of the protein) using a Zeba desalting column or a concentration spin column. Protein concentration was determined by SDS-PAGE electrophoresis (2.5. mu.g protein load per sample) and NanoDrop2000 using BSA as standard.

After elution, the column was washed successively with 20 column volumes of distilled water, and then the column was washed with 10 column volumes of 20% ethanol. Finally, the ethanol solution was immersed in the gel medium and stored at4 ℃.

Example 3 in vivo antitumor Activity of fusion proteins

Systemic injection of IL12-Fc completely abolished MC38 tumors, and Het IL12 vs Homo IL12 is more effective

To determine whether IL12-Fc was able to effectively eliminate tumors during systemic administration and to compare the therapeutic effects of the two forms of IL12-Fc, the MC38 mouse model was used, and mice and cohorts were treated with different doses by systemic administration. The test results are shown in fig. 9.

FIG. 9A: WT C57BL/6 mice (n-5/group) were treated with 5x 10 on day 0⁵MC38 cells were inoculated subcutaneously. PBS, 0.5. mu.g, 1. mu.g, 5. mu.g, 10. mu.g of Homo IL12 were injected intraperitoneally on days 13, 16, and 20, and tumor volumes were recorded for tumor bearing mice.

FIG. 9B: WT C57BL/6 mice (n-5/group) were treated with 5x 10 on day 0⁵MC38 cells were inoculated subcutaneously. PBS, 0.5. mu.g, 1. mu.g, 5. mu.g, 10. mu.g of Het IL12 were injected intraperitoneally on days 13, 16 and 20, and the tumor volume of tumor-bearing mice was recorded.

These results indicate that IL12-Fc is effective in eliminating tumors, and that Het IL12 is more effective than Homo IL 12.

Systemic use of IL12-Fc causes severe side effects, and Het IL12 vs Homo IL12 is more toxic

Since the IL12 receptor is widely present in T, B and NK cells, the use of IL12 is often associated with strong toxic side effects. Clinically, patients are characterized primarily by a variety of different hematological disorders and hepatotoxicity. Various inflammatory cytokines in mouse serum were experimentally measured as a primary indicator of toxicity caused by IL 12-Fc.

WT C57BL/6 mice (n-5/group) were treated with 5x 10 on day 0⁵MC38 cells were inoculated subcutaneously; PBS, 5. mu.g Homo IL12 or Het IL12 were injected intraperitoneally on days 13, 16 and 20. Blood was collected from the ocular vein at 6hrs after the administration on day 20 to test the inflammatory factors IL12p70, I in serumLevels of FN-gamma, TNF, MCP-1, IL-10 and IL-6.

As a result, as shown in fig. 10, both Homo IL12 and Het IL12 caused strong cytotoxicity, and Het IL12 was more cytotoxic than Homo IL 12.

Het IL12 receptor prodrugs effectively abrogated MC38 tumors

The prodrug of IL12 was constructed by linking the IL12 decoy receptor with a substrate sequence sensitive to certain proteases, in order to remove the decoy from IL-12 in the tumor microenvironment. Proteolytic enzymes capable of cleaving the substrate are more highly expressed in certain tumor types than in normal tissues, and therefore localization of active IL12 at the tumor site is increased, while systemic toxicity of IL12 is reduced. In vivo experiments, Het IL12 produced a stronger antitumor effect against MC38 tumors than Homo IL12 and was also more toxic when used in amounts below 5 μ g/mouse. To test the prodrug concept as a means to reduce the in vivo toxicity of IL12 therapy, Het IL12 (described in example 1) was linked to the decoy IL12 receptor in several configurations (e.g., Het-R1, Het-R2, Het-R1/R2). Also tested were Homo IL12 (e.g. Homo-R1, Homo-R2) linked to decoy IL12 receptors.

FIG. 11A: WT C57BL/6 mice (n-5/group) were treated with 5x 10 on day 0⁵MC38 cells were inoculated subcutaneously and 5 μ g Het IL12, Het-R1, Het-R2 or Het-R1/R2 were administered on days 10, 13 and 16, and the control group was treated with PBS.

FIG. 11B: WT C57BL/6 mice (n-5/group) were treated with 5x 10 on day 0⁵MC38 cells were inoculated subcutaneously. On days 10, 13 and 16, 2.5 μ g of Het IL12, Het-R1, Het-R2 or Het-R1/R2 were injected intraperitoneally and the control group was treated with PBS.

The results show that the three forms of prodrugs of Het IL12 are effective in eliminating tumors. The three forms of the Het IL12 prodrug have better anti-tumor effects on MC38 than Homo IL12 at a dose of 5 μ g/mouse, and the Het IL12 prodrug (Het R1, Het R2 or Het-R1/R2) is still effective in controlling tumors when the dose is reduced to 2.5 μ g/mouse.

Het linked to the IL12 receptor Prodrugs of IL12 have fewer side effects when administered systemically

Body weight changes of mice were recorded after systemic administration of different drugs/prodrugs, and serum was also collected from the ocular veins of mice and the expression level of inflammatory cytokines therein was measured.

FIG. 12A: WT C57BL/6 mice (n-5/group) were treated with 5x 10 on day 0⁵MC38 cells were inoculated subcutaneously and dosed with 2.5 μ g Het IL12, Het-R1, Het-R2, Het-R1/R2 on days 10, 13 and 16, and PBS was dosed to the control group. Body weight of mice was measured at the time of treatment.

FIGS. 12B-G: blood was obtained from the mouse eye vein at the time of treatment. Serum levels of the inflammatory cytokines IL12p70, TNF, IFN-. gamma.MCP-1, IL-10 and IL-6 were measured.

The results show that at a dose of 2.5 μ g/mouse, Het IL12(Het-R1, Het-R2) linked to a prodrug of IL12 receptor has fewer toxic side effects than Het IL12 not linked to the IL12 receptor and Het-R2 is less toxic than the other classes of prodrugs.

In summary, prodrugs of IL12-Fc linked to the IL12 receptor maintain antitumor efficacy and improve the safety of IL12-Fc in the mouse MC38 model. Low dose (2.5. mu.g) of Het IL12 prodrug linked to IL12 receptor completely abrogated tumor volume 130-150mm³The MC38 tumor of (1), and the tumor does not recur. The former (Het IL12-Fc prodrug linked to the IL12 receptor) gave lower toxicity during systemic injection, which is reflected in a significant reduction in weight loss and lower levels of inflammatory cytokines in the blood, compared to the same dose of IL12-Fc not linked to the IL12 receptor. In particular, Het-R2 is the safest construct.

Similar human versions of the various IL15 fusion proteins and prodrugs disclosed herein were also generated and tested in vitro. The production of human proteins follows the same cloning, transfection and purification protocols as described above.

The results of SDS-PAGE electrophoresis of purified human fusion proteins with and without 24 hours incubation with MMP14 at 37 ℃ are shown in FIG. 13.

HEK-Blue is used for human Het-R1 and Het-R1/R2 functions^TMIL12 reporter cell line assay (Invivogen). HEK-Blue^TMIL-12 cells were designed to detect biologically active human IL-12 by expressing STAT 4-inducible SEAP reporter genes. IL-12 and HEK-Blue^TMBinding of IL-12R on the surface of IL-12 cells triggers a signaling cascade leading to activation of STAT-4 and subsequent production of SEAP. HEK-Blue^TMIL-12 cell activation Using QUANTI-Blue^TMSEAP in cell supernatants was detected for measurement.

The following HEK-Blue was used^TMIL-12 cell reporter cell line assay:

(1) HEK-Blue^TMIL-12 cells in PBS soft rinse, and 1x 10⁶The density of individual cells/ml was suspended in fresh pre-warmed test medium (DMEM, 4.5g/L glucose, 2mM L-glutamine, 10% (v/v) heat-inactivated PBS) for 30mins at 56 ℃.

(2) Samples were serially diluted in flat bottom 96-well plates and 50. mu.l of cell suspension (. about.50,000 cells) per well in CO was used₂Incubate at 37 ℃ for 20-24 hours in a warm bath.

(3) 20 μ L of induced HEK-Blue per well in flat bottom 96-well plates^TMIL-12 cell supernatants were resuspended in 100. mu.L/well of QUANTI-Blue^TMThe solution was incubated in a 37 ℃ incubator for 15-min to 1 hour.

(4) SEAP levels were determined using a spectrophotometer at 650 nm.

As a result: figure 14A shows that IL12 activity of Het-R1 prodrugs after MMP14 digestion is comparable to or higher than Het IL 12. In contrast, Het-R1, which is not subjected to MMP14 digestion, is less active, indicating that the prodrug blocking mechanism functions as expected. FIG. 14B shows that the Het-R1/R2 prodrug after digestion of MMP14 is comparable in activity to Het IL12 and recombinant IL 12. In contrast, Het-R1/R2, which had not undergone MMP14 digestion, was less active, indicating that the prodrug blocking mechanism functioned as expected.

Sequence listing

SEQ ID No. 1: mouse P40 subunit (No Signal peptide)

SEQ ID No. 2: human P40 subunit (without signal peptide)

WELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

SEQ ID No. 3: mouse P35 subunit (No Signal peptide)

SEQ ID No. 4: human P35 subunit (without signal peptide)

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

SEQ ID No. 5: human Fc mortar

SEQ ID No. 6: human Fc pestle

SEQ ID No. 7: fc of human IgG1

SEQ ID No. 8: mouse R beta 1

SEQ ID No. 9: human R beta 1

MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRRLGSQGSSWSKWSSPVCVPPEN

SEQ ID No. 10: mouse R beta 2

SEQ ID No. 11: human R beta 2

MAHTFRGCSLAFMFIITWLLIKAKIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEP

SEQ ID No. 12: linker segment L1

GGGGSGGGGSGGGGS

SEQ ID No. 13: linker segment L2(MMP14)

GGGGSSGARYRWLTAGGGGS

SEQ ID No. 14: linker segment L2

GGGGSSGRSENIRTAGGGGS

SEQ ID No. 15: linker segment L2(MMP14)

GGGGSSGRAMHMYTAGGGGS

SEQ ID No. 16: linker segment L2(MMP14)

GGGGSSGAAMHMYTAGGGGS

SEQ ID No. 17: linker segment L2(MMP14)

GGGGSSGAIGFLRTAGGGGS

SEQ ID No. 18: linker segment L2(MMP14)

GGGGSSGASENIRTAGGGGS

SEQ ID No. 19: linker segment L2(MMP14)

GGGGSSGRPENIRTAGGGGS

SEQ ID No. 20: linker segment L2(MMP14)

GGGGSSGAPENIRTAGGGGS

SEQ ID No. 21: linker segment L2(MMP14)

GGGGSSGLISHSITAGGGGS

SEQ ID No. 22: linker segment L2(MMP14)

GGGGSSGNLRSKLTAGGGGS

SEQ ID No. 23: linker segment L2(MMP14)

GGGGSSGVFSIPLTAGGGGS

SEQ ID No. 24: linker segment L2(MMP14)

GGGGSSGIKYHSLTAGGGGS

SEQ ID No. 25: linker segment L2(MMP14)

SEQ ID No. 26: linker segment L2(MMP14)

GGGGSSGRIGFLRTAGGGGS

SEQ ID No. 27: mouse P35 signal peptide SP1

MCQSRYLLFL ATLALLNHLS LA

SEQ ID No. 28: human P35 signal peptide SP1

MCPARSLLLVATLVLLDHLSLA

SEQ ID No. 29: mouse P40 signal peptide SP2

MCPQKLTISW FAIVLLVSPL MA

SEQ ID No. 30: human P40 signal peptide SP2

MCHQQLVISWFSLVFLASPLVAI

SEQ ID No. 31: mouse Homo IL-12

SEQ ID No. 32: human Homo IL12

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

SEQ ID No. 33: mouse Het IL-12 subunit 1

SEQ ID No. 34: human Het IL12 subunit 1

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 35: mouse Het IL12 subunit 2

SEQ ID No. 36: human Het IL12 subunit 2

MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 37: mouse Homo-R1

SEQ ID No. 38: human Homo-R1

MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRRLGSQGSSWSKWSSPVCVPPENGGGGSSGRSENIRTAGGGGSWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

SEQ ID No. 39: mouse Homo-R2

SEQ ID No. 40: human Homo-R2

MAHTFRGCSLAFMFIITWLLIKAKIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPGGGGSSGRSENIRTAGGGGSWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

SEQ ID No. 41: mouse Het-R1/R2 subunit 1

SEQ ID No. 42: human Het-R1/R2 subunit 1

MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRRLGSQGSSWSKWSSPVCVPPENGGGGSSGRSENIRTAGGGGSWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 43: mouse Het-R1/R2 subunit 2

SEQ ID No. 44: human Het-R1/R2 subunit 2

mahtfrgcslafmfiitwllikaKIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPGGGGSSGRSENIRTAGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 45: mouse Het-R1 subunit 1

SEQ ID No. 46: human Het-R1 subunit 1

MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRRLGSQGSSWSKWSSPVCVPPENGGGGSSGRSENIRTAGGGGSWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 47: mouse Het-R1 subunit 2

SEQ ID No. 48: human Het-R1 subunit 2

MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 49: mouse Het-R2 subunit 1

SEQ ID No. 50: human Het-R2 subunit 1

MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 51: mouse Het-R2 subunit 2

SEQ ID No. 52: human Het-R2 subunit 2

MAHTFRGCSLAFMFIITWLLIKAKIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPGGGGSSGRSENIRTAGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Applicants' disclosure is described herein in a preferred embodiment with reference to the drawings, wherein like numerals represent the same or similar elements. Reference throughout this specification to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

The described features, structures, or characteristics of the applicants' disclosure may be combined in any suitable manner in one or more embodiments. In the description herein, numerous specific details are set forth in order to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that applicants' compositions and/or methods can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. The methods recited herein may be performed in any order that is logically possible, other than the specific order disclosed.

Is incorporated by reference

Other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, are referenced and cited in this disclosure. All of these documents are incorporated by reference herein in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the material of the present disclosure. In the event of a conflict, the conflict will be resolved in favor of the present disclosure as a preferred disclosure.

Equivalents of

The representative examples are intended to aid in the description of the invention and are not intended, nor should they be construed, to limit the scope of the invention. Indeed, various modifications of the invention and many other embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including the examples and references to the scientific and patent literature included herein. The described embodiments contain important additional information, examples and guidance that can be adapted to the practice of the invention in its various embodiments and equivalents thereof.