阅读说明：本技术 白介素15融合蛋白及其组合物和治疗方法 (Interleukin 15 fusion proteins and compositions and methods of treatment thereof ) 是由 傅阳心 彭华 郭静雅 于 2019-05-03 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15的融合蛋白及其前体药物、组合物和制备方法。(The present invention provides novel interleukin-15 fusion proteins and prodrugs, compositions, and methods of preparation thereof, 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: a subunit of the interleukin 15 receptor (IL15R) or a fragment thereof;

a second structural unit: active interleukin 15(IL 15);

a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; and

a first linker segment covalently linking the first, second and third building blocks,

wherein the first building block is at the N-terminus of the fusion protein and the second building block is located between the first and third building blocks.

2. A fusion protein comprising:

a first structural unit: a subunit of the interleukin 15 receptor (IL15R) or a fragment thereof;

a second structural unit: active IL 15;

a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; and

a linker segment L1 covalently linking the first, second and third structural units.

Wherein the second building block is at the N-terminus of the fusion protein and the first building block is between the second and third building blocks.

3. The fusion protein of claim 1 or 2, wherein the subunit of IL15R is selected from the group consisting of an alpha subunit, a beta subunit, and a gamma subunit.

4. The fusion protein of claim 3, wherein the subunit of IL15R is an alpha subunit.

5. The fusion protein of any one of claims 1-3, wherein the fragment is the sushi domain of the alpha subunit of IL15R having the amino acid sequence set forth in SEQ ID No. 4.

6. The fusion protein of any one of claims 1-3, wherein the IL15 is human or murine IL 15.

7. The fusion protein of claim 6, wherein the IL15 is mouse IL 15.

8. The fusion protein of claim 7, wherein the mouse IL15 has the amino acid sequence set forth in SEQ ID No. 1.

9. The fusion protein of any one of claims 1-8, wherein the antibody Fc fragment comprises a human Fc fragment.

10. The fusion protein of claim 9, wherein the antibody Fc fragment comprises human IgG1-Fc having the amino acid sequence set forth in SEQ ID No. 3.

11. The fusion protein of any one of claims 1-10, wherein the linker segment L1 comprises a plurality of GGGS.

12. The fusion protein of any one of claims 1-10, wherein the first linker segment linked to the third building block comprises the amino acid sequence set forth in SEQ ID No. 9.

13. The fusion protein of any one of claims 1-10, wherein the linker segment L1 linking the first and second building blocks comprises the amino acid sequence set forth in SEQ ID No. 8.

14. The fusion protein of any one of claims 1-13, further comprising:

a fourth structural unit located at the N-terminus of the fusion protein: the extracellular domain of the IL15 receptor beta subunit (RB);

a linker segment L2 covalently linking the fourth building block and the remaining building blocks of the fusion protein,

wherein the first building block is covalently linked to the C-terminus of the fourth building block and the second building block is located between the first and third building blocks, and

wherein the linker segment L2 is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

15. The fusion protein of any one of claims 1-13, further comprising:

a fourth structural unit located at the N-terminus of the fusion protein: the extracellular domain of the IL15 receptor beta subunit (RB);

a linker segment L2 covalently linking the fourth building block and the remaining building blocks of the fusion protein,

wherein the second building block is covalently linked to the C-terminus of the fourth building block and the first building block is located between the second and third building blocks, and

wherein the linker segment L2 is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

16. The fusion protein of claim 14 or 15, wherein the amino acid sequence of RB has the amino acid sequence set forth in SEQ ID No. 6.

17. The fusion protein of any one of claims 14-16, wherein the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase.

18. The fusion protein of claim 17, wherein the matrix metalloproteinase is matrix metalloproteinase 9(MMP 9).

19. The fusion protein of claim 17, wherein the matrix metalloproteinase is matrix metalloproteinase 14(MMP 14).

20. The fusion protein of any one of claims 14-18, wherein the linker segment L2 comprises the amino acid sequence set forth in SEQ ID nos. 10-23.

21. A homodimeric or heterodimeric protein comprising the fusion protein of any one of claims 1-19.

22. The homodimeric or heterodimeric protein of claim 20 comprising a monomer of RA-IL 15-Fc: a sushi domain of the IL15 receptor alpha subunit, linker segment L1, murine IL15, linker segment L2, human IgG1 Fc, and having the amino acid sequence set forth in SEQ ID No. 24.

23. The homodimeric or heterodimeric protein of claim 20 comprising a monomer of IL 15-RA-Fc: a fusion protein of murine IL15, linker segment L1, the sushi domain of the IL15 receptor alpha subunit, linker segment L1, human IgG1 Fc and having the amino acid sequence set forth in SEQ ID No. 26.

24. The homodimeric or heterodimeric protein of claim 20 comprising a monomer of IL 15-RA-Fc: human IL15, linker segment L1, sushi domain of IL15 receptor alpha subunit, linker segment L1, human IgG1 Fc, and having the amino acid sequence set forth in SEQ ID No. 27.

25. The homodimeric or heterodimeric protein of claim 20 comprising a monomer of RB-IL 15-RA-Fc: a fusion protein of the extracellular domain of the beta subunit of the IL15 receptor, the linker segment L2, murine IL15, the linker segment L1, the sushi domain of the alpha subunit of the IL15 receptor, the linker segment L1, human IgG1 Fc and having the amino acid sequence set forth in SEQ ID No. 28.

26. The homodimeric or heterodimeric protein of claim 20 comprising a monomer of RB-IL 15-RA-Fc: a fusion protein of the extracellular domain of the beta subunit of the IL15 receptor, linker segment L2, human IL15, linker segment L1, the sushi domain of the alpha subunit of the IL15 receptor, linker segment L1, human IgG1 Fc and having the amino acid sequence set forth in SEQ ID nos. 29-41.

27. The homodimeric or heterodimeric protein of any one of claims 21-26 that is hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

28. A substantially purified protein of any one of claims 1-27.

29. A polynucleotide encoding the protein of any one of claims 1-28.

30. An expression vector comprising the polynucleotide of claim 29.

31. A pharmaceutical composition comprising a protein according to any one of claims 1-28 and a pharmaceutically acceptable excipient, carrier or diluent.

32. 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 according to any one of claims 1 to 28 or a pharmaceutical composition according to claim 31,

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

33. The method of claim 32, further comprising administering to the subject one or more of chemotherapy and radiation therapy.

34. The method of claim 33, comprising administering a chemotherapeutic agent.

35. The method of claim 33, comprising administering radiation therapy.

36. Use of a protein according to any one of claims 1 to 28 for the treatment or alleviation of a disease or a disorder.

37. Use of a protein according to any one of claims 1 to 28 and a pharmaceutically acceptable excipient, carrier or diluent in the manufacture of a medicament for treating or ameliorating a disease or disorder.

38. The use of claim 35 or 36, wherein the disease or disorder is selected from head and neck cancer, endometrial cancer, colorectal cancer, ovarian cancer, breast cancer, melanoma, lung cancer, kidney cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain tumor, stomach cancer, testicular cancer, pancreatic cancer, thyroid cancer.

39. Use of a protein according to any one of claims 1 to 28 for the preparation of a medicament.

40. The use of claim 39, wherein the medicament is an anti-neoplastic drug.

41. The use of claim 40, wherein the anti-neoplastic agent is effective in treating B-cell lymphoma or anti-colorectal cancer.

42. A cell line comprising a polynucleotide encoding the protein of any one of claims 1-28.

43. A method of making a protein, the method comprising culturing the cell line of claim 42.

44. The method of claim 43, further comprising purifying or isolating the produced protein.

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

providing an expression vector encoding the protein of any one of claims 1-28;

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.

46. The method of claim 45, wherein the host cell is selected from the group consisting of 293F and CHO cells.

47. The method of claim 45 or 46, wherein the introduction of the expression vector is by transient transfection.

48. The method of any one of claims 45-47, wherein the purification of the protein is by affinity chromatography or pore size exclusion of protein A/G.

49. An isolated protein produced by the method of any one of claims 45-48.

50. The isolated protein of claim 49, 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 15 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 15(IL15) this 14-15 kDa glycoprotein is the first soluble cytokine discovered in 1994 (Grabstein et al, 1994Science 264: 965-8). Similar to interleukin 2, IL15 belongs to the four-helix bundle cytokine family. The human IL15 gene was mapped to chromosome 4 in the q 25-35 region. Mature IL15 is composed of 112 amino acids and contains 3N-glycosylation sites. The expression of IL15 is tightly regulated. Although IL-15mRNA can be found in many tissues and cells including fibroblasts, muscle cells, keratinocytes, kidney cells, lymphocytes, mast cells, and tumor cells, the mature protein is produced primarily by dendritic cells, monocytes, macrophages, and stromal cells but not by T cells. IL-15 expression is stimulated by cytokines such as granulocyte-macrophage colony stimulating Factor (GM-CSF), interferons, and agonists of Toll-like receptors (TLRs) (Markek et al, 2011Cytokine & Growth Factor Reviews 22: 99-108).

The IL15 receptor (IL15R) belongs to the hematopoietic superfamily. Heterotrimeric IL15R comprises α, β (CD122) and γ (CD132, common γ chain, yc) subunits. The beta subunit (IL15R beta) is shared with the IL2 receptor. Human IL15R α belongs to the type I transmembrane protein. Both IL2R α and IL15R α contain conserved sushi domains. IL15 has some similar functions to IL2, e.g. promoting proliferation of T and NK cells^[3](Thomas et al, 2006J of Immunology 177:6072-6080)。

IL15R α is expressed predominantly in Dendritic Cells (DCs) and monocytes. In most cases, IL15 binds to the receptor in trans-presented form. In the trans-presentation model, IL15 and IL15R α were synthesized in the same cell. IL15 and IL 15R. alpha. sushi domains bind to each other with high affinity in the cytoplasm and transport IL-15 to the cell membrane. IL15R α can then present IL-15 in trans to responsive cells such as T cells and NK cells.

IL15 exhibits the following pleiotropic functions in homeostasis and activation of both innate and adaptive immunity:

(1) IL15 plays an important role in the activation, proliferation and survival of CD8+ T cells;

(2) IL15 plays an important role in the activation and homeostasis of memory CD8+ T cells;

(3) IL15 plays an important role in the development, activation and proliferation of NK cells and NKT cells;

(4) IL15 plays an important role in the production of anti-tumor antibodies;

(5) IL15 plays an important role in activation, proliferation and differentiation of DCs through autocrine models, promoting expression of MHC II and CD80/CD86 on DCs, and enhancing presentation of DCs to CD8+ T cells;

(6) IL15 plays an important role in the activation of monocytes and macrophages; and is

(7) IL15 plays an important role in the inhibition of AICD, protecting T cells from Treg inhibition and overcoming resistance to tumor antigens.

IL2 has been approved by the FDA for the treatment of metastatic renal cell carcinoma and malignant melanoma. However, since at CD4⁺CD25⁺The critical role in T-regulatory cell maintenance and activation-induced cell death (AICD), the effectiveness of IL-2 as an anti-cancer therapeutic, has been questioned. This process results in the elimination of stimulated T cells and the induction of T-cell tolerance, limiting the therapeutic efficacy.

Unlike IL2, IL15 is not involved in activation-induced cell death (AICD) and maintenance of regulatory T cells. Therefore, IL15 may have significant advantages in the treatment of cancer compared to IL 2. Recent reports have shown that administration of a preformed complex of IL15 with its soluble receptor IL15R α increases the half-life of IL15 and increases proliferation of T and NK cells (Thomas et al, 2006J of Immunology 177: 6072-6080).

Importantly, soluble fusion proteins of IL15R α sushi domain and IL15 linked by a flexible peptide showed increased half-life of IL15 and proliferation of T and NK cells. In mouse B16F10 and DEN-induced HCC tumor models, the fusion protein can inhibit tumor growth and inhibit tumor metastasis. Furthermore, IL15 showed enhanced antitumor effect or inhibited tumor Growth in combination studies (Cheng et al 2014J of Hepatology 61: 1297-1303; Guo et al 2017Cytokine and Growth Factor Reviews 38: 10-21).

Various side effects have been associated with IL15 therapy, for example:

(1) inducing a cytokine cascade including TNF α, IL1, IL6, GM-CSF, and proinflammatory cytokines;

(2) promoting proliferation, survival and metastasis of certain tumor cells;

(3) activating autoimmune T cells and participating in autoimmune diseases;

(4) inducing coronary heart disease; and

(5) induces the expression of the inhibitory molecule PD1/PDL 1.

There are currently deficiencies in the available therapeutics and methods for, for example, hyperplasias, solid tumors, or hematopoietic malignancies. 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 IL15 and prodrugs thereof, compositions and methods of preparation thereof, useful for the treatment of various diseases and disorders, such as hyperplasia, solid tumors or hematopoietic malignancies.

In one aspect, the invention relates generally to a fusion protein. The fusion protein comprises: a first structural unit: a subunit of interleukin 15 receptor alpha (IL15R alpha) or a fragment thereof; a second structural unit: active IL 15; a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; and a first linker segment (L1) covalently linking the first, second and third building blocks, wherein the first building block is at the N-terminus of the fusion protein and the second building block is located between the first and third building blocks.

In another aspect, the invention relates generally to a fusion protein. The fusion protein comprises: a first structural unit: a subunit of interleukin 15 receptor alpha (IL15R alpha) or a fragment thereof; a second structural unit: active IL 15; a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; and a first linker segment (L1) covalently linking the first, second and third building blocks, wherein the second building block is N-terminal to the fusion protein and the first building block is located between the second and third building blocks.

In another aspect, the invention relates generally to a fusion protein. The fusion protein comprises a first structural unit: a subunit of interleukin 15 receptor alpha (IL15R alpha) or a fragment thereof; a second structural unit: active IL 15; a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; a fourth structural unit: a subunit of interleukin 15 receptor beta (IL15R beta), or a fragment thereof; and a first linker segment (L1) covalently linking the first, second, third and fourth building blocks, wherein the fourth building block is N-terminal to the fusion protein, the second building block is located between the fourth and first building blocks, and the first building block is located between the second and third building blocks.

In another aspect, the invention relates generally to a fusion protein. The fusion protein comprises: a first structural unit: a subunit of the interleukin 15 receptor (IL15R) or a fragment thereof; a second structural unit: active IL 15; a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; a fourth structural unit: a subunit of interleukin 15 receptor beta (IL15R beta), or a fragment thereof; a first linker segment (L1) covalently linking the first, second and third building units, wherein the first building unit is located between the second and third building units, and a second linker segment (L2) covalently links the fourth building unit to the second building unit, wherein the fourth building unit is at the N-terminus of the fusion protein.

In another aspect, the invention relates generally to a homodimeric or heterodimeric protein comprising the 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 representation of the structure of a fusion protein. FIG. 1A shows a schematic diagram of the structure of IL 15-Fc. Fig. 1B and 1C show schematic representations of two IL15 superagonists (both referred to as super IL 15): RA-IL15-Fc and IL 15-RA-Fc. FIG. 1D shows a schematic representation of the fusion protein RB-IL 15-RA-Fc.

FIG. 2 shows an exemplary SDS-PAGE electrophoresis of three fusion proteins.

FIG. 3 shows exemplary results of lymphocyte proliferation assays performed by IL15-Fc and super IL 15.

FIG. 4 shows exemplary results of lymphocyte proliferation assays performed by RB-IL15-RA-Fc and super IL 15.

Fig. 5 shows exemplary results of the therapeutic effect of super IL15 in an a20 tumor model. FIG. 5A shows exemplary data for therapeutic efficacy of super IL15 by intratumoral injection; fig. 5B shows exemplary data for survival of mice within tumors and following intraperitoneal injection; fig. 5C shows exemplary data for mice cured with tumors re-challenged with a20 tumor cells.

Fig. 6 shows exemplary results of the therapeutic effect of super IL15 in the MC38 tumor model. Fig. 6A shows exemplary data for therapeutic efficacy of super IL15 by intratumoral and intravenous injection. Fig. 6B shows exemplary data for survival of mice after treatment.

Figure 7 shows exemplary data for the therapeutic effect of super IL15 in the a20 mouse model with lower doses administered.

FIG. 8 shows an exemplary comparison of the therapeutic effect of RB-IL15-RA-Fc and super IL15 in an A20 tumor model following intravenous injection. Fig. 8A shows an exemplary tumor growth curve of mice after treatment. Figure 8B shows exemplary levels of cytokines in serum after treatment.

FIG. 9 shows an exemplary comparison of the therapeutic effect of RB-IL15-RA-Fc and super IL15 in a mouse A20 tumor model following intraperitoneal injection. Figure 9A shows exemplary survival rates of mice after treatment. Figure 9B shows exemplary levels of cytokines in serum after treatment.

Figure 10 shows an exemplary SDS-PAGE electrophoresis of purified human IL15 fusion protein with or without digestion with MMP 14. RB-IL15-RA-Fc is shown as RB-L1-15RA-Fc or RB-L2-15RA-Fc to emphasize whether L1 or L2 is used as a linker segment attached to RB. IL15-RA-Fc is shown as 15 RA-Fc.

FIG. 11 shows the use of HEK-Blue^TMIL2 reports exemplary results of cellular assays assessing the activity of human IL15 fusion proteins with or without incubation with MMP 14.

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 IL15 or IL15R a sequence) 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 algorithm 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 15" or "IL 15" refers to a polypeptide having biological activity with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the native mammalian IL15 amino acid sequence, meaning that the mutated protein ("mutein") has a similar (75% or greater) functionality to the native IL15 protein in at least one functional assay. Functionally, IL15 is a cytokine that regulates the activation and proliferation of T cells and natural killer cells.

IL15 and IL2 share many biological activities, including binding to IL2 β/IL15 β receptor subunit CD 122. The number of CD8+ memory cells is controlled by the balance between IL15 and IL 2. IL15 induces the activation of JAK kinases and the phosphorylation and activation of the transcriptional activators STAT3, STAT5 and STAT 6. IL15 also increased the expression of the apoptosis inhibitor BCL2L1/BCL-x (L), probably by the transcriptional activation activity of STAT6, thus preventing apoptosis. Two alternatively spliced transcript variants of the IL15 gene encoding the same mature protein have been reported.

Exemplary functional assays for IL15 polypeptides include T-cellsProliferation (e.g., Montes et al, 2005Clin Exp Immunol 142:292) and activation of NK cells, macrophages and neutrophils. For isolation and proliferation of particular subpopulations of immune cells (i.e., detection of proliferation of particular subpopulations of immune cells³H-thymidine incorporation) are well known in the art. Cell-mediated cytotoxicity assays can be used to measure NK cell, macrophage and neutrophil activation. Cell-mediated cytotoxicity assay comprising the isotope(s) ((iii))⁵¹Cr), dyes (e.g., tetrazolium salts, neutral red), or enzyme release, also well known in the art, may be achieved using commercially available kits (Oxford Biomedical Research, Oxford, M; cambrex, walker, Md.; invitrogen, Carlsbad, Calif). IL15 has also been shown to inhibit Fas-mediated apoptosis (e.g., Demirci et al, 2004Cell Mol Immunol 1: 123). Apoptosis assays including, for example, the TUNEL assay and the annexin V assay are well known in the art, and commercially available kits (R) can be used&D Systems, Minneapolis, Minn.) (e.g., Coliga et al, 1991-&Sons)。

As used herein, the term "interleukin 15 receptor alpha" or "IL 15R alpha" refers to the interleukin 15 receptor alpha amino acid sequence from a mammalian species. One skilled in the art will recognize that interleukin-15 receptor alpha nucleic acid and amino acid sequences may be publicly available in gene databases such as GenBank, by the National Center for Biotechnology Information, at the world wide web site ncbi. Exemplary native mammalian IL-15 receptor alpha nucleic acid or amino acid sequences can be from, for example, humans, primates, canines, felines, porcines, equines, bovines, ovines, rodents, murines, rats, hamsters, guinea pigs, and the like. Exemplary native mammalian IL-15 nucleic acid sequence accession numbers include NM _172200.1 (human subtype 2) and NM _002189.2 (human subtype 1 precursor). Exemplary accession numbers for the native mammalian IL-15 amino acid sequence include NP _751950.1 (human subtype 2) and NP _002180.1 (human subtype 1 precursor).

As used herein, "interleukin 15 receptor alpha" or "IL 15R alpha" may also refer to a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the native mammalian IL15R alpha amino acid sequence, has biological activity, and has similar (75% or greater) functionality in at least one functional assay as the native IL15R alpha protein. IL15R α is a cytokine receptor that specifically binds IL15 with high affinity. One functional assay is the specific binding to native IL15 protein.

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 (powdered tragacanth); 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 "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 "soluble IL15 receptor alpha" refers to a form of IL15 receptor alpha that lacks the transmembrane anchoring portion of the receptor and is therefore capable of being secreted outside the cell rather than anchored to the plasma membrane.

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 IL15 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: a subunit of the interleukin 15 receptor (IL15R) or a fragment thereof; a second structural unit: active IL 15; a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; and a first linker segment (L1) covalently linking the first, second and third building blocks, wherein the first building block is at the N-terminus of the fusion protein and the second building block is located between the first and third building blocks.

In another aspect, the invention relates generally to a fusion protein. The fusion protein comprises: a first structural unit: a subunit of the interleukin 15 receptor (IL15R) or a fragment thereof; a second structural unit: active IL 15; a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; and a first linker segment (L1) covalently linking the first, second and third building blocks, wherein the second building block is N-terminal to the fusion protein and the first building block is located between the second and third building blocks.

In certain embodiments of the fusion protein, the subunit of IL15R is selected from the group consisting of an alpha subunit, a beta subunit, and a gamma subunit.

In certain embodiments of the fusion protein, the subunit of IL15R is an alpha subunit.

In certain embodiments of the fusion protein, the fragment is the sushi domain of the alpha subunit of mouse IL15R, having the amino acid sequence set forth in SEQ ID No. 4.

In certain embodiments of the fusion protein, the fragment is the sushi domain of the alpha subunit of human IL15R, having the amino acid sequence set forth in SEQ ID No. 5.

In certain embodiments of the fusion protein, the IL15 is human or murine IL 15.

In certain embodiments of the fusion protein, the IL15 is mouse IL 15. In certain embodiments of the fusion protein, the mouse IL15 has the amino acid sequence set forth in SEQ ID No. 1.

In certain embodiments of the fusion protein, the IL15 is human IL 15. In certain embodiments of the fusion protein, the human IL15 has the amino acid sequence set forth in SEQ ID No. 2.

In certain embodiments of the fusion protein, the antibody Fc fragment comprises a human Fc fragment.

In certain embodiments of the fusion protein, the human Fc fragment comprises human IgG1-Fc having the amino acid sequence set forth in SEQ ID No. 3.

In certain embodiments of the fusion protein, the linker segment L1 comprises a plurality of GGGS.

In certain embodiments of the fusion protein, the linker segment L1 linking the first building block to the third building block comprises the amino acid sequence set forth in SEQ ID No. 9.

In certain embodiments of the fusion protein, the linker segment L1 linking the first and second building blocks comprises the amino acid sequence set forth in SEQ ID No. 8.

In certain embodiments, the fusion protein further comprises: a fourth structural unit located at the N-terminus of the fusion protein: the extracellular domain of the IL15 receptor beta subunit (R β); and a linker segment L2 covalently linking the fourth building block and the remaining building blocks of the fusion protein, wherein the first building block is covalently linked to the C-terminus of the fourth building block and the second building block is located between the first and third building blocks, and wherein the linker segment L2 is capable of being recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

In certain embodiments, the fusion protein further comprises: a fourth structural unit located at the N-terminus of the fusion protein: the extracellular domain of the IL15 receptor beta subunit (R β); and a linker segment L2 covalently linking the fourth building block and the remaining building blocks of the fusion protein, wherein the second building block is covalently linked to the C-terminus of the fourth building block and the first building block is located between the second and third building blocks, and wherein the linker segment L2 is capable of being recognized and hydrolysed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

In certain embodiments, the amino acid sequence of the mouse R β has the amino acid sequence set forth in SEQ ID No. 6.

In certain embodiments, the amino acid sequence of human R β has the amino acid sequence set forth in SEQ ID No. 7.

In certain embodiments of the fusion protein, the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase.

In certain embodiments of the fusion protein, the matrix metalloproteinase is matrix metalloproteinase 9(MMP 9).

In certain embodiments of the fusion protein, the matrix metalloproteinase is matrix metalloproteinase 14(MMP 14).

In certain embodiments of the fusion protein, the linker segment L2 comprises the amino acid sequence set forth in SEQ ID nos. 10-23.

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

In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of RA-IL 15-Fc: a sushi domain of the murine IL15 receptor alpha subunit, linker segment L1, murine IL15, linker segment L1, human IgG1 Fc and having the amino acid sequence as set forth, for example, in SEQ ID No. 24.

In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of IL 15-RA-Fc: a sushi domain of the human IL15 receptor alpha subunit, a linker segment L1, human IL15, a linker segment L1, a fusion protein of human IgG1 Fc and having an amino acid sequence as set forth in, for example, SEQ ID No. 25.

In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of IL 15-RA-Fc: a fusion protein of murine IL15, linker segment L1, the sushi domain of the IL15 receptor alpha subunit, linker segment L1, human IgG1 Fc and having the amino acid sequence as set forth, for example, in SEQ ID No. 26.

In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of IL 15-RA-Fc: human IL15, linker segment L1, sushi domain of IL15 receptor alpha subunit, linker segment L1, human IgG1 Fc, and having an amino acid sequence as set forth in SEQ ID No.27, for example.

In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of RB-IL 15-RA-Fc: a fusion protein of the extracellular domain of the mouse IL15 receptor beta subunit, the linker segment L2, the murine IL15, the linker segment L1, the sushi domain of the IL15 receptor alpha subunit, the linker segment L1, the human IgG1 Fc and having the amino acid sequence set forth, for example, in SEQ ID No. 28.

In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of RB-IL 15-RA-Fc: a fusion protein of the extracellular domain of the beta subunit of the human IL15 receptor, the linker segment L2, human IL15, the linker segment L1, the sushi domain of the alpha subunit of the IL15 receptor, the linker segment L1, human IgG1 Fc and having an amino acid sequence as set forth, for example, in SEQ ID nos. 29-41.

In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of RB-IL 15-RA-Fc: a fusion protein of the extracellular domain of the beta subunit of the human IL15 receptor, the linker segment L1, human IL15, the linker segment L1, the sushi domain of the alpha subunit of the IL15 receptor, the linker segment L1, human IgG1 Fc and having an amino acid sequence as set forth, for example, in SEQ ID No. 42.

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) Sotan (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, alkylsulfonates such as busulfan, improsulfan and piposulfan, aziridines such as benzodidopa, carboquone, miltdopa (meteedopa) and metopa, ethyleneimine and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine, annonaceous acetogenins (especially brazzein and brazzein), camptothecins (including the synthetic analogues topotecan), bryostatin, cathitin (calastatin), CC-1065 (including its adolesin, cabalen and bizelesin synthetic analogues), nostalgins (especially candidin 1 and 8), urocorticoid, duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1), eleutherobin, coprinus, broom cypress alcohol, spongistatin, nitrogen mustards such as chlorambucil, chlorophosphamide, estramustine, ifosfamide, dichloromethyl diethylamine oxide hydrochloride, melphalan, neoentin, benzene mustard cholesterol, prednimustine, trofosfamide, uracil mustard, nitrosoureas such as carmustine, chlorouramicin, fotemustine, lomustine, nimustine, and ramustine, antibiotics such as enediyne antibiotics (e.g., calicheamicin particular calicheamicin γ 11 and calicheamicin ω 11 (angelam. chel. ed. engl. 186) (1994)33:183-, darcinomycins including daptomycin a, bisphosphonates such as clodronate, esperamicin (esperamicin); and a novel oncostatin chromophore and related chromoprotein enediyne antibiotic chromophore), aclacinomycin (aclacinomysin), actinomycin, antromycin, azaserine, bleomycin, actinomycin C, karabicin, carminomycin, carzinophilin, tryptomycin, actinomycin D, daunomycin, ditobicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, sisomicin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, pelomomycin, podoficin, puromycin, doxycycline (quelamycin), Rodocitabine, streptonigrin, streptozotocin, tubercidin, ubenimex, netostatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogs such as dimethylfolic acid, methotrexate, pteropterin, trimetrexate, purine analogs such as fludarabine, 6-mercaptopurine, thioimidine, thioguanine, pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine, floxuridine, androgens such as dimethyltestosterone, drotasone propionate, epithiandrol, meindrotane, testolactone, antiadrench such as aminoglutethimide, mitotane, trostan, folic acid supplements such as folinic acid,acetyl glucuronate, aldphosphoramide glycoside, aminoacetylpropionic acid, eniluracil, amsacrine, betanidine (bestrebuil), bison, edatrexate, difofamine (defofamine), dimethoxine, diazequinone, efonide (elfomitehine), ethanamine, epothilone, etogrel, gallium nitrate, hydroxyurea, lentinan, lonidamine (lonidamine), maytansinoids such as maytansine and ansamitocin, mitoguazone, mitoxantrone, mupidamol (mopinnol), nitrazine (nitriarine), pentostatin, mechlorethamine (phenomedeit), pirarubicin, losoxantrone, podophyllic acid, 2-ethyl hydrazide, procarbazine,polysaccharide complexes (JHS Natural Products, Eugene, OR), Razoxan, Rhizomycin, Sizopyran (sizofian), germanium spire (spirogemanium), alternospora-ketonic acid, triimidyl quinone, 2,2' -trichlorotriethylamine, trichothecenes (in particular T-2 toxin, Virasurin A (verracutin A), Myrothecin A and serpentin (anguidine)), uratan, vindesine, dacarbazine, mechlorethamine, dibromomannitol, dibromodulcitol, pipobroman, gelitin (gacytosine), 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, cimeprimab), PD-L1 inhibitors (atelizumab, ovuzumab, delutamab), CTLA4 antagonists, 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, 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 IL15, IL15R, 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 wash conditions of 40-50 deg.C, 6 XSSC (sodium chloride/sodium citrate buffer) and 0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology, hybridization and wash conditions of 50-65 deg.C, 1 XSSC and 0.1% SDS indicate about 82-97% specificity, and hybridization and wash conditions of 52 deg.C, 0.1 XSSC and 0.1% SDS indicate about 99-100% specificity. 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, host cell growth may be limited. 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.; pEE12.4, Lonza Biologics, Basel, Switzerland).

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. Sci 21: 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-d.). 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 construction of fusion proteins

Construction of A.4 fusion proteins

Construction of control protein: IL15-Fc

Mouse IL15 was fused to the N-terminus of hIgG Fc (designated IL15-Fc), fig. 1A. The amino acid sequence of mouse IL15 is SEQ ID No. 1. The amino acid sequence of hIgG Fc is SEQ ID No. 3.

Two formats of super IL 15:

schematic representations of IL15R α sushi-IL15-Fc (designated RA-IL15-Fc) and IL15-IL15R α sushi-Fc (designated IL15-RA-Fc) are shown in FIGS. 1B and 1C. Similar in biological activity, the two structures are interchangeably referred to as super IL 1. The amino acid sequence of mouse IL15R α sushi is SEQ ID No. 4. The amino acid sequence of human IL15R α sushi is SEQ ID No. 5. The sequence of the linker between IL15R α sushi and IL15 is SEQ ID No. 8. Mouse RA-IL15-Fc and IL15-RA-Fc are represented in their entirety by SEQ ID No.24 and SEQ ID No. 26. Human RA-IL15-Fc and IL15-RA-Fc are represented in their entirety by SEQ ID No.25 and SEQ ID No. 27.

Prodrug:

the ECD (extracellular domain) of IL15R β was fused to the N-terminus of IL15-RA-Fc by linker segment L2.

A schematic representation of IL15R β ECD-L2-IL15-IL15R α sushi-Fc (designated RB-IL15-RA-Fc) is shown in FIG. 1D. The amino acid sequence of mouse IL15R β ECD is SEQ ID No. 6. The amino acid sequence of human IL15R β ECD is SEQ ID No. 7. Linker segment L2 is a substrate for MMP9 or MMP 14. The amino acid sequence of the MMP9 substrate linker is SEQ ID No. 10. The amino acid sequence of the MMP14 substrate linker is SEQ ID Nos. 11-23.

B. Construction, transfection, expression and purification of fusion proteins

The gene is cloned into an expression vector such as pEE12.4. The plasmid was transiently transfected into 293F cells. Supernatants were collected at day 4-7 post-transfection. The fusion protein was purified using protein a sepharose gel. All proteins were quantified by ELISA and SDS-PAGE.

The detailed procedure is as follows.

Construction of fusion proteins

IL15, IL15RA and IL15RB ECD were synthesized and cloned into pEE12.4-IgG kappa-hIgG 1 Fc plasmid containing mouse IgG kappa leader and human IgG1 Fc. Plasmids were extracted using a standard commercial plasmid extraction kit and stored at-80 ℃.

Transfection of fusion proteins

293F cells Using CD OptiCHO^TMCulturing in culture medium at 37 deg.C and 8% CO₂The incubators were incubated with shaking at 135 rpm. Cells were plated at 0.6-0.8X 10 days before transfection⁶Density of individual cells/mL plated. In the range of about 2.5-3.5X 10⁶Cells were harvested at density of individual cells/mL, washed with Freestyle 293 media, and resuspended in 200mL Freestyle 293. Will be provided withDNA (600. mu.g) was diluted with 5mL Freestyle 293 and filtered through a 0.22 μm filter. PEI (1.8mg) was diluted with 5mL Freestyle 293 and filtered through a 0.22 μm filter. The DNA and PEI were mixed and incubated for 5min at room temperature and then mixed with the cells in shake flasks. The flask was placed at 37 ℃ in 8% CO₂In the incubator, the flask was shaken at 85 rpm. 200mL EX-CELL was added 4 hours after transfection^TM293 medium, shaking at 135 rpm. At 20 hours post transfection, 3.8mM VPA was added. Supernatants were collected at days 4 to 7 post transfection, at which time cell viability was greater than 70%.

Purification of fusion proteins

The fusion protein was purified using a protein A-Sepharose column according to the manual (Repligen Corporation).

Binding buffer: 20mM sodium phosphate, pH 7.0

Elution buffer: 0.1M Glycine, pH2.7

Regeneration of buffer solution: 1M NaOH

Neutralization buffer: 1M Tris-HCl, pH 9.0

All buffers were filtered through a 0.45 μm filter.

(1) The sample was centrifuged at 8000 Xrpm for 2hr to remove cells, and then filtered through a 0.45 μm filter. Addition of NaN₃To a final concentration of 0.05% to prevent bacterial growth.

(2) If the column is preserved with 20% ethanol, it is washed with 5 column volumes of distilled water at a linear flow rate of 50 to 100 cm/h.

(3) The column is washed with 5 to 10 column volumes of elution buffer to wash away impurities.

(4) The column is equilibrated with 5 to 10 column volumes of binding buffer at a linear flow rate of 50 to 100 cm/hr.

(5) Applying the pre-treated sample to the column.

(6) The column is washed with 5 to 10 column volumes of binding buffer.

(7) The column was eluted in a 1.5mL elution tube.

The results of SDS-PAGE electrophoresis of the purified fusion proteins are shown in FIG. 2, in which lane S1 was loaded with IL15-Fc, lane S2 was loaded with super IL15, and lane S3 was loaded with RB-IL 15-RA-Fc.

Example 2 biological function of super IL15 fusion proteins

A. Function of promoting lymphocyte proliferation

The ability of interleukin 15(IL-15) to stimulate proliferation of the murine T cell line CTLL-2 was first characterized. In this protocol, CTLL-2 cells were cultured in the presence of serial dilutions of murine IL-15 and their growth measured by CCK 8.

The following procedure was used:

(1) CTLL2 cells were cultured using CTLL-2 assay medium supplemented with 100U/mL recombinant human IL-2.

(2) CTLL-2 cells in log phase growth were collected 24 to 48h after passage and washed twice to remove residual IL-2. Cells were resuspended in 5 to 10mL CTLL-2 assay medium, counted, and adjusted to a concentration of 2X 10⁴Individual cells/mL.

(3) Samples were diluted with CTLL-2 assay medium. The initial maximum concentration was 10. mu.g/mL. A1: 10 serial dilution was performed in 7 tubes.

(4) Add 100. mu.L of cell suspension (2X 10) to each flat bottom 96-well plate³Individual cells/well); to each well 100. mu.L of sample was added. A row of wells containing only 200. mu.L of assay medium was included as a negative control.

(5) The plate was covered and incubated for 48 to 72 hrs.

(6) 20 μ L of CCK8 was added. After 2-4 hrs, each well was read for OD450 and OD630 using a microtiter plate reader.

The results are shown in fig. 3, which indicates (1) that the biological activities of the two forms of super IL15 are similar, that is, that in the fusion protein, neither IL15 fragment nor IL15R α sushi fragment precedes, the function of super IL15 is unaffected; and (2) super IL15 has about 100-fold increased biological activity compared to IL 15-Fc.

Fusion fragments of IL15R beta ECD can block biological function of super IL15

The proliferation capacity of murine RB-IL15-RA-Fc and super IL15 on CTLL2 was examined by the CCK8 assay. The results shown in FIG. 4 indicate that the biological activity of RB-IL15-RA-Fc was 100-fold reduced. This suggests that the extracellular domain of IL-15R β may block the biological function of the super IL-15.

C. Antitumor effect and systemic toxicity in different tumor models

Model A20

Experiment 1(25 μ g): a20 cells (3X 10)⁶) Injected subcutaneously into the right flank of Balb/c mice. Will carry tumors (60-80 mm) on days 10 and 13³) Mice were treated intratumorally (i.t.) and intravenously (i.v.) with 25 μ g of super IL 15. The control group was treated with PBS. Tumor volume is length x width x height/2. Tumor growth curves were recorded.

As a result: (1) in the intratumoral treatment group, tumors from all mice showed complete regression (fig. 5A). Mice that had undergone complete tumor regression were re-challenged with a lethal amount of a20 cells. All mice rejected the re-challenged tumor, indicating a strong memory response (fig. 5C). (2) All mice died on the second dose iv treatment, indicating severe systemic toxicity (fig. 5B).

Experiment 2(12.5 μ g): a20 cells (3X 10)⁶) Injected subcutaneously into the right flank of Balb/c mice. Will carry tumors (60-80 mm) on days 10 and 13³) Mice were treated intratumorally (i.t.) and intravenously (i.v.) with 12.5 μ g of super IL 15. The control group was treated with PBS. Tumor volume was defined as length x width x height/2. Tumor growth curves were recorded.

Results (fig. 7): in the treatment group given intratumorally, 100% of mice achieved complete regression. In contrast, complete regression was obtained in 20% of the intravenously treated mice, with the remaining mice having partially controlled tumors.

MC38 model

Experiment: MC38 cells (5X 10)⁵) Subcutaneously injected into the right flank of C57 mice. Will bear tumors (60 mm) on days 7 and 10³) Mice were treated intratumorally (i.t.) and intravenously (i.v.) with 25 μ g of super IL 15. The control group was treated with PBS. Tumor volume is length x widthDegree x height/2. Tumor growth curves were recorded.

As a result: in the treatment group given intratumorally, 50% of mice achieved complete regression (fig. 6A) and survival was significantly improved (fig. 6B). In contrast, mice given intravenously did not achieve complete tumor regression (fig. 6A), and mice survived slightly higher (fig. 6B).

The above results indicate that the super IL15 appears to play a role locally in the Tumor Microenvironment (TME).

Example 3 comparison of tumor treatment Effect and side effects of super IL15 and RB-IL15-RA-Fc

A20 cells (3X 10)⁶) Injected subcutaneously into the right flank of Balb/c mice. Will carry tumors (60-80 mm) on days 10 and 13³) Mice were treated intraperitoneally (i.p.) with 12.5 μ g of either super IL15 or R β -IL 15-RA-Fc. Tumor growth was measured twice weekly. Serum was collected 20hr after the second injection. Cytokine levels in serum were measured by Cytometric Bead Array (CBA) and tumor curves were recorded.

The following CBA protocol was used:

(1) serum was collected from the ocular vein and stored at-80 ℃.

(2) Serum was evaluated for IL12p70, IL-6, IFN-. gamma., TNF. alpha., MCP1, and IL-10 using a CBA kit from BD.

(3) Standards were reconstituted with 2.0mL assay diluent and then recalibrated at room temperature for at least 15 min. The standard was serially diluted in the following proportions: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1: 256.

(4) Th1/Th2/Th17 cytokine capture beads were mixed. The number of assay tubes (including standards and controls) required for the experiment was determined. Each capture bead suspension was vortexed vigorously for 3 to 5 seconds prior to mixing. For each assay tube to be analyzed, a2 μ Ι _ aliquot of each capture bead was added to a single tube. To the tube, 10 μ L aliquots of mouse Th1/Th2/Th17 PE detection reagent were added and vortexed extensively.

(5) Th1/Th2/Th17 cytokine assay: the mixed capture beads were vortexed and 20 μ Ι _ was added to all assay tubes. To the control tube, 50. mu.L of mouse Th1/Th2/Th17 cytokine standard dilution was added. To a sample assay tube with appropriate label 50 μ L of each unknown sample was added. The assay tubes were incubated at room temperature for 2hr in the dark.

(6) Add 1mL of wash buffer to each assay tube and centrifuge at 300g for 5 min.

(7) The supernatant was carefully aspirated from each assay tube and discarded. To each assay tube 300. mu.L of wash buffer was added to resuspend the bead pellet.

(8) Samples were analyzed by flow cytometry and cytokine levels were calculated from standards.

The therapeutic effect is shown in FIG. 8A, indicating that the therapeutic effect of RB-IL15-RA-Fc administered intravenously is similar to that of super IL 15.

The results of the comparison of serum inflammatory factor levels are shown in fig. 8B, indicating that the toxic side effects of RB-IL15-RA-Fc are significantly reduced compared to super IL 15.

A20 cells (3X 10)⁶) Injected subcutaneously into the right flank of Balb/c mice. Will carry tumors (60-80 mm) on days 10 and 13³) Mice were treated intraperitoneally (i.p.) with 25 μ g of either super IL15 or RB-IL 15-RA-Fc. Tumor growth was measured twice weekly. Serum was collected 20hr after the second injection. Cytokine levels in serum were measured by Cytometric Bead Array (CBA) and tumor curves were recorded.

As a result: after tumor reinjection and treatment with super IL15, tumor-bearing mice were clearly ill, had severe weight loss, decreased mobility, cockled hairs, and all died within one day after the second treatment. In contrast, none of the mice treated with RB-IL15-RA-Fc died and none of the mice appeared unhealthy. The survival curve for RB-IL15-RA-Fc was significantly longer than that for the super IL 15. The survival curves are shown in fig. 9A and serum inflammatory factor levels are shown in fig. 9B.

Taken together, RB-IL15-RA-Fc reduced the toxic side effects of super IL 15.

Similar human versions of various IL15 fusion proteins and prodrugs were also generated and tested in vitro. Human protein production was performed following the cloning, transfection and purification protocols described previously for murine protein production.

Recombinant human MMP-14/MT1-MMP (R & D Systems) was activated and incubated with IL15 fusion protein at 37 ℃ for 24hrs to confirm prodrug activation and cleavage at the L2 junction site.

The results of the SDS-PAGE electrophoresis of the purified human fusion protein with or without incubation with MMP14 for 24hrs at 37 ℃ are shown in FIG. 10.

Functional use of HEK-Blue for human RB-IL15-RA-Fc^TMIL2 reporter cell assay (Invivogen). HEK-Blue after IL-2 stimulation^TMIL-2 cells initiate the activation of STAT5 and subsequent secretion of SEAP. STAT 5-induced SEAP levels QUANTI-Blue can be used^TMIs easily monitored. HEK-Blue because IL15 binds to and signals through a complex consisting of the beta and common gamma chains of the IL-2/IL-15 receptor^TMIL-2 cell lines may also be used to measure the functional activity of IL15 and/or pro-IL 15.

The following HEK-Blue IL-2 reporter cell assay was used:

(1) HEK-Blue IL-2 cells were gently rinsed in PBS and washed at-1X 10⁶The cells/mL density were 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 in CO₂The incubator was incubated with 50. mu.L of cell suspension (. about.50,000 cells) per well for 20-24hrs at 37 ℃.

(3) mu.L of induced HEK-Blue IL-2 cell supernatant was mixed with 100. mu.L of resuspended QUANTI-Blue per well in a flat bottom 96 well plate^TMThe solution is incubated in an incubator at 37 ℃ for 15-min to 1 hr.

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

As a result: FIG. 11 demonstrates that RB-L2-15RA-Fc, which was constructed with the MMP14 substrate sequence embedded in the linker segment (L2) and incubated with MMP14, exhibited the same level of function as 15RA-Fc with or without MMP14 incubation. Consistently, RB-L1-15RA-Fc, which was constructed without the substrate sequence for MMP14 embedded in the linker segment (L1), performed similarly to the sample without MMP14 incubation. The construct symbol 15RA is an abbreviation for IL 15-L1-RA.

Sequence listing

SEQ ID No. 1: mouse IL15

NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS

SEQ ID No. 2: human IL15

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

SEQ ID No. 3: human IgG1-Fc

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 4: mouse R alpha-sushi structural domain

GTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT

SEQ ID No. 5: human R alpha-sushi structural domain

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP

SEQ ID No. 6: mouse R beta extracellular domain

AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKE

SEQ ID No. 7: human R β extracellular domain

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT

SEQ ID No. 8: linker segment L1

SGGGSGGGGSGGGGSGGGGSGGGSLQ

SEQ ID No. 9: linker segment L1

GGGGS

SEQ ID No. 10: linker segment L2(MMP9)

GGGGSPVGLIGGGGGS

SEQ ID No. 11: linker segment L2(MMP14)

GGGGSSGARYRWLTAGGGGS

SEQ ID No. 12: linker segment L2(MMP14)

GGGGSSGRIGFLRTAGGGGS

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

GGGGSSGAIGFLRTAGGGGS

SEQ ID No. 14: linker segment L2(MMP14)

GGGGSSGRAMHMYTAGGGGS

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

GGGGSSGAAMHMYTAGGGGS

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

GGGGSSGRSENIRTAGGGGS

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

GGGGSSGASENIRTAGGGGS

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

GGGGSSGRPENIRTAGGGGS

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

GGGGSSGAPENIRTAGGGGS

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

GGGGSSGLISHSITAGGGGS

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

GGGGSSGNLRSKLTAGGGGS

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

GGGGSSGVFSIPLTAGGGGS

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

GGGGSSGIKYHSLTAGGGGS

SEQ ID No. 24: mouse RA-IL15-Fc

GTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTSGGGSGGGGSGGGGSGGGGSGGGSLQNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 25: human RA-IL15-Fc

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSGGGGSGGGGSGGGGSGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 26: mouse IL15-RA-Fc

NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 27: human IL15-RA-Fc

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 28: mouse RB-L2-IL15-RA-Fc

AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKEGGGGSPVGLIGGGGGSNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

SEQ ID No. 29: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGARYRWLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 30: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRIGFLRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 31: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGAIGFLRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 32: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRAMHMYTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 33: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGAAMHMYTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 34: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRSENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 35: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGASENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 36: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRPENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 37: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGAPENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 38: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGLISHSITAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 39: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGNLRSKLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 40: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGVFSIPLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 41: human RB-L2-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGIKYHSLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No. 42: human RB-L1-IL15-RA-Fc

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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.